Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

MiR-122-5p inhibits cell migration and invasion in gastric cancer by down-regulating DUSP4

MiR-122-5p inhibits cell migration and invasion in gastric cancer by down-regulating DUSP4 CANCER BIOLOGY & THERAPY 2018, VOL. 19, NO. 5, 427–435 https://doi.org/10.1080/15384047.2018.1423925 RESEARCH PAPER MiR-122-5p inhibits cell migration and invasion in gastric cancer by down-regulating DUSP4 a b b c a a a d Xiaofeng Xu , Feng Gao , Jianjiang Wang , Lan Tao , Jinsong Ye , Li Ding , Wei Ji , and Xing Chen a b Department of Laboratory Medicine, Jingjiang People’s Hospital, Jingjiang, Jiangsu, China; Department of Gastroenterology, Jingjiang People’s c d Hospital, Jingjiang, Jiangsu, China; Department of Central Laboratory, Jingjiang People’s Hospital, Jingjiang, Jiangsu, China; Department of Science and Education, Jingjiang People’s Hospital, Jingjiang, Jiangsu, China ABSTRACT ARTICLE HISTORY Received 20 November 2017 Objective: To explore the relationship between miR-122-5p and DUSP4 and their effects on gastric cancer Accepted 29 December 2017 (GC) cell mobility and invasiveness. Methods: Abnormally expressed miRNAs and mRNAs were analyzed using microarrays. The miR-122-5p KEYWORDS and DUSP4 mRNA expression levels in GC tissues and cells were determined by RT-qPCR. The target DUSP4; Gastric cancer; relationship between miR-122-5p and DUSP4 was validated by dual luciferase reporter assay. GC cell invasion; migration; miR-122- mobility and invasiveness were respectively observed by wound healing assay and transwell invasion 5p assay. Western blot and immunohistochemistry were used for detection of the expressions of DUSP4 protein and MMP2 and MMP9 proteins related to cell invasion and migration. The migration and invasion abilities of gastric cancer cells in vivo were evaluated according to the number of lung metastatic nodules in mice. Results: The expression of miR-122-5p in GC tissues and cells was significantly down-regulated, whereas DUSP4 expression was up-regulated. Bioinformatics prediction strategies and dual luciferase reporter assay verified the binding sites of miR-122-5p on 3 UTR of DUSP4 and the target relationship between miR-122- 5p and DUSP4. Overexpression of miR-122-5p and knockdown of DUSP4 in BGC-823 cells observantly suppressed GC cell mobility and invasiveness, whereas downregulation of miR-122-5p expression promoted cell metastasis. MiR-122-5p inhibited GC cell mobility and invasiveness and pulmonary tumor metastasis via downregulation of DUSP4. Conclusion: MiR-122-5p restrained migration and invasion abilities of GC cells by repressing DUSP4. Introduction with 3 -UTR. Previous studies substantiated that dysregulation Gastric cancer (GC) is identified as one of the most prevalent of miRNAs often occurs in multiple cancers. Furthermore, aber- malignancies, with over 1 million death cases annually world- rant expressions of miRNAs could interfere with tumorgenesis 12,13 wide. Up to now, smoking, alcohol consumption and helico- and development. In a large family of miRNAs, miR-122 has bacter pylori infection have been found to be main culprits of been thought to be down-regulated in several types of cancers, 2,3 14 15 16 GC. Due to its high metastasis allowing the neoplasm to including GC, gallbladder carcinoma, bladder cancer and so rapidly progress into advanced stages, early diagnosis of GC on. Furthermore, Ergun € et al. has demonstrated the suppressive remains difficult. Although enormous advancement has been function of miR-122-5p on human breast cancer by targeting achieved in the treatment of GC, multiple therapeutic strate- ADAM10, indicating that miR-122-5p may act as a therapeutic gies still cannot cure such a malignant cancer. Moreover, target in breast cancer. Bai et al. revealed that miR-122-5p is despite traditional TNM staging system is widely applied to commonly repressed in primary hepatocellular carcinomas clinical treatment, fewer biomarkers can accurately predict (HCCs) and can inhibit somatotropin-releasing factor (SRF) the metastasis and recurrence of GC. However, more and activities and tumorgenesis, suggesting that miR-122-5p can act more scientists start to focus on the potential mechanisms of as a tumor inhibitor through different molecular pathways. In the GC and explore new therapeutic targets for the treatment of present study, we delve deeper into the molecular mechanisms of GC. miR-122-5p in GC. MicroRNAs (miRNAs), as a class of small, single stranded and Dual-specificity phosphatases (DUSPs), as a heterogeneous non-coding RNAs, play critical parts in post-transcriptional regu- group of protein phosphatases, can dephosphorylate tyrosine and lation and function as either tumor inhibitor or facilitator in mul- serine or threonine residues. Currently, 25 human DUSPs genes 8,9 tiple cancers. It have been reported that these small molecules have already been explored, of which approximately 11 genes are usually repress mRNAs and prevent transcription by combining found to result in MAPK signaling inactivation and others play a CONTACT Feng Gao [email protected] Department of Gastroenterology, Jingjiang People’s Hospital, No.28 East Zhongzhou Road, Jingjiang 214500, Jiangsu, China; Xing Chen [email protected] Department of Science and Education, Jingjiang People’s Hospital, No. 28 East Zhongzhou Road, Jingjiang 214500, Jiangsu, China. © 2018 Taylor & Francis Group, LLC 428 X. XU ET AL. regulatory role in the dephosphorylation of diverse targets. Sev- Table 1. Primer sequences for RT-qPCR. eral DUSPs involved in the development of specifichuman can- 0 0 Primer sequence (5 to 3 ) cers have been reported over the past decade. Zhang et al. has 0 0 MiR-122-5p (F) 5 -TATTCGCACTGGATACGACACAAAC-3 revealed the inhibitory effects on sanguinarine on growth and 0 0 MiR-122-5p (R) 5 -GCCCGTGGAGTGTGACAATGGT-3 invasiveness of human gastric cancer cells via down-regulation of 0 0 U6 (F) 5 -GCTTCGGCAGCACATATACTAAAAT-3 0 0 DUSP4/ERK pathway. De et al. verified that higher expression U6 (R) 5 -CGCTTCACGAATTTGCGTGTCAT-3 0 0 DUSP4 (F) 5 -TGGTTCATGGAAGCCATAGAG-3 of DUSP4 was associated with a worse overall survival and with 0 0 DUSP4 (R) 5 -CCCGTTTCTTCATCATCAGG-3 clinical characteristics of colorectal cancer patients. Wei et al. 0 0 GAPDH (F) 5 -CCTGCCTCTACTGGCGCTGC-3 0 0 substantiated that miR-101 inhibited macrophage-induced cell GAPDH (R) 5 -GCAGTGGGGACACGGAAGGC-3 growth of hepatocellular carcinoma through targeting DUSP1. F: forward primer; R: reverse primer. Samsonov et