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miR-192, miR-194 and miR-215: a convergent microRNA network suppressing tumor progression in renal cell carcinoma

miR-192, miR-194 and miR-215: a convergent microRNA network suppressing tumor progression in... Abstract MicroRNAs (miRNAs) play a crucial role in tumor progression and metastasis. We, and others, recently identified a number of miRNAs that are dysregulated in metastatic renal cell carcinoma compared with primary renal cell carcinoma. Here, we investigated three miRNAs that are significantly downregulated in metastatic tumors: miR-192, miR-194 and miR-215. Gain-of-function analyses showed that restoration of their expression decreases cell migration and invasion in renal cell carcinoma cell line models, whereas knockdown of these miRNAs resulted in enhancing cellular migration and invasion abilities. We identified three targets of these miRNAs with potential role in tumor aggressiveness: murine double minute 2, thymidylate synthase, and Smad Interacting protein 1/zinc finger E-box binding homeobox 2. We observed a convergent effect (the same molecule can be targeted by all three miRNAs) and a divergent effect (the same miRNA can control multiple targets) for these miRNAs. We experimentally validated these miRNA–target interactions using three independent approaches. First, we observed that miRNA overexpression significantly reduces the mRNA and protein levels of their targets. In the second, we observed significant reduction of the luciferase signal of a vector containing the 3'UTR of the target upon miRNA overexpression. Finally, we show the presence of inverse correlation between miRNA changes and the expression levels of their targets in patient specimens. We also examined the prognostic significance of miR-215 in renal cell carcinoma. Lower expression of miR-215 is associated with significantly reduced disease-free survival time. These findings were validated on an independent data set from The Cancer Genome Atlas. These results can pave the way to the clinical use of miRNAs as prognostic markers and therapeutic targets. Introduction Renal cell carcinoma (RCC) accounts for about 90% of the adult kidney cancers ( 1 ) and is one of the top 10 prevalent cancers in North America. The incidence of RCC is steadily rising in the past few decades ( 2 ). It is also an aggressive tumor with 35% overall mortality and 30% metastatic potential. Favorable prognosis of RCC is associated with early diagnosis and treatment, whereas patients diagnosed at the metastatic stage have poor prognosis with only 9% 5 year survival rate. Ninety percent of cancer associated mortality in RCC is due to metastasis ( 3 ). Unfortunately, there are no biomarkers available to accurately predict the prognosis of RCC. Therefore, there is an urgent need for more understanding of the pathogenesis of RCC metastasis as a crucial step towards identification of prognostic markers and development of targeted therapies for this aggressive tumor. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression by binding to their target genes. They regulate critical biological processes, including development, cell differentiation, proliferation and apoptosis ( 4 ). They also play important roles in tumor development and metastasis ( 5 ). miRNAs were found to be differentially expressed in cancer and were shown to target key molecules involved in tumor progression ( 6 ). miRNA dysregulation in RCC is recently reported ( 7 , 8 ), and their involvement in RCC pathogenesis is also documented ( 9–12 ). They have potential utilities as cancer biomarkers and therapeutic targets ( 13 , 14 ). We, and others, identified a number of miRNAs that are differentially expressed in metastatic RCC compared with primary tumors ( 15–17 ). miR-192, miR-194 and miR-215 were among the most significantly downregulated in metastatic clear cell renal cell carcinoma (ccRCC). These three miRNAs are highly enriched in the normal kidney ( 18 ), and miR-192 and miR-194 were reported to be strongly expressed in renal cortex ( 19 ). All three miRNAs can be induced by p53 ( 20 ) and they are also reported to be p53 positive regulators through an autoregulatory loop ( 21 ). A recent study reported the role of these miRNAs in endometrial cancer progression and suggested their potential therapeutic utility ( 22 ). Also, miR-194 overexpression is reported to suppress liver cancer metastasis. The three miRNAs are found in two clusters: the miR-215/miR-194-1 cluster on chromosome 1 (1q41) and the miR-192/miR-194-2 cluster on chromosome 11 (11q13.1). miR-194-1 and miR-194-2 have the same mature sequence that are derived from two different precursors on two chromosomal locations. miR-192 and miR-215 are closely related with similar seed sequence. In this study, we delineate the functional involvement of miR-192, miR-194 and miR-215 in RCC progression. We provide experimental evidence that these miRNAs affect cell migration and invasion abilities. We also identified three targets of these miRNAs that are related to cancer aggressiveness; Murine double minute 2 (MDM2), thymidylate synthase (TYMS) and Smad Interacting protein 1/zinc finger E-box binding homeobox 2 (SIP1/ZEB2). We experimentally validated the miRNA–target interactions using three independent approaches in vitro and in vivo . We finally provide preliminary evidence on the potential significance of miR-215 as a prognostic marker in RCC. Our findings document the active involvement of miRNAs in kidney cancer progression. We also uncover an miRNA network with convergent properties (where multiple miRNAs target the same molecule). We further document the presence of a divergent effect of these miRNAs with the same miRNA being able to simultaneously control a number of targets. Materials and methods Specimen collection Twenty primary ccRCC cancerous tissue specimens and 61 formalin-fixed paraffin-embedded (FFPE) tissues were collected from St. Michael’s Hospital, and the Ontario Tumor Bank, Toronto, Canada. Expression data from additional 218 patients with primary ccRCC were obtained from The Cancer Genome Atlas (TCGA) Database. Fresh specimens were collected immediately after resection, snap frozen in liquid nitrogen, and stored at −80ºC until total RNA extraction. Areas of pure tumor tissues were identified by a pathologist. Samples were taken from areas with no hemorrhage or necrosis and multiple sections were mixed from the same tumor to compensate for tumor heterogeneity. All the procedures were approved by the Research Ethics Board at St. Michael’s Hospital, Toronto, Canada. RNA extraction and quantitative reverse transcription–polymerase chain reaction Two milligrams of fresh frozen ccRCC tissues were used for nucleic acid isolation. For formalin-fixed tumors, nucleic acid isolation was done using six cores of pure tumor areas from formalin-fixed paraffin-embedded tissues of ccRCC (each core was 1.0mm in diameter). Total RNA was extracted using the miRNeasy Kit (Qiagen, Mississauga, Canada) according to the manufacturer’s protocol. RNA quality and concentration were determined spectrophotometrically. For miRNA analyses, miRNA-specific reverse transcription was performed with 5 ng total RNA using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) as described by the manufacturer for miR-192, miR-194 and miR-215. Thermal cycling conditions are shown in Supplementary Table 1A . Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) was performed using the TaqMan microRNA Assay® Kit on the Step One™ Plus Real-Time PCR System (Applied Biosystems). Thermal cycling conditions were according to the manufacturer’s fast protocol, and all reactions were performed in triplicate. Relative expression was determined using the ΔΔC T method, and expression values were normalized to small nuclear RNA, RNU48 (Applied Biosystems), which was shown to be stably expressed in ccRCC tissues ( 23 ). Thermal cycling conditions are shown in Supplementary Table 1B . For the expression analyses of target genes, the primer sequences are shown in Supplementary Table 2 . Reverse transcription was performed with High capacity RNA-to-cDNA kit (Applied Biosystems) as per the manufacturer’s instructions. Thermal cycling conditions are shown in Supplementary Table 1C . qRT–PCR was performed using the Fast Syber Green Master Mix (Applied Biosystems). Peptidylprolyl isomerase A (cyclophilin A) or hypoxanthine phosphoribosyltransferase 1 was used as endogenous controls. Thermal cycling conditions are shown in Supplementary Table 1D . Cell culture and miRNA transfection RCC cell lines 786-O, ACHN and CAKI-1 were obtained from American Type Culture Collection (ATCC; Manassas, VA) and were grown according to manufacturer’s protocol. Pre-miR™ precursors and anti-miR™ miR inhibitors for miR-192, miR-194 and miR-215 were purchased from Applied Biosystems. Cells were transfected using SiPORT™ NeoFX ™ transfection agent (Ambion, Austin, TX) as recommended by the manufacturer and described in our previous publications ( 24 , 25 ). Briefly, ‘transfection agent’ was diluted in Opti-MEM® Reduced Serum Media (Invitrogen, Carlsbad, CA) and incubated for 10 min at room temperature. miRNA precursors and inhibitors were diluted in the same Media to a final concentration of 30 nM and then combined with transfection agent and incubated for 10 min at room temperature. Transfection mixtures were added to the cell culture plate and overlaid with cell suspensions. Cells were then incubated at 37ºC and 5% CO 2 . Three separate transfections were performed, and each was analyzed in triplicate. Transfection efficiency was confirmed using BLOCK-IT Fluorescent Oligo (Invitrogen). Migration assay 786-O cells were seeded in a 12-well plate, and transfected with SiPORT™ NeoFX ™ transfection agent, scrambled miRNA, miR-192, miR-194 and miR-215 or their inhibitors, or co-transfected with the miRNA and its inhibitor. Twenty four hours after transfection, the cell monolayer was wounded using a 200 μl pipette tip. Hydroxyurea (100 mM) was added to the cell culture to inhibit cell proliferation. Photomicrographs were taken every 30 min starting at the time of wounding (0 h) and continued up to 9 h using a microscope in an incubation chamber with 37ºC and 5% CO 2 . This microscope was programmed to take a series of photomicrographs at the exact place. Image J Software (National Institutes of Health, Bethesda, MD, http://rsbweb.nih.gov/ij/ ) was used for cell migration analysis. Percent cell-free area was calculated as [(cell-free area 9h /cell-free area 0h ) × 100] and cell migration rate was displayed as the percent of cell covered area (100− percent cell-free area). Each experiment was performed in triplicate. Invasion assay The effect of miR-192, miR-194 and miR-215 on cellular invasion was examined using BD BioCoat Matrigel Invasion Chamber (BD Biosciences, Bedford, MA). 786-O and ACHN cells were transfected with SiPORT™ NeoFX ™ transfection agent, scrambled miRNA, miR-192, miR-194, miR-215 or their inhibitors, or co-transfected with the miRNA and its inhibitor. Twenty four hours later, cells were trypsinized and resuspended in low serum media (Dulbecco’s modified Eagle’s medium supplemented with 0.5% fetal bovine serum). Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum was used as a chemoattractant, added to the bottom chamber, and cells (5.0 × 10 4 cells/ml) were plated on the upper chamber. Cells were incubated for 22 h at 37ºC and 5% CO 2 in a humidified tissue culture incubator. After incubation, non-invading cells were removed from the upper surface of the membrane and cells on the lower surface were stained with Diff-Quick. The experiment was performed in triplicate. Photomicrographs at ×40 magnification in three fields were taken and invading cells were counted. The percentage invasion was calculated [(mean number of cells that invaded through Matrigel insert/mean number of cells that migrated through the control insert membrane) × 100]. Cell proliferation assay Cellular proliferation was measured by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (Roche, Mississauga, Ontario) assay. Cells were plated at 6.0 × 10 3 cells per well in a 96-well plate and transfected with SiPORT™ NeoFX ™ transfection agent, miR-192, miR-194 and miR-215, or co-transfected with the miRNA and its inhibitor. Cells were incubated for 3 days after which 10 μl of 5mg/ml solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in phosphate-buffered saline was added and incubated for 4 h at 37°C. The precipitate was then solubilized in 100 μl solubilization solution (10% SDS in 0.01M HCl) and incubated at 37°C overnight. The absorbance of each well was measured at a wavelength of 550 nm. Each test was repeated in triplicate. Western blot CAKI-1 cells were transfected with miR-192, miR-194, or miR-215 or co-transfected with the miRNA and its inhibitor. Forty eight hours later, cells were lysed using NETN lysis buffer (pH 8.0) with protease inhibitor cocktail tablets (Roche). Proteins were collected after centrifugation at 21 000 g for 10 min at 4°C. BCA Protein Assay Reagent (Pierce Biotechnology, Rockford, IL) was used to determine protein concentrations. Total cellular proteins were separated in a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a nitrocellulose membrane. The membrane was blocked with 5% bovine serum albumin or 5% milk in Tris-buffered saline with Tween 20 and incubated with primary antibodies diluted in blocking solution overnight at 4ºC with shaking. The membranes were then washed with Tris-buffered saline with Tween 20 and incubated with appropriate secondary antibody diluted in blocking solution for 1 h. The following antibodies were used in this study: anti-Thymidylate Synthase (Millipore Corporation), MDM2 antibody (Santa Cruz Biotechnology) and anti-SIP1/ZEB2 (Abcam, Cambridge, MA). Membranes were stripped and re-probed for anti-α-tubulin (Cell Signalling Technology Inc.) as a loading control. Western blot photos were taken and quantified using the VersaDoc Imaging System (Bio-Rad, Hercules, CA), QuantityOne and Image Lab 3.0 programs. Luciferase assay Luciferase reporter plasmids containing the 3′UTR of TYMS, MDM2 and ZEB2 were obtained from SwitchGear Genomics (Menlo Park, CA). Empty vector was used as a positive control. 786-O cells were seeded in 96-well plates. On the second day, cells were transfected with the Luciferase reporter plasmids containing the 3′UTR of TYMS, MDM2 and ZEB2, the empty vector, or co-transfected with the plasmids and miRNAs (miR-192, miR-194 or miR-215) using the DharmaFect Duo transfection agent (Dharmacon, Thermoscientific) as recommended by the manufacturer. On the third day, luciferase activity was measured using the LightSwitch Luciferase Assay Reagents (SwitchGear Genomics, Menlo Park, CA). Luminescence was calculated for each construct (luminescence = specific miRNA/non-targeting control). Survival analysis miR-215 expression was measured using qRT–PCR in 61 FFPE tissues of primary ccRCC. Survival analysis was performed by constructing Kaplan–Meier disease-free survival (DFS) curve. DFS was defined as the time between the initial resection of the kidney tumor and the occurrence of recurrence or metastasis. Bioinformatic analysis ‘Level 3’ miRNA expression data (normalized gene expression generated using the Illumina GA miRNASeq platform) for miR-215 in 218 patients with primary ccRCC and overall survival data were obtained from The Cancer Genome Atlas (TCGA). The X-tile algorithm ( 26 ) was used to generate a prognostic optimal cutoff point to dichotomize miR-215 miRNA expression as ‘ miR-215 – High Expression’ and ‘ miR-215 – Low Expression’ using the lowest Monte Carlo P value <0.05. TCGA data types, platforms and methodologies are as described previously (The Cancer Genome Atlas Research Network 2008). Phylogenetic analysis The University of California Santa Cruz (UCSC) Genome Browser was used for sequence comparison of miR-192, miR-194 and miR-215. Conservation among species of these miRNAs was examined with sequence alignment in the genomes of 28 vertebrate species, including 17 mammalian species. Results Overexpression of miR-192, miR-194 and miR-215 decreases cellular migration rate We first checked the endogenous expression levels of miR-192, miR-194 and miR-215 in a number of kidney cancer cell lines. The 786-O, ACHN and CAKI-1 cell lines were found to have very low expression (compared with a pool of patient primary ccRCC tumor tissues) ( Supplementary Figure 1 ). The cells were then transfected with each of these miRNAs. Successful transfection was confirmed by qRT–PCR by comparing the pre- to post- transfection levels of the miRNA ( Supplementary Figure 2 ). The efficiency of the anti-miR decreased when the cell doubled. However, the cells maintained enough levels during the experimental procedures (up to 3 days). Next, we investigated the effect of these miRNAs on cell migration using wound healing assay. The 786-O cells were transfected with miR-192, miR-194, miR-215, or their inhibitors, or co-transfected with each of these miRNAs and its inhibitor. A number of controls were also used, including untransfected cells, cells transfected with transfection agent only, an miRNA with no effect on migration (as a negative control) and cells transfected with positive control (an miRNA with known effect on migration). Transfection with each of the three miRNAs resulted in significant reduction in the rate of cell migration compared with controls. As shown in Figure 1A and B , overexpression of miR-192 significantly reduced cellular migration compared with untransfected cells, cells transfected with transfection reagent only ( P = 0.0045), anti-miR-192 ( P = 0.0061), or scrambled miRNA control ( P = 0.0094). Also, miR-194 significantly slowed cellular migration compared with control cells transfected with transfection agent only or with anti-miR-194 ( P = 0.0105 and P = 0.0196, respectively) ( Figure 1C ). The same was observed for miR-215, where transfected cells showed significantly reduced cell migration rate compared with transfection reagent control ( P = 0.0143) ( Figure 1D ). The co-transfection of the miRNA and its inhibitor resulted in restoration of the rate of cellular migration, further confirming our results ( Figure 1). We were not able to identify other cell lines with overexpression of these miRNAs and thus knockdown experiments were not performed. Fig. 1. Open in new tabDownload slide Overexpression of miR-192, miR-194, or miR-215 has negative effect on cellular migration. ( A ) Representative photomicrographs showing the effect of miR-192 expression on the migration rate of the 786-O RCC cell line. The top row shows the cells at the time of wounding (0h), and the bottom row shows cellular migration after 9 h. Overexpression of miR-192 led to significant reduction in the rate of cell migration, with incomplete wound closure after 9 h, compared with controls. This was restored when co-transfecting miR-192 and its inhibitor. ( B ) Representative bar graph showing the effect of miR-192 on cell migration. Comparable results were obtained for miR-194 ( C ), and miR-215 ( D ). Overexpression of miR-192, miR-194 and miR-215 reduces cellular invasion We also examined the effect of miR-192, miR-194 and miR-215 on cellular invasion ability using 786-O and ACHN cell line models. Appropriate controls were used as above. Cells transfected with miR-192, miR-194 or miR-215 showed significant decrease in cellular invasion compared with untransfected cells, cells transfected with ‘transfe c tion agent’ only, scrambled miRNA or each of these miRNAs inhibitor. The rate of cellular invasion was partially restored when co-transfecting the miRNA and its inhibitor ( Figure 2 and Supplementary Figure 3 ). Fig. 2. Open in new tabDownload slide miR-192, miR-194 and miR-215 have negative effect on cellular invasion. ( A ) Representative photomicrographs showing the effect