al. demonstrated that miR-145 restrained papillary thyroid cancer cell reproduction by targeting DUSP6. However, treatment at Jingjiang People’s Hospital during the period there are fewer studies on the relationship between DUSP6 and between April 2015 and April 2017. All samples were assessed miR-122-5p and their underlying mechanism on GC. by clinicopathological examination and all subjects had been The current study probed into the impacts of miR-122-5p confirmed to receive no radiotherapy or chemotherapy. The and DUSP4 on GC and the association between them. We first samples were conserved in liquid nitrogen at ¡80 C for subse- conjectured that there was a target relationship between miR- 122-5p and DUSP4 and miR-122-5p exerted a certain influence quent experiments. All experiments were ratified by the Ethics on GC cell by modulating DUSP4. Next, the target relationship Committee of Jingjiang People’s Hospital, and informed con- between miR-122-5p and DUSP4 was validated by bioinformat- sents were provided by all subjects. ics prediction strategies and dual luciferase reporter. The impacts of miR-122-5p on GC cells were detected by Immuno- Microarray analysis histochemistry, HE staining, wound healing assay, transwell assay and pulmonary metastasis model assay. Differentially expressed miRNAs were screened out through the GSE78091 gene microarray hybridization, which con- tains 3 random pairs of GC tissues and adjacent tissues. Materials and methods Totally there were 3554 probes and 2095 human genes and some genes were measured by multiple probes. Abnormally Clinical specimens expressed mRNAs were screened out through the GSE2685 GC tissues and the adjacent tissues (at least 3 cm away from the gene microarray hybridization, which contains 22 primary tumor) were collected from 30 patients undergoing surgical human advanced gastric cancer tissues samples and 8 Figure 1. MiR-122-5p was low-expressed in gastric cancer tissues and cells (A) Aberrant expression miRNAs in gastric cancer tissues were reflected by the volcano plot. (B) MiR-122-5p was a lowly expressed miRNA in gastric cancer tissues than in adjacent tissues shown in the heat map. (C) MiR-122-5p expression was significantly lower expression in GC tissues than in adjacent tissues determined by qRT-PCR. (D) The expressions of miR-122-5p in human gastric cancer cell lines (BGC-823, SGC-7901, MGC- 803 and HGC-27) were conspicuously lower compared with human normal gastric mucosal cell line GES1 determined by qRT-PCR. P < 0.05, P < 0.01, compared with normal tissues or GES1 cell line. CANCER BIOLOGY & THERAPY 429 Figure 2. DUSP4 was high-expressed in gastric cancer tissues and cells (A) Abnormal expression mRNAs in gastric carcinoma tissues were reflected by the volcano plot. (B) DUSP4 was a highly expressed mRNA in gastric carcinoma tissues than in noncancerous gastric tissues shown in the heat map. (C) The expression of DUSP4 in GC tissues was dramatically higher than that in adjacent tissues determined by qRT-PCR. (D-E) The protein expression of DUSP4 experienced a significant increase in GC tissues detected by immunohistochemistry and western blot. P < 0.01, compared with noncancerous or adjacent tissues. adjacent gastric tissues samples. Totally there were 7129 (NC) group: GC cells transfected with scrambles and probes and 7129 human genes and each gene was measured siRNA control. (2) miR-122-5p mimics group: GC cells by one probe. Fold change value greater than 2 and P less transfected with miR-122-5p mimics. (3) miR-122-5p than 0.05 were regarded as the screening criteria for aber- inhibitor group: GC cells transfected with miR-122-5p rantly expressed miRNAs and mRNAs. inhibitor. (4) si-DUSP4 group: GC cells transfected with DUSP4 siRNA (si-DUSP4). (5) MIX group: GC cells co- transfected with miR-122-5p inhibitor and si-DUSP4. The Cell culture and cell transfection transfection efficiency was detected b under an inverted HEK-293T cells were acquired from the stem cell bank of fluorescence microscope. Shanghai Institutes for Biological Sciences, Chinese Acad- emy of Sciences (Shanghai, China). Human normal gastric mucosal cell line GES1 and human gastric cancer cell lines qRT-PCR BCG-823, SGC-7901, MGC-803, HGC-27 were both pro- cured from BeNa Culture Collection Company (Beijing The extraction of total RNA was implemented with TRIzol China). Cells were cultured in RPMI-1640 medium (Gibco- reagent (Invitrogen) following the protocol of the manufac- Invitrogen, USA) with 10% FBS (Hyclone, USA) at 37 C turer. Reverse transcription of extracted RNA and real-time and dissolved using 0.25% trypsin (Invitrogen, USA). quantitative PCR were successively performed according to Transfection was carried out through Lipofectamine 2000 instruction of RT-PCR kit (Invitrogen) and the fluorescence following the instruction of manufacturer (Invitrogen). GC quantitative PCR kit (Invitrogen). Primer sequences used were cells were assigned to five groups: (1) negative control presented at Table 1 with GAPDH as an internal reference for 430 X. XU ET AL. Figure 3. DUSP4 was a potential target of miR-122-5p (A) There was a potential binding site of miR-122-5p on the 3'-UTR of DUSP4 predicted by bioinformatics software. (B) MiR-122-5p could inhibit the luciferase activity of the cells transfected wild-type 3 UTR of DUSP4, but had little influence on that of those transfected with mutated- type 3 UTR of DUSP4. (C-D) There was a negative correlation between DUSP4 and miR-122-5p in HEK-293T cells and BCG-823 cells. P < 0.05, compared with NC group. mRNAs and U6 for miRNAs, and the relative expressions of primary antibody (1:100) and HRP-conjugated anti-rabbit sec- ¡ DDC miR-122-5p and DUSP4 were quantified by 2 method. ondary antibody (1:2000). Afterwards, immunoreactivity in the sections was detected using a horseradish peroxidase (3,3 -diami- nobenzidine substrate) kit (BioGenex, Fremont, CA, USA). The Western blot slides were then counterstained with hematoxylin (Beyotime, Shanghai, China), dehydrated and mounted. The sections were Total proteins were isolated by using 1 ml radioimmunopreci- evaluated by an optical microscopy. pitation assay buffer with 10 ml phenylmethanesulfonyl fluo- ride. Then protein concentration was examined by a bicinchoninic acid protein assay kit. Proteins were separated using 8% SDS-PAGE, then electro-transferred onto PVDF Hematoxylin-Eosin (HE) staining membranes, which were sealed with 5% non-fat milk for 2 h The tissues embedded in paraffin were first deparaffinized in and incubated