of miR-194 on cell migration in 786-O RCC cell line by comparing the number of cells invaded through the matrigel inserts to the number of cells invaded through the control inserts. Control inserts are shown in the top panel and matrigel inserts in the lower panel. miR-194 transfection resulted in significant reduction of cell invasion ability compared with untransfected cells and cells transfected with ‘transfection agent’ only, scrambled miRNA (negative control), and anti-miR-194. Partial restoration of cell invasion was obtained when co-transfection with miR-194 and its inhibitor. ( B ) A representative bar graph showing the effect of miR-194 on cell invasion. Comparable results were obtained for miR-192 ( C ) and miR-215 ( D ). Overexpression of miR-192, miR-194 and miR-215 has insignificant effects on cellular proliferation We assessed the effect of miR-192, miR-194 and miR-215 on cellular proliferation. Three kidney cancer cell lines; ACHN, CAKI-1 and 786-O, were transfected with miR-192, miR-194 or miR-215. These three miRNAs showed little insignificant reduction of cellular proliferation in ACHN cells ( Supplementary Figure 4 ). There was no significant effect on cell proliferation in CAKI-1 and 786-O cell lines. miR-192, miR-194 and miR-215 can target ZEB2, MDM2 and TYMS To elucidate the mechanisms by which 192, miR-194 and miR-215 can affect cellular processes involved in metastasis as cellular migration and invasion, we performed target prediction analysis and identified a number of pathways and targets that are known to be involved in tumor progression ( Supplementary Table 3 ). It is worth mentioning that targets of these three miRNAs are overlapping because of the high degree of similarity in the seed sequence of the miRNAs. We selected three potential targets: MDM2, TYMS and Smad Interacting protein 1/zinc finger E-box binding homeobox 2 (SIP1/ZEB2) for experimental validation. These molecules are reported in the literature to contribute to tumor progression and metastasis. We experimentally validated the miRNA–target interactions using three independent approaches. The first is by measuring the effect of miRNA overexpression on mRNA and protein levels of the predicted target. The second approach was to measure the effect of miRNA overexpression on a luciferase signal of a vector containing the 3'UTR of the predicted target. The third is to examine the presence of inverse correlation between miRNA expression and the expression levels of their targets in vivo in patient tissues. In the first approach, we examined the effect of overexpression of these three miRNAs on the expression of the targets. We screened a number of kidney cancer cell lines and identified three RCC cell line models (786-O, ACHN and CAKI-1) with high endogenous expression levels of ZEB2, MDM2 and TYMS ( Supplementary Figure 5 ). We then compared the level of expression of these targets, at both the mRNA and protein levels, before and after transfection of each of these miRNAs. At the mRNA level, overexpression of each of miR-192, miR-194 and miR-215 significantly decreases ZEB2 expression in 786-O ( Figure 3A–C ), CAKI ( Supplementary Figure 6A ) and ACHN cell lines ( Supplementary Figure 6B ) compared with control cells. Co-transfection of these cells with the miRNAs and their inhibitors was able to restore ZEB2 expression to almost normal levels. Fig. 3. Open in new tabDownload slide miR-192, miR-194 and miR-215 can target ZEB2. Representative bar graphs showing that ZEB2 expression was significantly decreased, at the mRNA level, in 786-O cells, upon transfection of miR-192 ( A ), miR-194 ( B ) and miR-215 ( C ). These effects were partially restored upon the co-transfection of the miRNA and its inhibitor. Expressions are shown as relative expression values compared with control untrasnfected cells to the far left. NC; negative control of scrambled miRNA sequences. We also validated miRNA–TYMS interactions in CAKI-1, 786-O and ACHN cells. Overexpression of any of the three miRNAs resulted in significant reduction in TYMS expression, at the mRNA level, in CAKI-1 ( Supplementary Figure 7A – Supplementary Data ), 786-O ( Supplementary Figure 7D ) and ACHN cells ( Supplementary Figure 7E ). The most significant drop in the level of TYMS was seen with miR-192. Transfection with miR-194 or miR-215 caused less reduction of TYMS levels, although this was still statistically significant. Similarly, MDM2 expression was significantly decreased after miR-192, miR-194 or miR-215 overexpression in ACHN ( Supplementary Figure 8A – Supplementary Data ), CAKI-1 and 786-O cells ( Supplementary Figure 8D – Supplementary Data ). It should be noted, however, that the degree of target suppression was variable among the different miRNAs in different cell lines. This can be due to a number of factors, including the number of recognition sites (miRNA response elements) in each target and other cell-specific factors. We also examined the effects of miR-192, miR-194 and miR-215 on ZEB2, MDM2 and TYMS expression at the protein level. MDM2 protein expression was significantly reduced when CAKI-1 cells were transfected with miR-192, miR-194, or miR-215 ( P < 0.0001, P = 0.0015 and P < 0.0001, respectively). This effect was largely overcome when co-transfecting the cells with each of these miRNA and its inhibitor ( Figure 4A and B ). The protein expression of TYMS was similarly significantly decreased in cells transfected with miR-192, miR-194 or miR-215 compared with control cells ( P = 0.0320, P = 0.0052 and P = 0.0070, respectively). Protein expression levels were partially restored in cells co-transfected with the miRNA and its inhibitor ( Supplementary Figure 9 ). Our results also show that overexpression of miR-192, miR-194, or miR-215 significantly reduced ZEB2 protein expression in CAKI-1 cells compared with control cells and that expression levels can be partially restored when cells are co-transfected with each of these miRNAs and its inhibitor ( Supplementary Figure 10 ). Fig. 4. Open in new tabDownload slide MDM2 is a target of miR-192, miR-194 and miR-215 (A and B). ( A ) Representative western blot analysis showing decreased MDM2 protein level in CAKI-1 cells transfected with miR-192, miR-194 or miR-215 compared with cells transfected with the transfection agent only or co-transfected with the miRNA and its inhibitor. ( B ) Bar graph showing the quantification of changes in protein expression levels in the gel. Expression values were compared with control untransfected cells. α-Tubulin was used as a loading control. MDM2, ZEB2 and TYMS are targets of miR-192, miR-194 and miR-215 (C–E). Co-transfection of 786-O cells with reporter vectors containing the 3'UTR of MDM2 ( C ), ZEB2 ( D ) or TYMS ( E ) and miR-192, miR-194 or miR-215 significantly decreased luciferase activity compared with cells transfected with the constructs only. In the second approach, we further validated these miRNA–target interactions by measuring the change of fluorescence signal of a luciferase vector containing the 3'UTR of the target upon miRNA transfection. This system provides more evidence about a ‘direct’ interaction between the miRNA and its target. The 786-O cell line was transfected with the Luciferase reporter plasmids containing the 3'UTR of TYMS, MDM2 or ZEB2 or co-transfected with the plasmids and the miRNA (miR-192, miR-194 or miR-215). Luciferase activity decreased significantly in cells co-transfected with plasmids of the target and the targeting miRNA compared with cells transfected with target plasmid only ( Figure 4C–E ). The third approach was to examine the presence of negative correlation between miR-192, miR-194 and miR-215 and their predicted targets—MDM2, ZEB2 and TYMS—by qRT–PCR in vivo in patient tissues. We examined the expression levels of the three miRNAs and their targets on 20 fresh frozen primary ccRCC tissues. As shown in Figure 5 , we observed a negative correlation between these three miRNAs and their targets, with lower expression levels of the miRNA associated with higher expression of its target in the same patient, and vice versa. This provides indirect evidence that MDM2, ZEB2 and TYMS are targets of miR-192, miR-194 and miR-215 in vivo . Fig. 5. Open in new tabDownload slide A negative correlation was observed between the expression levels of miR-192 and miR-215 and their predicted targets, ZEB2 and MDM2 in patient tissues. The graphs show pair-wise comparison between each of the miRNAs and its target for each patient. Our results show the presence of a negative correlation between these miRNAs and their predicted targets with lower expression of miR-192 and miR-215 associated with higher expression of MDM2 ( A and B ) and ZEB2 ( C and D ), and vice versa. This provides indirect evidence that ZEB2 and MDM2 are targets of miR-192 and miR-215. Patient cases are shown on the x -axes and the relative expression values are presented along the y -axes. Expression levels are shown as relative expression values of the miRNAs and their targets normalized against an internal control in the same specimen. An interesting observation was the high degree of overlap between targets of all three miRNAs. This was predicted by target prediction algorithms and experimentally validated. We further confirmed this overlap be performing sequence alignment of these miRNAs and the 3'UTR of their predicted targets (MDM2, ZEB2 and TYMS) using the European Molecular Biology Open Software Suite ( http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html ). Interestingly, miR-192 and miR-215 shared not only the same seed sequence but also their mature sequences, which are almost identical. They only differ in two nucleotides (the 1st and the 20th nucleotides). In addition, miR-194 showed partial complementarity with both miRNAs (data not shown). Also, all three miRNAs show partial complementarity with the 3'UTR of the three targets. We further validated the target predictions using an independent algorithm (the online microRNA prediction tool utilizing the PITA algorithm based on sequence analysis, http://132.77.150.113/pubs/mir07/mir07_prediction.html ), as a shown in Supplementary Table 4 . miR-215 as a prognostic marker for RCC As miR-215 was significantly differentially expressed between primary and metastatic ccRCC ( 17 ), we hypothesized that this miRNA can serve as a potential prognostic marker. We examined the expression of miR-215 with the ‘gold standard’ qRT–PCR using miRNA-specific probes in 61 formalin-fixed paraffin-embedded tissues of primary ccRCC. Kaplan–Meier survival curve ( Figure 6A ) showed that lower expression of miR-215 was associated with significantly decreased DFS time (patients with lower miR-215 expression = 26.4 months versus patients with higher miR-215 expression = 49.2 months, P = 0.0320). Fig. 6. Open in new tabDownload slide ( A ) Kaplan–Meier DFS plot comparing miR-215 expression in RCC patients. miR-215 expression was dichotomized into high and low expression categories. Patients with lower miR-215 expression had significantly lower DFS compared with those with high expression ( P = 0.032). ( B ) Kaplan–Meier overall survival (OS) plot comparing miR-215 expression in RCC patients. ‘Level 3’ miRNA expression data (normalized gene expression generated using the Illumina GA miRNASeq platform) for miR-215 in ccRCC and overall survival data were obtained from The Cancer Genome Atlas (TCGA). Lower expression of miR-215 is significantly associated with worse survival ( P = 0.0032). We further validated these data in silico on an independent data set of 218 primary ccRCC cases with available overall survival data from The Cancer Genome Atlas and observed lower expression of miR-215 to be associated with significantly worse survival ( P = 0.0032) ( Figure 6B ). miR-192, miR-194 and miR-215 are conserved among species We used the University of California Santa Cruz (UCSC) Genome Browser for sequence comparison of miR-192, miR-194 and miR-215 among species. Our analysis shows the high conservation of these three miRNAs among 28 species ( Supplementary Figure 11 ). Conservation among species indicates that these miRNAs may have vital functions that are maintained during their evolution. Discussion In our previous work, we identified an miRNA signature of metastatic ccRCC. miR-192, miR-194 and miR-215 were significantly differentially expressed in metastatic compared with primary ccRCC. In this study, we provide evidence that these three miRNAs can be involved in RCC progression and that miR-215 is a potential prognostic marker for ccRCC. We also identified and validated three of their target genes; ZEB2, MDM2 and TYMS. miR-192, miR-194 and miR-215 are highly enriched in the normal kidney ( 18 ), indicating that they play a role in kidney development and differentiation. In our previous work, we reported that these three miRNAs are significantly downregulated in primary ccRCC compared with normal kidney tissue ( 8 ). These three miRNAs are further downregulated in metastatic ccRCC ( 17 ). Taken together, this step-wise downregulation indicates their involvement in controlling tumor-suppressor pathways. This is also supported by the fact that these three miRNAs are present in two clusters. Members of the same cluster are usually co-expressed and co-regulated. The miR-215/miR-194-1 cluster is located within the common fragile site FRA1H (1q41–q42.1) that is deleted in many types of cancers ( 27 ). 1q41 was reported to be associated with breast and esophageal cancer metastasis. The downregulation of these three miRNAs was also reported in other cancers including nephroblastoma, myeloma, colon and gastric cancers ( 20 , 21 ). Our results show that the main effect of these three miRNAs is on tumor migration and invasion. Little effect was shown on tumor proliferation although this was not statistically significant. This is not unprecedented. Recent literature showing that the key contribution of some of these miRNAs is on tumor invasion and migration abilities, which are essential features for metastasis and epithelial to mesenchymal transition (EMT) ( 28 , 29 ). Moreover, our results should be interpreted with caution as it might be cell or tissue type specific. Others have also shown more significant effect on cell proliferation in other cell types. Our in silico analysis showed that miR-192, miR-194 and miR-215 can target ZEB2, MDM2 and TYMS. MDM2 is a key inhibitor of p53. It activates hypoxia inducible factor 1 alpha and vascular endothelial growth factor activity ( 30 ). MDM2 overexpression is reported to be associated with metastasis in many cancers. Its overexpression decreases E-cadherin levels with subsequent increase in cell motility in breast carcinoma ( 31 ). It was also reported to increase cell motility and invasiveness in RCC ( 32 ). TYMS is essential for DNA synthesis and its inhibition is reported to block DNA replication and repair ( 33 ). TYMS SNP variations are associated with increased risk of RCC ( 34 ). TYMS upregulation was reported in RCC and correlates with tumor progression ( 35 ). It is also a target of the 5-fluorouracil used in advanced RCC ( 36 ). It was also shown to have a prognostic significance in bladder cancer. SIP1/ZEB2 represses E-cadherin and was shown to be involved in EMT, which is a key process in tumor progression. ZEB2 can mediate the hypoxia inducible factor 1 repression effect of E-cadherin in RCC. We experimentally validated our in silico predictions using independent approaches. These results, however, should be interpreted with caution, due to the possibility of indirect targeting and off-target effects. The luciferase system can provide more evidence of direct miRNA–target interactions. Finally, we provide in vivo evidence by documenting the negative correlation between the expression of these three miRNAs and these targets in ccRCC patients’ tissues. Our findings are also consistent with recent reports showing that miR-192 and miR-215 can target TYMS in colon cancer ( 37 ). MDM2 is also a target of these miRNAs in myeloma ( 21 ). Interestingly, these miRNAs occur in two clusters. Members of the same cluster can have coordinated effects. They may target the same molecule (convergent targeting) or may hit several molecules in the same or related biological pathways (divergent targeting), as demonstrated in recent reports ( 38 , 39 ). The remarkable target overlap can be explained by the fact that miR-192 and miR-215 have similar seed sequence which is highly important for miRNA–target interaction. It should be also noted that 3′ supplementary sites can enhance the seed pairing. Pairing to the 3′ region includes mainly the nucleotides 13–16. Furthermore, this 3′ pairing can also compensate for nucleotide mismatch in the seed region ( 40 ). miRNAs were reported to target not only the 3'UTR but also 5'UTR, coding regions, promoters and gene termini. Our results are not unprecedented; Senanayake et al. identified ACVR2B to be a common target for miR-192, miR-194 and miR-215 in renal childhood neoplasms ( 41 ). The involvement of these miRNAs in tumor progression is not surprising and is supported by previous reports. miR-192, miR-194 and miR-215 are induced by p53 and also they were reported to be p53 positive regulators ( 20 , 21 ). Kim et al. demonstrated that p53 can regulate EMT through targeting ZEB2 by miR-192 family. Also, Krishnamachary et al. showed that E-cadherin repression by hypoxia inducible factor 1 can be mediated by ZEB2 in RCC ( 42 ). Also, MDM2 overexpression correlates with tumor progression and metastasis in different cancers, including RCC ( 43–47 ). Our findings can also have therapeutic implications. TYMS is a target of the 5-fluorouracil anticancer agents that is now being considered in combination therapies for advanced RCC. Recently, using RCC xenograft model, 5-fluorouracil was shown to enhance the Sorafenib and Sunitinib antitumor effect ( 48 ). Recently, MDM2 is being investigated as a potential new therapeutic target in colon cancer ( 49 ). Also, Vastsyayan et al. demonstrated that MDM2 inhibitor (Nutlin-3) can enhance the effect of Sorafenib in RCC ( 50 ). The fact that one miRNA can hit multiple targets attracted the attention to the miRNAs as promising therapies in cancer. The different strategies for using miRNA therapy were recently reviewed including miRNA replacement using adeno-associated viral vectors and synthetic miRNA precursors in hepatocellular carcinoma and prostate cancer, respectively ( 51 ). Also, targeting miRNAs using synthetic molecules can be of therapeutic interest. In conclusion, we showed that miR-192, miR-194 and miR-215 have tumor suppressor effects on RCC by reducing the cellular migration and invasion abilities. We identified potential mechanisms through which these three miRNAs can negatively affect these biological processes by targeting key molecules involved in metastasis. These three miRNAs can have hundreds of targets and a global analysis of the overall spectrum of changes of miRNAs would be more suitable. However, due to limited resources we did a targeted approach to select the targets that are of clinical significance that was shown in other cancers. This however does not exclude the presence of other significant targets that are yet to be identified and validated. This is a net effect of miRNAs and there might be a number of underlined, direct and indirect, and sometimes being even opposing target effects. Also, we demonstrated that miR-215 can be potential prognostic marker in RCC. Our results can be a step forward towards developing a new therapeutic target in RCC. Funding Canadian Institute of Health Research (119606); Canadian Cancer Society (20185); Kidney Foundation of Canada. Conflict of Interest Statement: None declared. 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miR-192, miR-194 and miR-215: a convergent microRNA network suppressing tumor progression in renal cell carcinoma