in the primary antibodies rabbit polyclonal anti- xylene, and then soaked in alcohol with gradient concentration DUSP4 (ab72593, 1:500, Abcam Trading, Shanghai, China), (100%, 95%, 90%, 80%, 70%) for 10 min each. Hematoxylin rabbit polyclonal anti-MMP2 (ab37150, 1:1000, Abcam Trad- was instilled to stain the tissues for 15 min after washed with ing), rabbit polyclonal anti-MMP9 (1:1000, ab38898, Abcam distilled water for 2 min. The stained tissues were disposed in Trading), rabbit polyclonal anti-b-actin (ab8227, 1:1000, 1% hydrochloric acid ethanol to separate color for 20 s, and Abcam Trading) and rabbit polyclonal anti-GAPDH (ab9485, then soaked in warm water (50 C) for 5 min. The 0.25% eosin 1:2500, Abcam Trading). After rinsed by tris buffer saline con- dye solution was instilled for counterstaining for 2 min, and taining 20% Tween twice, the PVDF membrane was incubated the stained tissues were dehydrated by being soaked in gradient for 2 h at 37 C in the diluted HRP-labeled goat anti-rabbit IgG concentration of alcohol (70%, 80%, 90%, 95%, 100% ethyl H&L secondary antibodies (1:2000). Then the membranes were alcohol) for 10 min each. The samples were observed by using rinsed using TBST for 20 min and visualized using an ECL Plus an optical microscope (200 £). kit (Life Technology Inc., USA). GAPDH and b-actin were respectively considered as internal references for DUSP4 and MMP2/ MMP9. Wound healing assay GC cells BCG-823 were inoculated on 6-well plates, and the Immunohistochemistry germfree pipette was utilized to create a linear scratch in cell The primary and secondary antibodies used in this assay were monolayer when cell confluence was up to 80%. The cells were same as those in western blot. The tissues were fixed in 4% formal- then washed with PBS for 2»3 times and cultivated in the dehyde buffered with phosphate-buffered saline, then dehydrated, serum-free medium. Lastly, we observed the wound distance at embedded in paraffin and serial section cut (4 mm thick). DUSP4 0 h and 24 h by using image-Pro Plus 6.0 (Media Cybernetics, protein was detected after incubation with the rabbit polyclonal Inc., Rockville, MD, USA) and measure the cell mobility. CANCER BIOLOGY & THERAPY 431 Figure 4. MiR-122-5p inhibited gastric cancer cell migration and invasion ability by targeting DUSP4 (A) The transfection efficiency was confirmed by qRT-PCR. (B) The wound distance and cell mobility of the experimental groups at different time points were measured by wound healing assay. The migration ability of the cells in miR- 122-5p group or si-DUSP4 group was significantly lower than in NC group. The NC group and the Mix group could not be significantly distinguished (£ 200). (C) The num- ber of invasion cells in miR-122-5p mimics group and si-DUSP4 group was remarkably fewer compared with NC group, whereas that in miR-122-5p inhibitor group was considerably more than that in NC group detected by transwell invasion assay. Little difference could be found between NC group and the Mix group. P < 0.05, com- pared with NC group. Transwell assay including luciferase genes to construct pMIR-DUSP4-wt plasmids after recovery, purification and enzyme digestion. The transfected cells were seeded onto the upper chamber Likewise, the pMIR-DUSP4-mut plasmids were also con- along with serum-free medium. The medium containing 10% structed based on this method. The recombinant plasmids FBS was instilled into the lower chamber and incubated for were identified through PCR, double-restrict-enzyme diges- 24 h. The cells on the upper chamber were gently removed by tion of EarI (CTCTTC) and PspOMI (GGGCCC) and DNA cotton swabs. The migrating or invading cells in the lower sequence. The HEK-293T cells were first carefully seeded chamber were fixed with alcohol for 15 min and stained using into 24-well plates. Following transfection, RIPA lysis buffer crystal violet for 10 min. Lastly, the cells were detected under (Thermo Fisher Scientific) was added into the 24-well plates an optical microscope (200 £). (100 mL / well). Then the mixture was centrifuged and the supernatant was collected into a 96-well plate. Renilla lucif- erase activity was properly normalized to fireflyluciferase Dual luciferase reporter assay activity. Finally, the relative luciferase activities were was determined following the instructions of Dual-Luciferase Thesequence3 UTR of DUSP4 was cloned into pMIR Reporter Assay kit (Promega). eukaryotic expression vectors (Promega, Madison, USA) 432 X. XU ET AL. Figure 5. MiR-122-5p influenced the expression of MMP2 and MMP9 proteins in gastric cancer by regulating DUSP4 (A-B) The protein expressions of MMP2 and MMP9 were observantly down-regulated in BGC-823 cells transfected with miR-122-5p mimics or si-DUSP detected by western blot and immunohistochemistry. Conversely, the expressions of MMP2 and MMP9 were significantly up-regulated in BGC-823 cells transfected with miR-122-5p inhibitor. The expressions of MMP2 and MMP9 in MIX group were equivalent to those in NC group. P < 0.05, compared with NC group. Murine pulmonary metastasis models procedures were approved by Animal Ethics Committee in C57BL/6nudemice(4-5weeksold,18–20 g, male and Jingjiang People’sHospital. 12 C57BL/6 micewereran- female) were bought from Shanghai Experimental Animal domly divided into two groups and labeled. 5 £ 10 trans- Center (Shanghai, China) and maintained in specificpatho- fected cells in 100 ml of PBS were first injected into the tail th gen free (SPF) level environment. All the experimental vein of the mice. All mice were sacrificed at 24 day after Figure 6. MiR-122-5p inhibited pulmonary tumor metastasis by repressing DUSP4 in vivo (A) The number of murine pulmonary nodules determined by dissecting micro- scope. The number of mice lung metastasis nodules in miR-122-5p mimics group or si-DUSP4 group remarkably decreased, while that in miR-122-5p inhibitor group con- spicuously increased. The number of mice lung metastasis nodules in Mix group was equivalent to that in NC group. (B) HE staining was conducted to detect the number of lung metastatic nodules after tail vein injection. The lungs of mice injected with transfected cells in miR-122-5p mimics group or si-DUSP4 group had almost no visible metastatic nodules, while that of mice in miR-122-5p inhibitor group was fully covered with nodules. (C) The lung weight of mice in miR-122-5p mimics group or si- DUSP4 group decreased significantly, whereas that of mice in miR-122-5p inhibitor increased sharply. There was no significant