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Oxford University Press
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10.1093/carcin/bgt184
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Abstract

Abstract MicroRNAs (miRNAs) play a crucial role in tumor progression and metastasis. We, and others, recently identified a number of miRNAs that are dysregulated in metastatic renal cell carcinoma compared with primary renal cell carcinoma. Here, we investigated three miRNAs that are significantly downregulated in metastatic tumors: miR-192, miR-194 and miR-215. Gain-of-function analyses showed that restoration of their expression decreases cell migration and invasion in renal cell carcinoma cell line models, whereas knockdown of these miRNAs resulted in enhancing cellular migration and invasion abilities. We identified three targets of these miRNAs with potential role in tumor aggressiveness: murine double minute 2, thymidylate synthase, and Smad Interacting protein 1/zinc finger E-box binding homeobox 2. We observed a convergent effect (the same molecule can be targeted by all three miRNAs) and a divergent effect (the same miRNA can control multiple targets) for these miRNAs. We experimentally validated these miRNA–target interactions using three independent approaches. First, we observed that miRNA overexpression significantly reduces the mRNA and protein levels of their targets. In the second, we observed significant reduction of the luciferase signal of a vector containing the 3'UTR of the target upon miRNA overexpression. Finally, we show the presence of inverse correlation between miRNA changes and the expression levels of their targets in patient specimens. We also examined the prognostic significance of miR-215 in renal cell carcinoma. Lower expression of miR-215 is associated with significantly reduced disease-free survival time. These findings were validated on an independent data set from The Cancer Genome Atlas. These results can pave the way to the clinical use of miRNAs as prognostic markers and therapeutic targets. Introduction Renal cell carcinoma (RCC) accounts for about 90% of the adult kidney cancers ( 1 ) and is one of the top 10 prevalent cancers in North America. The incidence of RCC is steadily rising in the past few decades ( 2 ). It is also an aggressive tumor with 35% overall mortality and 30% metastatic potential. Favorable prognosis of RCC is associated with early diagnosis and treatment, whereas patients diagnosed at the metastatic stage have poor prognosis with only 9% 5 year survival rate. Ninety percent of cancer associated mortality in RCC is due to metastasis ( 3 ). Unfortunately, there are no biomarkers available to accurately predict the prognosis of RCC. Therefore, there is an urgent need for more understanding of the pathogenesis of RCC metastasis as a crucial step towards identification of prognostic markers and development of targeted therapies for this aggressive tumor. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression by binding to their target genes. They regulate critical biological processes, including development, cell differentiation, proliferation and apoptosis ( 4 ). They also play important roles in tumor development and metastasis ( 5 ). miRNAs were found to be differentially expressed in cancer and were shown to target key molecules involved in tumor progression ( 6 ). miRNA dysregulation in RCC is recently reported ( 7 , 8 ), and their involvement in RCC pathogenesis is also documented ( 9–12 ). They have potential utilities as cancer biomarkers and therapeutic targets ( 13 , 14 ). We, and others, identified a number of miRNAs that are differentially expressed in metastatic RCC compared with primary tumors ( 15–17 ). miR-192, miR-194 and miR-215 were among the most significantly downregulated in metastatic clear cell renal cell carcinoma (ccRCC). These three miRNAs are highly enriched in the normal kidney ( 18 ), and miR-192 and miR-194 were reported to be strongly expressed in renal cortex ( 19 ). All three miRNAs can be induced by p53 ( 20 ) and they are also reported to be p53 positive regulators through an autoregulatory loop ( 21 ). A recent study reported the role of these miRNAs in endometrial cancer progression and suggested their potential therapeutic utility ( 22 ). Also, miR-194 overexpression is reported to suppress liver cancer metastasis. The three miRNAs are found in two clusters: the miR-215/miR-194-1 cluster on chromosome 1 (1q41) and the miR-192/miR-194-2 cluster on chromosome 11 (11q13.1). miR-194-1 and miR-194-2 have the same mature sequence that are derived from two different precursors on two chromosomal locations. miR-192 and miR-215 are closely related with similar seed sequence. In this study, we delineate the functional involvement of miR-192, miR-194 and miR-215 in RCC progression. We provide experimental evidence that these miRNAs affect cell migration and invasion abilities. We also identified three targets of these miRNAs that are related to cancer aggressiveness; Murine double minute 2 (MDM2), thymidylate synthase (TYMS) and Smad Interacting protein 1/zinc finger E-box binding homeobox 2 (SIP1/ZEB2). We experimentally validated the miRNA–target interactions using three independent approaches in vitro and in vivo . We finally provide preliminary evidence on the potential significance of miR-215 as a prognostic marker in RCC. Our findings document the active involvement of miRNAs in kidney cancer progression. We also uncover an miRNA network with convergent properties (where multiple miRNAs target the same molecule). We further document the presence of a divergent effect of these miRNAs with the same miRNA being able to simultaneously control a number of targets. Materials and methods Specimen collection Twenty primary ccRCC cancerous tissue specimens and 61 formalin-fixed paraffin-embedded (FFPE) tissues were collected from St. Michael’s Hospital, and the Ontario Tumor Bank, Toronto, Canada. Expression data from additional 218 patients with primary ccRCC were obtained from The Cancer Genome Atlas (TCGA) Database. Fresh specimens were collected immediately after resection, snap frozen in liquid nitrogen, and stored at −80ºC until total RNA extraction. Areas of pure tumor tissues were identified by a pathologist. Samples were taken from areas with no hemorrhage or necrosis and multiple sections were mixed from the same tumor to compensate for tumor heterogeneity. All the procedures were approved by the Research Ethics Board at St. Michael’s Hospital, Toronto, Canada. RNA extraction and quantitative reverse transcription–polymerase chain reaction Two milligrams of fresh frozen ccRCC tissues were used for nucleic acid isolation. For formalin-fixed tumors, nucleic acid isolation was done using six cores of pure tumor areas from formalin-fixed paraffin-embedded tissues of ccRCC (each core was 1.0mm in diameter). Total RNA was extracted using the miRNeasy Kit (Qiagen, Mississauga, Canada) according to the manufacturer’s protocol. RNA quality and concentration were determined spectrophotometrically. For miRNA analyses, miRNA-specific reverse transcription was performed with 5 ng total RNA using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) as described by the manufacturer for miR-192, miR-194 and miR-215. Thermal cycling conditions are shown in Supplementary Table 1A . Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) was performed using the TaqMan microRNA Assay® Kit on the Step One™ Plus Real-Time PCR System (Applied Biosystems). Thermal cycling conditions were according to the manufacturer’s fast protocol, and all reactions were performed in triplicate. Relative expression was determined using the ΔΔC T method, and expression values were normalized to small nuclear RNA, RNU48 (Applied Biosystems), which was shown to be stably expressed in ccRCC tissues ( 23 ). Thermal cycling conditions are shown in Supplementary Table 1B . For the expression analyses of target genes, the primer sequences are shown in Supplementary Table 2 . Reverse transcription was performed with High capacity RNA-to-cDNA kit (Applied Biosystems) as per the manufacturer’s instructions. Thermal cycling conditions are shown in Supplementary Table 1C . qRT–PCR was performed using the Fast Syber Green Master Mix (Applied Biosystems). Peptidylprolyl isomerase A (cyclophilin A) or hypoxanthine phosphoribosyltransferase 1 was used as endogenous controls. Thermal cycling conditions are shown in Supplementary Table 1D . Cell culture and miRNA transfection RCC cell lines 786-O, ACHN and CAKI-1 were obtained from American Type Culture Collection (ATCC; Manassas, VA) and were grown according to manufacturer’s protocol. Pre-miR™ precursors and anti-miR™ miR inhibitors for miR-192, miR-194 and miR-215 were purchased from Applied Biosystems. Cells were transfected using SiPORT™ NeoFX ™ transfection agent (Ambion, Austin, TX) as recommended by the manufacturer and described in our previous publications ( 24 , 25 ). Briefly, ‘transfection agent’ was diluted in Opti-MEM® Reduced Serum Media (Invitrogen, Carlsbad, CA) and incubated for 10 min at room temperature. miRNA precursors and inhibitors were diluted in the same Media to a final concentration of 30 nM and then combined with transfection agent and incubated for 10 min at room temperature. Transfection mixtures were added to the cell culture plate and overlaid with cell suspensions. Cells were then incubated at 37ºC and 5% CO 2 . Three separate transfections were performed, and each was analyzed in triplicate. Transfection efficiency was confirmed using BLOCK-IT Fluorescent Oligo (Invitrogen). Migration assay 786-O cells were seeded in a 12-well plate, and transfected with SiPORT™ NeoFX ™ transfection agent, scrambled miRNA, miR-192, miR-194 and miR-215 or their inhibitors, or co-transfected with the miRNA and its inhibitor. Twenty four hours after transfection, the cell monolayer was wounded using a 200 μl pipette tip. Hydroxyurea (100 mM) was added to the cell culture to inhibit cell proliferation. Photomicrographs were taken every 30 min starting at the time of wounding (0 h) and continued up to 9 h using a microscope in an incubation chamber with 37ºC and 5% CO 2 . This microscope was programmed to take a series of photomicrographs at the exact place. Image J Software (National Institutes of Health, Bethesda, MD, http://rsbweb.nih.gov/ij/ ) was used for cell migration analysis. Percent cell-free area was calculated as [(cell-free area 9h /cell-free area 0h ) × 100] and cell migration rate was displayed as the percent of cell covered area (100− percent cell-free area). Each experiment was performed in triplicate. Invasion assay The effect of miR-192, miR-194 and miR-215 on cellular invasion was examined using BD BioCoat Matrigel Invasion Chamber (BD Biosciences, Bedford, MA). 786-O and ACHN cells were transfected with SiPORT™ NeoFX ™ transfection agent, scrambled miRNA, miR-192, miR-194, miR-215 or their inhibitors, or co-transfected with the miRNA and its inhibitor. Twenty four hours later, cells were trypsinized and resuspended in low serum media (Dulbecco’s modified Eagle’s medium supplemented with 0.5% fetal bovine serum). Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum was used as a chemoattractant, added to the bottom chamber, and cells (5.0 × 10 4 cells/ml) were plated on the upper chamber. Cells were incubated for 22 h at 37ºC and 5% CO 2 in a humidified tissue culture incubator. After incubation, non-invading cells were removed from the upper surface of the membrane and cells on the lower surface were stained with Diff-Quick. The experiment was performed in triplicate. Photomicrographs at ×40 magnification in three fields were taken and invading cells were counted. The percentage invasion was calculated [(mean number of cells that invaded through Matrigel insert/mean number of cells that migrated through the control insert membrane) × 100]. Cell proliferation assay Cellular proliferation was measured by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (Roche, Mississauga, Ontario) assay. Cells were plated at 6.0 × 10 3 cells per well in a 96-well plate and transfected with SiPORT™ NeoFX ™ transfection agent, miR-192, miR-194 and miR-215, or co-transfected with the miRNA and its inhibitor. Cells were incubated for 3 days after which 10 μl of 5mg/ml solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in phosphate-buffered saline was added and incubated for 4 h at 37°C. The precipitate was then solubilized in 100 μl solubilization solution (10% SDS in 0.01M HCl) and incubated at 37°C overnight. The absorbance of each well was measured at a wavelength of 550 nm. Each test was repeated in triplicate. Western blot CAKI-1 cells were transfected with miR-192, miR-194, or miR-215 or co-transfected with the miRNA and its inhibitor. Forty eight hours later, cells were lysed using NETN lysis buffer (pH 8.0) with protease inhibitor cocktail tablets (Roche). Proteins were collected after centrifugation at 21 000 g for 10 min at 4°C. BCA Protein Assay Reagent (Pierce Biotechnology, Rockford, IL) was used to determine protein concentrations. Total cellular proteins were separated in a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a nitrocellulose membrane. The membrane was blocked with 5% bovine serum albumin or 5% milk in Tris-buffered saline with Tween 20 and incubated with primary antibodies diluted in blocking solution overnight at 4ºC with shaking. The membranes were then washed with Tris-buffered saline with Tween 20 and incubated with appropriate secondary antibody diluted in blocking solution for 1 h. The following antibodies were used in this study: anti-Thymidylate Synthase (Millipore Corporation), MDM2 antibody (Santa Cruz Biotechnology) and anti-SIP1/ZEB2 (Abcam, Cambridge, MA). Membranes were stripped and re-probed for anti-α-tubulin (Cell Signalling Technology Inc.) as a loading control. Western blot photos were taken and quantified using the VersaDoc Imaging System (Bio-Rad, Hercules, CA), QuantityOne and Image Lab 3.0 programs. Luciferase assay Luciferase reporter plasmids containing the 3′UTR of TYMS, MDM2 and ZEB2 were obtained from SwitchGear Genomics (Menlo Park, CA). Empty vector was used as a positive control. 786-O cells were seeded in 96-well plates. On the second day, cells were transfected with the Luciferase reporter plasmids containing the 3′UTR of TYMS, MDM2 and ZEB2, the empty vector, or co-transfected with the plasmids and miRNAs (miR-192, miR-194 or miR-215) using the DharmaFect Duo transfection agent (Dharmacon, Thermoscientific) as recommended by the manufacturer. On the third day, luciferase activity was measured using the LightSwitch Luciferase Assay Reagents (SwitchGear Genomics, Menlo Park, CA). Luminescence was calculated for each construct (luminescence = specific miRNA/non-targeting control). Survival analysis miR-215 expression was measured using qRT–PCR in 61 FFPE tissues of primary ccRCC. Survival analysis was performed by constructing Kaplan–Meier disease-free survival (DFS) curve. DFS was defined as the time between the initial resection of the kidney tumor and the occurrence of recurrence or metastasis. Bioinformatic analysis ‘Level 3’ miRNA expression data (normalized gene expression generated using the Illumina GA miRNASeq platform) for miR-215 in 218 patients with primary ccRCC and overall survival data were obtained from The Cancer Genome Atlas (TCGA). The X-tile algorithm ( 26 ) was used to generate a prognostic optimal cutoff point to dichotomize miR-215 miRNA expression as ‘ miR-215 – High Expression’ and ‘ miR-215 – Low Expression’ using the lowest Monte Carlo P value <0.05. TCGA data types, platforms and methodologies are as described previously (The Cancer Genome Atlas Research Network 2008). Phylogenetic analysis The University of California Santa Cruz (UCSC) Genome Browser was used for sequence comparison of miR-192, miR-194 and miR-215. Conservation among species of these miRNAs was examined with sequence alignment in the genomes of 28 vertebrate species, including 17 mammalian species. Results Overexpression of miR-192, miR-194 and miR-215 decreases cellular migration rate We first checked the endogenous expression levels of miR-192, miR-194 and miR-215 in a number of kidney cancer cell lines. The 786-O, ACHN and CAKI-1 cell lines were found to have very low expression (compared with a pool of patient primary ccRCC tumor tissues) ( Supplementary Figure 1 ). The cells were then transfected with each of these miRNAs. Successful transfection was confirmed by qRT–PCR by comparing the pre- to post- transfection levels of the miRNA ( Supplementary Figure 2 ). The efficiency of the anti-miR decreased when the cell doubled. However, the cells maintained enough levels during the experimental procedures (up to 3 days). Next, we investigated the effect of these miRNAs on cell migration using wound healing assay. The 786-O cells were transfected with miR-192, miR-194, miR-215, or their inhibitors, or co-transfected with each of these miRNAs and its inhibitor. A number of controls were also used, including untransfected cells, cells transfected with transfection agent only, an miRNA with no effect on migration (as a negative control) and cells transfected with positive control (an miRNA with known effect on migration). Transfection with each of the three miRNAs resulted in significant reduction in the rate of cell migration compared with controls. As shown in Figure 1A and B , overexpression of miR-192 significantly reduced cellular migration compared with untransfected cells, cells transfected with transfection reagent only ( P = 0.0045), anti-miR-192 ( P = 0.0061), or scrambled miRNA control ( P = 0.0094). Also, miR-194 significantly slowed cellular migration compared with control cells transfected with transfection agent only or with anti-miR-194 ( P = 0.0105 and P = 0.0196, respectively) ( Figure 1C ). The same was observed for miR-215, where transfected cells showed significantly reduced cell migration rate compared with transfection reagent control ( P = 0.0143) ( Figure 1D ). The co-transfection of the miRNA and its inhibitor resulted in restoration of the rate of cellular migration, further confirming our results ( Figure 1). We were not able to identify other cell lines with overexpression of these miRNAs and thus knockdown experiments were not performed. Fig. 1. Open in new tabDownload slide Overexpression of miR-192, miR-194, or miR-215 has negative effect on cellular migration. ( A ) Representative photomicrographs showing the effect of miR-192 expression on the migration rate of the 786-O RCC cell line. The top row shows the cells at the time of wounding (0h), and the bottom row shows cellular migration after 9 h. Overexpression of miR-192 led to significant reduction in the rate of cell migration, with incomplete wound closure after 9 h, compared with controls. This was restored when co-transfecting miR-192 and its inhibitor. ( B ) Representative bar graph showing the effect of miR-192 on cell migration. Comparable results were obtained for miR-194 ( C ), and miR-215 ( D ). Overexpression of miR-192, miR-194 and miR-215 reduces cellular invasion We also examined the effect of miR-192, miR-194 and miR-215 on cellular invasion ability using 786-O and ACHN cell line models. Appropriate controls were used as above. Cells transfected with miR-192, miR-194 or miR-215 showed significant decrease in cellular invasion compared with untransfected cells, cells transfected with ‘transfe c tion agent’ only, scrambled miRNA or each of these miRNAs inhibitor. The rate of cellular invasion was partially restored when co-transfecting the miRNA and its inhibitor ( Figure 2 and Supplementary Figure 3 ). Fig. 2. Open in new tabDownload slide miR-192, miR-194 and miR-215 have negative effect on cellular invasion. ( A ) Representative photomicrographs showing the effect of miR-194 on cell migration in 786-O RCC