difference between Mix group and NC group in terms of lung weight. P < 0.05, P < 0.01, compared with NC group. CANCER BIOLOGY & THERAPY 433 injection. Next, the lung, liver and brain tissues were iso- DUSP4 was a potential target of miR-122-5p lated and the number of lung metastatic nodules was The candidate target gene of miR-122-5p was predicted by counted under anatomical microscope. At last, 4% parafor- bioinformatics software, of which the results suggested that maldehyde was used to fix lung, liver and brain tissues for there was a potential binding site of miR-122-5p on the 3'- HE staining and weighting. UTR of DUSP4 (Fig. 3A). Meanwhile, dual luciferase reporter assay also illustrated that after transfected with pMIR-DUSP4-wild type plasmids, the luciferase activity of Statistical analysis BGC-823 cells decreased by about 50% in miR-122-5p Statistical software SPSS version 19.0 (SPSS Inc., USA) and mimics group compared with NC group (P < 0.05, Graphpad 6.0 (GraphPad Software, Inc., USA) were applied to Fig. 3B). However, no significant difference was found data analysis. Results were presented in form of mean § stan- between miR-122-5p mimics group and NC group in terms dard deviation. The differences between two groups were com- of luciferase activity of the cells after transfection with pared by Student’s t-test, while that among three or more pMIR-DUSP4-mutated type plasmids (P > 0.05), suggesting groups was analyzed using one-way ANOVA method. All that miR-122-5p could directly bind to 3'-UTR of DUSP4 experiments were conducted in triplicate and P < 0.05 was and hinder the transcriptional activity of DUSP4.Further- considered as an indicator of statistical significance. more, the results of qRT-PCR indicated that the relative expression of DUSP4 mRNA was negatively correlated to that of miR-122-5p both in HEK-293T cells (P < 0.05, Results Fig. 3C) and BCG-823 cells (P < 0.05, Fig. 3D), which fur- MiR-122-5p was low-expressed in gastric cancer tissues ther validated the above results. and cells We took fold change over 2 and P < 0.05 as the filtration MiR-122-5p suppressed gastric cancer cell mobility and standard to screen out abnormally expressed miRNAs invasiveness by targeting DUSP4 (Fig. 1A) and found that there were 111 up-regulated miR- The mRNA expressions of miR-122-5p and DUSP4 after cell NAs and 76 down-regulated miRNAs in GC tissues in com- transfection were also examined by qRT-PCR. As shown in parison with the adjacent tissues. Among these miRNAs, Fig. 4A, the expression of miR-122-5p was dramatically up-reg- miR-122-5p was found to be down-regulated and miR-122-5p ulated in miR-122-5p mimics group, whereas that in miR-122- expression in GC tissues was averagely 2.99 times lower than 5p inhibitor group was remarkably down-regulated (both that in the adjacent tissues (P D 0.001249428). The top 10 P < 0.05). Conversely, DUSP4 expression levels in miR-122-5p highly expressed miRNAs and the first 10 lowly expressed mimics group and si-DUSP4 group drastically decreased, while miRNAs were selected for the heat map (Fig. 1B). Further- those in miR-122-5p inhibitor group observantly increased (all more, qRT-PCR results exhibited that miR-122-5p expression P < 0.05). There was no conspicuous distinction between Mix was observantly lower in GC tissues than in adjacent tissues group and NC group in terms of the mRNA expressions of (P < 0.01, Fig. 1C). Beyond that, the expressions of miR-122- miR-122-5p and DUSP4 (both P > 0.05). Additionally, wound 5p in human gastric cancer cell lines (BGC-823, SGC-7901, healing assay results exhibited that the cell mobility ability was MGC-803 and HGC-27) were conspicuously lower compared conspicuously attenuated after transfection with miR-122-5p with human normal gastric mucosal cell line GES1 (P < 0.05, mimics or DUSP4 siRNA, while the migration ability of the Fig. 1D). BCG-823 cell line was chosen for subsequent experi- cells transfected with miR-122-5p inhibitor was significantly ments as presented the most significant difference of expres- sion in contrast to GES1 cell line (P < 0.01). enhanced (all P < 0.05, Fig. 4B). No significant difference of cell mobility was detected between Mix group and NC group (P > 0.05). Furthermore, the GC cell invasion was determined DUSP4 was high-expressed in gastric cancer tissues and by transwell invasion assay, of which the results suggested that cells the number of invasion cells in miR-122-5p mimics group and si-DUSP4 group was remarkably fewer compared with NC Likewise, we screened out aberrantly expressed mRNAs based group, whereas that in miR-122-5p inhibitor group was consid- on fold change over 2 and P < 0.05 (Fig. 2A), finding that 245 erably more than that in NC group (all P < 0.05, Fig. 4C). Little mRNAs were up-regulated and 336 mRNAs were down-regu- difference could be observed between NC group and Mix group lated in GC tissues. DUSP4 was found in the up-regulated (P > 0.05). Overall, miR-122-5p inhibited GC cell mobility and mRNAs, and the DUSP4 expression level was averagely invasiveness by down-regulating DUSP4. 1.01 times higher in gastric carcinoma tissues than in noncan- cerous tissues (P D 0.003334112). The top 10 highly or lowly expressed mRNAs were selected for the heat map (Fig. 2B). In MiR-122-5p influenced the expression of MMP2 and MMP9 addition, qRT-PCR also exhibited that DUSP4 expression in proteins by regulating DUSP4 GC tissues was dramatically higher compared with adjacent tissues (P < 0.01, Fig. 2C). In addition, the results from Western blot and immunohistochemistry were both immunohistochemistry (Fig. 2D) and western blot (Fig. 2E) employed to identify the expressions of MMP2 and MMP9 manifested that the expression of DUSP4 protein displayed a proteins related to cell metastasis. Results from western blot considerable increase in GC tissues (P < 0.01). (Fig. 5A) immunohistochemistry (Fig. 5B)bothindicated 434 X. XU ET AL. that the expressions of MMP2 and MMP9 proteins were For instance, miR-122-5p could play key roles in diagnosis and 29, 30 observantly down-regulated in BGC-823 cells transfected specific early prognosis of acute myocardial infarction. In with miR-122-5p mimics or si-DUSP (P < 0.05). the present research, we substantiated that miR-122-5p could Conversely, the expressions of MMP2 and MMP9 were sig- exerted inhibitory influence in GC cell metastasis through nificantly up-regulated in BGC-823 cells transfected with down-regulating DUSP4, suggesting that miR-122-5p could miR-122-5p inhibitor (P < 0.05). The