cell line by comparing the number of cells invaded through the matrigel inserts to the number of cells invaded through the control inserts. Control inserts are shown in the top panel and matrigel inserts in the lower panel. miR-194 transfection resulted in significant reduction of cell invasion ability compared with untransfected cells and cells transfected with ‘transfection agent’ only, scrambled miRNA (negative control), and anti-miR-194. Partial restoration of cell invasion was obtained when co-transfection with miR-194 and its inhibitor. ( B ) A representative bar graph showing the effect of miR-194 on cell invasion. Comparable results were obtained for miR-192 ( C ) and miR-215 ( D ). Overexpression of miR-192, miR-194 and miR-215 has insignificant effects on cellular proliferation We assessed the effect of miR-192, miR-194 and miR-215 on cellular proliferation. Three kidney cancer cell lines; ACHN, CAKI-1 and 786-O, were transfected with miR-192, miR-194 or miR-215. These three miRNAs showed little insignificant reduction of cellular proliferation in ACHN cells ( Supplementary Figure 4 ). There was no significant effect on cell proliferation in CAKI-1 and 786-O cell lines. miR-192, miR-194 and miR-215 can target ZEB2, MDM2 and TYMS To elucidate the mechanisms by which 192, miR-194 and miR-215 can affect cellular processes involved in metastasis as cellular migration and invasion, we performed target prediction analysis and identified a number of pathways and targets that are known to be involved in tumor progression ( Supplementary Table 3 ). It is worth mentioning that targets of these three miRNAs are overlapping because of the high degree of similarity in the seed sequence of the miRNAs. We selected three potential targets: MDM2, TYMS and Smad Interacting protein 1/zinc finger E-box binding homeobox 2 (SIP1/ZEB2) for experimental validation. These molecules are reported in the literature to contribute to tumor progression and metastasis. We experimentally validated the miRNA–target interactions using three independent approaches. The first is by measuring the effect of miRNA overexpression on mRNA and protein levels of the predicted target. The second approach was to measure the effect of miRNA overexpression on a luciferase signal of a vector containing the 3'UTR of the predicted target. The third is to examine the presence of inverse correlation between miRNA expression and the expression levels of their targets in vivo in patient tissues. In the first approach, we examined the effect of overexpression of these three miRNAs on the expression of the targets. We screened a number of kidney cancer cell lines and identified three RCC cell line models (786-O, ACHN and CAKI-1) with high endogenous expression levels of ZEB2, MDM2 and TYMS ( Supplementary Figure 5 ). We then compared the level of expression of these targets, at both the mRNA and protein levels, before and after transfection of each of these miRNAs. At the mRNA level, overexpression of each of miR-192, miR-194 and miR-215 significantly decreases ZEB2 expression in 786-O ( Figure 3A–C ), CAKI ( Supplementary Figure 6A ) and ACHN cell lines ( Supplementary Figure 6B ) compared with control cells. Co-transfection of these cells with the miRNAs and their inhibitors was able to restore ZEB2 expression to almost normal levels. Fig. 3. Open in new tabDownload slide miR-192, miR-194 and miR-215 can target ZEB2. Representative bar graphs showing that ZEB2 expression was significantly decreased, at the mRNA level, in 786-O cells, upon transfection of miR-192 ( A ), miR-194 ( B ) and miR-215 ( C ). These effects were partially restored upon the co-transfection of the miRNA and its inhibitor. Expressions are shown as relative expression values compared with control untrasnfected cells to the far left. NC; negative control of scrambled miRNA sequences. We also validated miRNA–TYMS interactions in CAKI-1, 786-O and ACHN cells. Overexpression of any of the three miRNAs resulted in significant reduction in TYMS expression, at the mRNA level, in CAKI-1 ( Supplementary Figure 7A – Supplementary Data ), 786-O ( Supplementary Figure 7D ) and ACHN cells ( Supplementary Figure 7E ). The most significant drop in the level of TYMS was seen with miR-192. Transfection with miR-194 or miR-215 caused less reduction of TYMS levels, although this was still statistically significant. Similarly, MDM2 expression was significantly decreased after miR-192, miR-194 or miR-215 overexpression in ACHN ( Supplementary Figure 8A – Supplementary Data ), CAKI-1 and 786-O cells ( Supplementary Figure 8D – Supplementary Data ). It should be noted, however, that the degree of target suppression was variable among the different miRNAs in different cell lines. This can be due to a number of factors, including the number of recognition sites (miRNA response elements) in each target and other cell-specific factors. We also examined the effects of miR-192, miR-194 and miR-215 on ZEB2, MDM2 and TYMS expression at the protein level. MDM2 protein expression was significantly reduced when CAKI-1 cells were transfected with miR-192, miR-194, or miR-215 ( P < 0.0001, P = 0.0015 and P < 0.0001, respectively). This effect was largely overcome when co-transfecting the cells with each of these miRNA and its inhibitor ( Figure 4A and B ). The protein expression of TYMS was similarly significantly decreased in cells transfected with miR-192, miR-194 or miR-215 compared with control cells ( P = 0.0320, P = 0.0052 and P = 0.0070, respectively). Protein expression levels were partially restored in cells co-transfected with the miRNA and its inhibitor ( Supplementary Figure 9 ). Our results also show that overexpression of miR-192, miR-194, or miR-215 significantly reduced ZEB2 protein expression in CAKI-1 cells compared with control cells and that expression levels can be partially restored when cells are co-transfected with each of these miRNAs and its inhibitor ( Supplementary Figure 10 ). Fig. 4. Open in new tabDownload slide MDM2 is a target of miR-192, miR-194 and miR-215 (A and B). ( A ) Representative western blot analysis showing decreased MDM2 protein level in CAKI-1 cells transfected with miR-192, miR-194 or miR-215 compared with cells transfected with the transfection agent only or co-transfected with the miRNA and its inhibitor. ( B ) Bar graph showing the quantification of changes in protein expression levels in the gel. Expression values were compared with control untransfected cells. α-Tubulin was used as a loading control. MDM2, ZEB2 and TYMS are targets of miR-192, miR-194 and miR-215 (C–E). Co-transfection of 786-O cells with reporter vectors containing the 3'UTR of MDM2 ( C ), ZEB2 ( D ) or TYMS ( E ) and miR-192, miR-194 or miR-215 significantly decreased luciferase activity compared with cells transfected with the constructs only. In the second approach, we further validated these miRNA–target interactions by measuring the change of fluorescence signal of a luciferase vector containing the 3'UTR of the target upon miRNA transfection. This system provides more evidence about a ‘direct’ interaction between the miRNA and its target. The 786-O cell line was transfected with the Luciferase reporter plasmids containing the 3'UTR of TYMS, MDM2 or ZEB2 or co-transfected with the plasmids and the miRNA (miR-192, miR-194 or miR-215). Luciferase activity decreased significantly in cells co-transfected with plasmids of the target and the targeting miRNA compared with cells transfected with target plasmid only ( Figure 4C–E ). The third approach was to examine the presence of negative correlation between miR-192, miR-194 and miR-215 and their predicted targets—MDM2, ZEB2 and TYMS—by qRT–PCR in vivo in patient tissues. We examined the expression levels of the three miRNAs and their targets on 20 fresh frozen primary ccRCC tissues. As shown in Figure 5 , we observed a negative correlation between these three miRNAs and their targets, with lower expression levels of the miRNA associated with higher expression of its target in the same patient, and vice versa. This provides indirect evidence that MDM2, ZEB2 and TYMS are targets of miR-192, miR-194 and miR-215 in vivo . Fig. 5. Open in new tabDownload slide A negative correlation was observed between the expression levels of miR-192 and miR-215 and their predicted targets, ZEB2 and MDM2 in patient tissues. The graphs show pair-wise comparison between each of the miRNAs and its target for each patient. Our results show the presence of a negative correlation between these miRNAs and their predicted targets with lower expression of miR-192 and miR-215 associated with higher expression of MDM2 ( A and B ) and ZEB2 ( C and D ), and vice versa. This provides indirect evidence that ZEB2 and MDM2 are targets of miR-192 and miR-215. Patient cases are shown on the x -axes and the relative expression values are presented along the y -axes. Expression levels are shown as relative expression values of the miRNAs and their targets normalized against an internal control in the same specimen. An interesting observation was the high degree of overlap between targets of all three miRNAs. This was predicted by target prediction algorithms and experimentally validated. We further confirmed this overlap be performing sequence alignment of these miRNAs and the 3'UTR of their predicted targets (MDM2, ZEB2 and TYMS) using the European Molecular Biology Open Software Suite ( http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html ). Interestingly, miR-192 and miR-215 shared not only the same seed sequence but also their mature sequences, which are almost identical. They only differ in two nucleotides (the 1st and the 20th nucleotides). In addition, miR-194 showed partial complementarity with both miRNAs (data not shown). Also, all three miRNAs show partial complementarity with the 3'UTR of the three targets. We further validated the target predictions using an independent algorithm (the online microRNA prediction tool utilizing the PITA algorithm based on sequence analysis, http://132.77.150.113/pubs/mir07/mir07_prediction.html ), as a shown in Supplementary Table 4 . miR-215 as a prognostic marker for RCC As miR-215 was significantly differentially expressed between primary and metastatic ccRCC ( 17 ), we hypothesized that this miRNA can serve as a potential prognostic marker. We examined the expression of miR-215 with the ‘gold standard’ qRT–PCR using miRNA-specific probes in 61 formalin-fixed paraffin-embedded tissues of primary ccRCC. Kaplan–Meier survival curve ( Figure 6A ) showed that lower expression of miR-215 was associated with significantly decreased DFS time (patients with lower miR-215 expression = 26.4 months versus patients with higher miR-215 expression = 49.2 months, P = 0.0320). Fig. 6. Open in new tabDownload slide ( A ) Kaplan–Meier DFS plot comparing miR-215 expression in RCC patients. miR-215 expression was dichotomized into high and low expression categories. Patients with lower miR-215 expression had significantly lower DFS compared with those with high expression ( P = 0.032). ( B ) Kaplan–Meier overall survival (OS) plot comparing miR-215 expression in RCC patients. ‘Level 3’ miRNA expression data (normalized gene expression generated using the Illumina GA miRNASeq platform) for miR-215 in ccRCC and overall survival data were obtained from The Cancer Genome Atlas (TCGA). Lower expression of miR-215 is significantly associated with worse survival ( P = 0.0032). We further validated these data in silico on an independent data set of 218 primary ccRCC cases with available overall survival data from The Cancer Genome Atlas and observed lower expression of miR-215 to be associated with significantly worse survival ( P = 0.0032) ( Figure 6B ). miR-192, miR-194 and miR-215 are conserved among species We used the University of California Santa Cruz (UCSC) Genome Browser for sequence comparison of miR-192, miR-194 and miR-215 among species. Our analysis shows the high conservation of these three miRNAs among 28 species ( Supplementary Figure 11 ). Conservation among species indicates that these miRNAs may have vital functions that are maintained during their evolution. Discussion In our previous work, we identified an miRNA signature of metastatic ccRCC. miR-192, miR-194 and miR-215 were significantly differentially expressed in metastatic compared with primary ccRCC. In this study, we provide evidence that these three miRNAs can be involved in RCC progression and that miR-215 is a potential prognostic marker for ccRCC. We also identified and validated three of their target genes; ZEB2, MDM2 and TYMS. miR-192, miR-194 and miR-215 are highly enriched in the normal kidney ( 18 ), indicating that they play a role in kidney development and differentiation. In our previous work, we reported that these three miRNAs are significantly downregulated in primary ccRCC compared with normal kidney tissue ( 8 ). These three miRNAs are further downregulated in metastatic ccRCC ( 17 ). Taken together, this step-wise downregulation indicates their involvement in controlling tumor-suppressor pathways. This is also supported by the fact that these three miRNAs are present in two clusters. Members of the same cluster are usually co-expressed and co-regulated. The miR-215/miR-194-1 cluster is located within the common fragile site FRA1H (1q41–q42.1) that is deleted in many types of cancers ( 27 ). 1q41 was reported to be associated with breast and esophageal cancer metastasis. The downregulation of these three miRNAs was also reported in other cancers including nephroblastoma, myeloma, colon and gastric cancers ( 20 , 21 ). Our results show that the main effect of these three miRNAs is on tumor migration and invasion. Little effect was shown on tumor proliferation although this was not statistically significant. This is not unprecedented. Recent literature showing that the key contribution of some of these miRNAs is on tumor invasion and migration abilities, which are essential features for metastasis and epithelial to mesenchymal transition (EMT) ( 28 , 29 ). Moreover, our results should be interpreted with caution as it might be cell or tissue type specific. Others have also shown more significant effect on cell proliferation in other cell types. Our in silico analysis showed that miR-192, miR-194 and miR-215 can target ZEB2, MDM2 and TYMS. MDM2 is a key inhibitor of p53. It activates hypoxia inducible factor 1 alpha and vascular endothelial growth factor activity ( 30 ). MDM2 overexpression is reported to be associated with metastasis in many cancers. Its overexpression decreases E-cadherin levels with subsequent increase in cell motility in breast carcinoma ( 31 ). It was also reported to increase cell motility and invasiveness in RCC ( 32 ). TYMS is essential for DNA synthesis and its inhibition is reported to block DNA replication and repair ( 33 ). TYMS SNP variations are associated with increased risk of RCC ( 34 ). TYMS upregulation was reported in RCC and correlates with tumor progression ( 35 ). It is also a target of the 5-fluorouracil used in advanced RCC ( 36 ). It was also shown to have a prognostic significance in bladder cancer. SIP1/ZEB2 represses E-cadherin and was shown to be involved in EMT, which is a key process in tumor progression. ZEB2 can mediate the hypoxia inducible factor 1 repression effect of E-cadherin in RCC. We experimentally validated our in silico predictions using independent approaches. These results, however, should be interpreted with caution, due to the possibility of indirect targeting and off-target effects. The luciferase system can provide more evidence of direct miRNA–target interactions. Finally, we provide in vivo evidence by documenting the negative correlation between the expression of these three miRNAs and these targets in ccRCC patients’ tissues. Our findings are also consistent with recent reports showing that miR-192 and miR-215 can target TYMS in colon cancer ( 37 ). MDM2 is also a target of these miRNAs in myeloma ( 21 ). Interestingly, these miRNAs occur in two clusters. Members of the same cluster can have coordinated effects. They may target the same molecule (convergent targeting) or may hit several molecules in the same or related biological pathways (divergent targeting), as demonstrated in recent reports ( 38 , 39 ). The remarkable target overlap can be explained by the fact that miR-192 and miR-215 have similar seed sequence which is highly important for miRNA–target interaction. It should be also noted that 3′ supplementary sites can enhance the seed pairing. Pairing to the 3′ region includes mainly the nucleotides 13–16. Furthermore, this 3′ pairing can also compensate for nucleotide mismatch in the seed region ( 40 ). miRNAs were reported to target not only the 3'UTR but also 5'UTR, coding regions, promoters and gene termini. Our results are not unprecedented; Senanayake et al. identified ACVR2B to be a common target for miR-192, miR-194 and miR-215 in renal childhood neoplasms ( 41 ). The involvement of these miRNAs in tumor progression is not surprising and is supported by previous reports. miR-192, miR-194 and miR-215 are induced by p53 and also they were reported to be p53 positive regulators ( 20 , 21 ). Kim et al. demonstrated that p53 can regulate EMT through targeting ZEB2 by miR-192 family. Also, Krishnamachary et al. showed that E-cadherin repression by hypoxia inducible factor 1 can be mediated by ZEB2 in RCC ( 42 ). Also, MDM2 overexpression correlates with tumor progression and metastasis in different cancers, including RCC ( 43–47 ). Our findings can also have therapeutic implications. TYMS is a target of the 5-fluorouracil anticancer agents that is now being considered in combination therapies for advanced RCC. Recently, using RCC xenograft model, 5-fluorouracil was shown to enhance the Sorafenib and Sunitinib antitumor effect ( 48 ). Recently, MDM2 is being investigated as a potential new therapeutic target in colon cancer ( 49 ). Also, Vastsyayan et al. demonstrated that MDM2 inhibitor (Nutlin-3) can enhance the effect of Sorafenib in RCC ( 50 ). The fact that one miRNA can hit multiple targets attracted the attention to the miRNAs as promising therapies in cancer. The different strategies for using miRNA therapy were recently reviewed including miRNA replacement using adeno-associated viral vectors and synthetic miRNA precursors in hepatocellular carcinoma and prostate cancer, respectively ( 51 ). Also, targeting miRNAs using synthetic molecules can be of therapeutic interest. In conclusion, we showed that miR-192, miR-194 and miR-215 have tumor suppressor effects on RCC by reducing the cellular migration and invasion abilities. We identified potential mechanisms through which these three miRNAs can negatively affect these biological processes by targeting key molecules involved in metastasis. These three miRNAs can have hundreds of targets and a global analysis of the overall spectrum of changes of miRNAs would be more suitable. However, due to limited resources we did a targeted approach to select the targets that are of clinical significance that was shown in other cancers. This however does not exclude the presence of other significant targets that are yet to be identified and validated. This is a net effect of miRNAs and there might be a number of underlined, direct and indirect, and sometimes being even opposing target effects. Also, we demonstrated that miR-215 can be potential prognostic marker in RCC. Our results can be a step forward towards developing a new therapeutic target in RCC. Funding Canadian Institute of Health Research (119606); Canadian Cancer Society (20185); Kidney Foundation of Canada. Conflict of Interest Statement: None declared. 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Journal

Carcinogenesis – Oxford University Press

Published: Oct 1, 2013

Keywords: thymidylate synthase

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