expressions of MMP2 function as a tumor inhibitor in GC. and MMP9 in MIX group were equivalent to those in NC Additionally, the regulation function of DUSP4 was reported 31–33 group (P > 0.05), suggesting that miR-122-5p affected the in many previous study reports, which indicated that expression of MMP2 and MMP9 proteins related to cell DUSP4 had been thought to be a critical signaling regulator of migration and invasiveness by regulating DUSP4. multiple cellular processes, such as cell invasion, migration, dif- ferentiation and so forth. Zhang et al. found that sanguinarine exerted a inhibitive influence on GC cell growth and invasion MiR-122-5p inhibited pulmonary tumor metastasis by via regulation of the DUSP4/ERK pathway. Lai et al. reported repressing DUSP4 in vivo that the increased phosphorylation of ERK, correlated with To further confirm the effects of miR-122-5p and DUSP4 on decreased abundance of the phosphatases DUSP4 and DUSP6, GC cell metastasis, murine pulmonary metastasis model assay might counteract resistance to c-Met inhibitors in GC. Our was performed, from which the results displayed that the num- study verified that DUSP4 could be repressed by miR-122-5p, ber of mice lung metastasis nodules in miR-122-5p mimics hence impeding the progression of GC. Besides, a few signaling group or si-DUSP4 group remarkably decreased, while that in pathways have been found to be involved in DUSP-mediated 35 36 miR-122-5p inhibitor group conspicuously increased (Fig. 6A, regulation like ERK1/2/JNK pathway and MAPK pathways. P < 0.05). The number of mice lung metastasis nodules in Mix However, a lack of study on signaling pathway was one of the group was equivalent to that in NC group (P > 0.05). HE stain- limitations in this study. Apart from it, the small number of tis- ing assay also indicated that the lungs of mice injected with sue samples might make our results not completely convincing. transfected cells in miR-122-5p mimics group or si-DUSP4 In conclusion, over-expression of miR-122-5p could inhibit group had almost no visible metastatic nodules, while that of the cell mobility and invasiveness in GC through targeting mice in miR-122-5p inhibitor group was fully covered with DUSP4. The finding not only laid a foundation for an intensive nodules (Fig. 6B, P < 0.05). The number of mice lung nodules study on GC, but also provided two promising therapeutic tar- in Mix group was equivalent to that in NC group (P > 0.05). gets in GC treatment. Additionally, the lung weight of mice in miR-122-5p mimics group or si-DUSP4 group decreased significantly (P < 0.05), while that of mice in miR-122-5p inhibitor group increased Conflict of interest sharply (Fig. 6C, P < 0.01). There was no significant difference The authors declare no potential conflicts of interest. between Mix group and NC group in terms of lung weight (P > 0.05). Taken together, miR-122-5p restrained pulmonary metastasis by repressing DUSP4 in vivo. Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed. Discussion The current experiments have already identified that miR-122- Funding 5p was low-expressed, while DUSP4 was high-expressed in GC tissues and cell lines. Over-expression of miR-122-5p or knock- None. down of DUSP4 could inhibit the metastasis of GC cells. We disclosed that miR-122-5p played a tumor-suppressive role in cell mobility and invasiveness by targeting DUSP4 in GC cells. References Meanwhile, we also validated that miR-122-5p impeded pul- 1. Van Cutsem E, Sagaert X, Topal B, Haustermans K, Prenen H. Gastric monary tumor metastasis by repressing DUSP4 in vivo. cancer. Lancet. 2016;388:2654–64. doi:10.1016/S0140-6736(16)30354-3. The molecular mechanism of GC was a process involving 2. Li L, Ying XJ, Sun TT, Yi K, Tian HL, Sun R, Tian JH, Yang KH. Over- multiple factor, including epigenetic factors and genetic factors. view of methodological quality of systematic reviews about gastric Up to now, considerable researches have substantiated that cancer risk and protective factors. Asian Pac J Cancer Prev: APJCP. 2012;13:2069–79. doi:10.7314/APJCP.2012.13.5.2069. miRNAs play a crucial function in the gene regulation in multi- 3. Kato M, Asaka M. Recent knowledge of the relationship between Heli- ple cancers. Zhang et al. unraveled that miR-200c suppressed cobacter pylori and gastric cancer and recent progress of gastroendo- GC cell reproduction and metastasis through directly targeting scopic diagnosis and treatment for gastric cancer. Jpn J Clin Oncol. FN1. Wang et al. demonstrated that miR-190b enhanced the 2010;40:828–37. doi:10.1093/jjco/hyq119. radio-sensitivity of GC cells through negatively regulated Bcl- 4. Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J. Cancer statistics in China, 2015. CA: a cancer journal for 2. Ding et al. revealed that miR-27a stimulated the tumorgen- clinicians. 2016;66:115–32. esis of GC by activating the AKT/GSK3b pathway. Mi et al. 5. Zheng L, Jiao W, Song H, Qu H, Li D, Mei H, Chen Y, Yang F, Li H, validated that miR-181a-5p facilitated the development of GC Huang K, et al. miRNA-558 promotes gastric cancer progression via RASSF6-modulated MAPK signaling pathway. Recently, through attenuating Smad4-mediated repression of heparanase miR-122-5p has been identified as a potential novel biomarker. expression. Cell Death Dis. 2016;7:e2382. doi:10.1038/cddis.2016.293. CANCER BIOLOGY & THERAPY 435 6. Chia NY, Tan P. Molecular classification of gastric cancer. Ann Oncol: 23. Wei X, Tang C, Lu X, Liu R, Zhou M, He D, Zheng D, Sun C, Wu Z. official journal of the European Society for Medical Oncology. MiR-101 targets DUSP1 to regulate the TGF-beta secretion in sorafe- 2016;27:763–9. doi:10.1093/annonc/mdw040. nib inhibits macrophage-induced growth of hepatocarcinoma. Onco- 7. Tsai MM, Wang CS, Tsai CY, Huang HW, Chi HC, Lin YH, Lu PH, target. 2015;6:18389–405. doi:10.18632/oncotarget.4089. Lin KH. Potential Diagnostic, Prognostic and Therapeutic Targets of 24. Samsonov R, Burdakov V, Shtam T, Radzhabovsmall a CZ, Vasilyev MicroRNAs in Human Gastric Cancer. Int J Mol Sci. 2016;17. D, Tsyrlina E, Titov S, Ivanov M, Berstein L, Filatov M, et al. Plasma doi:10.3390/ijms17060945. exosomal miR-21 and miR-181a differentiates follicular from papillary 8. Sundaram P, Hultine S, Smith LM, Dews M, Fox JL, Biyashev D, thyroid cancer. Tumour Biol: the journal of the International Society Schelter JM, Huang Q, Cleary MA, Volpert OV, et al. p53-responsive for Oncodevelopmental Biology and Medicine. 2016;37:12011–21. miR-194 inhibits thrombospondin-1 and promotes angiogenesis in doi:10.1007/s13277-016-5065-3. colon cancers. Cancer Res. 2011;71:7490–501. doi:10.1158/0008-5472. 25. Zhang H, Sun Z, Li Y, Fan D, Jiang H. MicroRNA-200c binding to CAN-11-1124. FN1 suppresses the proliferation, migration and invasion of gastric 9. Bar-Eli M. Searching for the ‘melano-miRs’: miR-214 drives melanoma cancer cells. Biomed Pharmacother D Biomedecine & pharmacothera- metastasis. EMBO J. 2011;30:1880–1. doi:10.1038/emboj.2011.132. pie. 2017;88:285–92. 10. An Y, Zhang Z, Shang Y, Jiang X, Dong J, Yu P, Nie Y, Zhao Q. miR- 26. Wang C, Qiao C. MicroRNA-190b confers radio-sensitivity through 23b-3p regulates the chemoresistance of gastric cancer cells by target- negative regulation of Bcl-2 in gastric cancer cells. Biotechnol Lett. ing ATG12 and HMGB2. Cell Death Dis. 2015;6:e1766. doi:10.1038/ 2017;39:485–90. doi:10.1007/s10529-016-2273-2. cddis.2015.123. 27. Ding L, ZhangS,XuM,Zhang R, SuiP,YangQ.MicroRNA-27a 11. Zhang X, Zeng J, Zhou M, Li B, Zhang Y, Huang T, Wang L, Jia J, contributes to the malignant behavior of gastric cancer cells by Chen C. The tumor suppressive role of miRNA-370 by targeting directly targeting PH domain and leucine-rich repeat protein FoxM1 in acute myeloid leukemia. Mol Cancer. 2012;11:56. phosphatase 2. J Exp Clin Cancer Res: CR. 2017;36:45. doi:10.1186/1476-4598-11-56. doi:10.1186/s13046-017-0516-2. 12. Zhu M, Zhang N, He S, Lui Y, Lu G, Zhao L. MicroRNA-106a targets 28. Mi Y, Zhang D, Jiang W, Weng J, Zhou C, Huang K, Tang H, Yu Y, TIMP2 to regulate invasion and metastasis of gastric cancer. FEBS Liu X, Cui W, et al. miR-181a-5p promotes the progression of gastric Lett. 2014;588:600–7. doi:10.1016/j.febslet.2013.12.028. cancer via RASSF6-mediated MAPK signalling activation. Cancer 13. Miko E, Margitai Z, Czimmerer Z, Varkonyi I, Dezso B, Lanyi A, Lett. 2017;389:11–22. doi:10.1016/j.canlet.2016.12.033. Bacso Z, Scholtz B. miR-126 inhibits proliferation of small cell lung 29. Cortez-Dias N, Costa MC, Carrilho-Ferreira P, Silva D, Jorge C, cancer cells by targeting SLC7A5. FEBS Lett. 2011;585:1191–6. Calisto C, Pessoa T, Robalo Martins S, de Sousa JC, da Silva PC, et al. doi:10.1016/j.febslet.2011.03.039. Circulating miR-122-5p/miR-133b Ratio Is a Specific Early Prognostic 14. Chen Q, Ge X, Zhang Y, Xia H, Yuan D, Tang Q, Chen L, Pang X, Biomarker in Acute Myocardial Infarction. Circ J: official journal of Leng W, Bi F. Plasma miR-122 and miR-192 as potential novel bio- the Japanese Circulation Society. 2016;80:2183–91. doi:10.1253/circj. markers for the early detection of distant metastasis of gastric cancer. CJ-16-0568. Oncol Rep. 2014;31:1863–70. doi:10.3892/or.2014.3004. 30. Yao XL, Lu XL, Yan CY, Wan QL, Cheng GC, Li YM. Circulating 15. Lu W, Zhang Y, Zhou L, Wang X, Mu J, Jiang L, Hu Y, Dong P, Liu Y. miR-122-5p as a potential novel biomarker for diagnosis of acute miR-122 inhibits cancer cell malignancy by targeting PKM2 in gall- myocardial infarction. Int J Clin Exp Pathol. 2015;8:16014–9. bladder carcinoma. Tumour Biol. 2015. doi:10.1007/s13277-015- 31. Barajas-Espinosa A, Basye A, Angelos MG, Chen CA. Modulation of 4308-z. p38 kinase by DUSP4 is important in regulating cardiovascular func- 16. Wang Y, Xing QF, Liu XQ, Guo ZJ, Li CY, Sun G. MiR-122 targets tion under oxidative stress. Free Radic Biol Med. 2015;89:170–81. VEGFC in bladder cancer to inhibit tumor growth and angiogenesis. doi:10.1016/j.freeradbiomed.2015.07.013. Am J Transl Res. 2016;8:3056–66. 32. Schmid CA, Robinson MD, Scheifinger NA, Muller S, Cogliatti S, 17. Ergun S, Ulasli M, Igci YZ, Igci M, Kirkbes S, Borazan E, Balik A, Tzankov A, Muller € A. DUSP4 deficiency caused by promoter hyper- Yumrutas ¸ O, Camci C, Cakmak EA, et al. The association of the expres- methylation drives JNK signaling and tumor cell survival in diffuse sion of miR-122-5p and its target ADAM10 with human breast cancer. large B cell lymphoma. J Exp Med. 2015;212:775–92. doi:10.1084/ Mol Biol Rep. 2015;42:497–505. doi:10.1007/s11033-014-3793-2. jem.20141957. 18. Bai S, Nasser MW, Wang B, Hsu SH, Datta J, Kutay H, Yadav A, Nuovo 33. Kim SY, Han YM, Oh M, Kim WK, Oh KJ, Lee SC, Bae KH, Han BS. G, Kumar P, Ghoshal K, et al. MicroRNA-122 inhibits tumorigenic prop- DUSP4 regulates neuronal differentiation and calcium homeostasis by erties of hepatocellular carcinoma cells and sensitizes these cells to sorafe- modulating ERK1/2 phosphorylation. Stem Cells Dev. 2015;24:686– nib. J Biol Chem. 2009;284:32015–27. doi:10.1074/jbc.M109.016774. 700. doi:10.1089/scd.2014.0434. 19. Patterson KI, Brummer T, O’Brien PM, Daly RJ. Dual-specificity 34. Lai AZ, Cory S, Zhao H, Gigoux M, Monast A, Guiot MC, Huang S, phosphatases: critical regulators with diverse cellular targets. Biochem Tofigh A, Thompson C, Naujokas M, et al. Dynamic reprogramming J. 2009;418:475–89. doi:10.1042/BJ20082234. of signaling upon met inhibition reveals a mechanism of drug resis- 20. Huang CY, Tan TH. DUSPs, to MAP kinases and beyond. Cell Biosci. tance in gastric cancer. Sci Signal. 2014;7:ra38. doi:10.1126/ 2012;2:24. doi:10.1186/2045-3701-2-24. scisignal.2004839. 21. Zhang R, Wang G, Zhang PF, Zhang J, Huang YX, Lu YM, Da W, Sun 35. Xu T, Wu X, Chen Q, Zhu S, Liu Y, Pan D, Chen X, Li D. The anti- Q, Zhu JS. Sanguinarine inhibits growth and invasion of gastric cancer apoptotic and cardioprotective effects of salvianolic acid a on rat car- cells via regulation of the DUSP4/ERK pathway. J Cell Mol Med. diomyocytes following ischemia/reperfusion by DUSP-mediated regu- 2017;21:1117–27. doi:10.1111/jcmm.13043. lation of the ERK1/2/JNK pathway. PloS One. 2014;9:e102292. 22. De Vriendt V, De Roock W, Di Narzo AF, Tian S, Biesmans B, Jacobs doi:10.1371/journal.pone.0102292. B, Budinska E, Sagaert X, Rossi S, D’Ario G, et al. DUSP 4 expression 36. Balko JM, Schwarz LJ, Bhola NE, Kurupi R, Owens P, Miller TW, identifies a subset of colorectal cancer tumors that differ in MAPK Gomez H, Cook RS, Arteaga CL. Activation of MAPK pathways due activation, regardless of the genotype. Biomarkers: biochemical indica- to DUSP4 loss promotes cancer stem cell-like phenotypes in basal-like tors of exposure, response, and susceptibility to chemicals. breast cancer. Cancer Res. 2013;73:6346–58. doi:10.1158/0008-5472. 2013;18:516–24. doi:10.3109/1354750X.2013.819038. CAN-13-1385. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cancer Biology & Therapy Taylor & Francis

MiR-122-5p inhibits cell migration and invasion in gastric cancer by down-regulating DUSP4

Download PDF
9 pages

Loading next page...
 
/lp/taylor-francis/mir-122-5p-inhibits-cell-migration-and-invasion-in-gastric-cancer-by-Ay8CjiMeGB

References (39)

Publisher
Taylor & Francis
Copyright
© 2018 Taylor & Francis Group, LLC
ISSN
1555-8576
eISSN
1538-4047
DOI
10.1080/15384047.2018.1423925
Publisher site
See Article on Publisher Site

Abstract

CANCER BIOLOGY & THERAPY 2018, VOL. 19, NO. 5, 427–435 https://doi.org/10.1080/15384047.2018.1423925 RESEARCH PAPER MiR-122-5p inhibits cell migration and invasion in gastric cancer by down-regulating DUSP4 a b b c a a a d Xiaofeng Xu , Feng Gao , Jianjiang Wang , Lan Tao , Jinsong Ye , Li Ding , Wei Ji , and Xing Chen a b Department of Laboratory Medicine, Jingjiang People’s Hospital, Jingjiang, Jiangsu, China; Department of Gastroenterology, Jingjiang People’s c d Hospital, Jingjiang, Jiangsu, China; Department of Central Laboratory, Jingjiang People’s Hospital, Jingjiang, Jiangsu, China; Department of Science and Education, Jingjiang People’s Hospital, Jingjiang, Jiangsu, China ABSTRACT ARTICLE HISTORY Received 20 November 2017 Objective: To explore the relationship between miR-122-5p and DUSP4 and their effects on gastric cancer Accepted 29 December 2017 (GC) cell mobility and invasiveness. Methods: Abnormally expressed miRNAs and mRNAs were analyzed using microarrays. The miR-122-5p KEYWORDS and DUSP4 mRNA expression levels in GC tissues and cells were determined by RT-qPCR. The target DUSP4; Gastric cancer; relationship between miR-122-5p and DUSP4 was validated by dual luciferase reporter assay. GC cell invasion; migration; miR-122- mobility and invasiveness were respectively observed by wound healing assay and transwell invasion 5p assay. Western blot and immunohistochemistry were used for detection of the expressions of DUSP4 protein and MMP2 and MMP9 proteins related to cell invasion and migration. The migration and invasion abilities of gastric cancer cells in vivo were evaluated according to the number of lung metastatic nodules in mice. Results: The expression of miR-122-5p in GC tissues and cells was significantly down-regulated, whereas DUSP4 expression was up-regulated. Bioinformatics prediction strategies and dual luciferase reporter assay verified the binding sites of miR-122-5p on 3 UTR of DUSP4 and the target relationship between miR-122- 5p and DUSP4. Overexpression of miR-122-5p and knockdown of DUSP4 in BGC-823 cells observantly suppressed GC cell mobility and invasiveness, whereas downregulation of miR-122-5p expression promoted cell metastasis. MiR-122-5p inhibited GC cell mobility and invasiveness and pulmonary tumor metastasis via downregulation of DUSP4. Conclusion: MiR-122-5p restrained migration and invasion abilities of GC cells by repressing DUSP4. Introduction with 3 -UTR. Previous studies substantiated that dysregulation Gastric cancer (GC) is identified as one of the most prevalent of miRNAs often occurs in multiple cancers. Furthermore, aber- malignancies, with over 1 million death cases annually world- rant expressions of miRNAs could interfere with tumorgenesis 12,13 wide. Up to now, smoking, alcohol consumption and helico- and development. In a large family of miRNAs, miR-122 has bacter pylori infection have been found to be main culprits of been thought to be down-regulated in several types of cancers, 2,3 14 15 16 GC. Due to its high metastasis allowing the neoplasm to including GC, gallbladder carcinoma, bladder cancer and so rapidly progress into advanced stages, early diagnosis of GC on. Furthermore, Ergun € et al. has demonstrated the suppressive remains difficult. Although enormous advancement has been function of miR-122-5p on human breast cancer by targeting achieved in the treatment of GC, multiple therapeutic strate- ADAM10, indicating that miR-122-5p may act as a therapeutic gies still cannot cure such a malignant cancer. Moreover, target in breast cancer. Bai et al. revealed that miR-122-5p is despite traditional TNM staging system is widely applied to commonly repressed in primary hepatocellular carcinomas clinical treatment, fewer biomarkers can accurately predict (HCCs) and can inhibit somatotropin-releasing factor (SRF) the metastasis and recurrence of GC. However, more and activities and tumorgenesis, suggesting that miR-122-5p can act more scientists start to focus on the potential mechanisms of as a tumor inhibitor through different molecular pathways. In the GC and explore new therapeutic targets for the treatment of present study, we delve deeper into the molecular mechanisms of GC. miR-122-5p in GC. MicroRNAs (miRNAs), as a class of small, single stranded and Dual-specificity phosphatases (DUSPs), as a heterogeneous non-coding RNAs, play critical parts in post-transcriptional regu- group of protein phosphatases, can dephosphorylate tyrosine and lation and function as either tumor inhibitor or facilitator in mul- serine or threonine residues. Currently, 25 human DUSPs genes 8,9 tiple cancers. It have been reported that these small molecules have already been explored, of which approximately 11 genes are usually repress mRNAs and prevent transcription by combining found to result in MAPK signaling inactivation and others play a CONTACT Feng Gao [email protected] Department of Gastroenterology, Jingjiang People’s Hospital, No.28 East Zhongzhou Road, Jingjiang 214500, Jiangsu, China; Xing Chen [email protected] Department of Science and Education, Jingjiang People’s Hospital, No. 28 East Zhongzhou Road, Jingjiang 214500, Jiangsu, China. © 2018 Taylor & Francis Group, LLC 428 X. XU ET AL. regulatory role in the dephosphorylation of diverse targets. Sev- Table 1. Primer sequences for RT-qPCR. eral DUSPs involved in the development of specifichuman can- 0 0 Primer sequence (5 to 3 ) cers have been reported over the past decade. Zhang et al. has 0 0 MiR-122-5p (F) 5 -TATTCGCACTGGATACGACACAAAC-3 revealed the inhibitory effects on sanguinarine on growth and 0 0 MiR-122-5p (R) 5 -GCCCGTGGAGTGTGACAATGGT-3 invasiveness of human gastric cancer cells via down-regulation of 0 0 U6 (F) 5 -GCTTCGGCAGCACATATACTAAAAT-3 0 0 DUSP4/ERK pathway. De et al. verified that higher expression U6 (R) 5 -CGCTTCACGAATTTGCGTGTCAT-3 0 0 DUSP4 (F) 5 -TGGTTCATGGAAGCCATAGAG-3 of DUSP4 was associated with a worse overall survival and with 0 0 DUSP4 (R) 5 -CCCGTTTCTTCATCATCAGG-3 clinical characteristics of colorectal cancer patients. Wei et al. 0 0 GAPDH (F) 5 -CCTGCCTCTACTGGCGCTGC-3 0 0 substantiated that miR-101 inhibited macrophage-induced cell GAPDH (R) 5 -GCAGTGGGGACACGGAAGGC-3 growth of hepatocellular carcinoma through targeting DUSP1. F: forward primer; R: reverse primer. Samsonov et al. demonstrated that miR-145 restrained papillary thyroid cancer cell reproduction by targeting DUSP6. However, treatment at Jingjiang People’s Hospital during the period there are fewer studies on the relationship between DUSP6 and between April 2015 and April 2017. All samples were assessed miR-122-5p and their underlying mechanism on GC. by clinicopathological examination and all subjects had been The current study probed into the impacts of miR-122-5p confirmed to receive no radiotherapy or chemotherapy. The and DUSP4 on GC and the association between them. We first samples were conserved in liquid nitrogen at ¡80 C for subse- conjectured that there was a target relationship between miR- 122-5p and DUSP4 and miR-122-5p exerted a certain influence quent experiments. All experiments were ratified by the Ethics on GC cell by modulating DUSP4. Next, the target relationship Committee of Jingjiang People’s Hospital, and informed con- between miR-122-5p and DUSP4 was validated by bioinformat- sents were provided by all subjects. ics prediction strategies and dual luciferase reporter. The impacts of miR-122-5p on GC cells were detected by Immuno- Microarray analysis histochemistry, HE staining, wound healing assay, transwell assay and pulmonary metastasis model assay. Differentially expressed miRNAs were screened out through the GSE78091 gene microarray hybridization, which con- tains 3 random pairs of GC tissues and adjacent tissues. Materials and methods Totally there were 3554 probes and 2095 human genes and some genes were measured by multiple probes. Abnormally Clinical specimens expressed mRNAs were screened out through the GSE2685 GC tissues and the adjacent tissues (at least 3 cm away from the gene microarray hybridization, which contains 22 primary tumor) were collected from 30 patients undergoing surgical human advanced gastric cancer tissues samples and 8 Figure 1. MiR-122-5p was low-expressed in gastric cancer tissues and cells (A) Aberrant expression miRNAs in gastric cancer tissues were reflected by the volcano plot. (B) MiR-122-5p was a lowly expressed miRNA in gastric cancer tissues than in adjacent tissues shown in the heat map. (C) MiR-122-5p expression was significantly lower expression in GC tissues than in adjacent tissues determined by qRT-PCR. (D) The expressions of miR-122-5p in human gastric cancer cell lines (BGC-823, SGC-7901, MGC- 803 and HGC-27) were conspicuously lower compared with human normal gastric mucosal cell line GES1 determined by qRT-PCR. P < 0.05, P < 0.01, compared with normal tissues or GES1 cell line. CANCER BIOLOGY & THERAPY 429 Figure 2. DUSP4 was high-expressed in gastric cancer tissues and cells (A) Abnormal expression mRNAs in gastric carcinoma tissues were reflected by the volcano plot. (B) DUSP4 was a highly expressed mRNA in gastric carcinoma tissues than in noncancerous gastric tissues shown in the heat map. (C) The expression of DUSP4 in GC tissues was dramatically higher than that in adjacent tissues determined by qRT-PCR. (D-E) The protein expression of DUSP4 experienced a significant increase in GC tissues detected by immunohistochemistry and western blot. P < 0.01, compared with noncancerous or adjacent tissues. adjacent gastric tissues samples. Totally there were 7129 (NC) group: GC cells transfected with scrambles and probes and 7129 human genes and each gene was measured siRNA control. (2) miR-122-5p mimics group: GC cells by one probe. Fold change value greater than 2 and P less transfected with miR-122-5p mimics. (3) miR-122-5p than 0.05 were regarded as the screening criteria for aber- inhibitor group: GC cells transfected with miR-122-5p rantly expressed miRNAs and mRNAs. inhibitor. (4) si-DUSP4 group: GC cells transfected with DUSP4 siRNA (si-DUSP4). (5) MIX group: GC cells co- transfected with miR-122-5p inhibitor and si-DUSP4. The Cell culture and cell transfection transfection efficiency was detected b under an inverted HEK-293T cells were acquired from the stem cell bank of fluorescence microscope. Shanghai Institutes for Biological Sciences, Chinese Acad- emy of Sciences (Shanghai, China). Human normal gastric mucosal cell line GES1 and human gastric cancer cell lines qRT-PCR BCG-823, SGC-7901, MGC-803, HGC-27 were both pro- cured from BeNa Culture Collection Company (Beijing The extraction of total RNA was implemented with TRIzol China). Cells were cultured in RPMI-1640 medium (Gibco- reagent (Invitrogen) following the protocol of the manufac- Invitrogen, USA) with 10% FBS (Hyclone, USA) at 37 C turer. Reverse transcription of extracted RNA and real-time and dissolved using 0.25% trypsin (Invitrogen, USA). quantitative PCR were successively performed according to Transfection was carried out through Lipofectamine 2000 instruction of RT-PCR kit (Invitrogen) and the fluorescence following the instruction of manufacturer (Invitrogen). GC quantitative PCR kit (Invitrogen). Primer sequences used were cells were assigned to five groups: (1) negative control presented at Table 1 with GAPDH as an internal reference for 430 X. XU ET AL. Figure 3. DUSP4 was a potential target of miR-122-5p (A) There was a potential binding site of miR-122-5p on the 3'-UTR of DUSP4 predicted by bioinformatics software. (B) MiR-122-5p could inhibit the luciferase activity of the cells transfected wild-type 3 UTR of DUSP4, but had little influence on that of those transfected with mutated- type 3 UTR of DUSP4. (C-D) There was a negative correlation between DUSP4 and miR-122-5p in HEK-293T cells and BCG-823 cells. P < 0.05, compared with NC group. mRNAs and U6 for miRNAs, and the relative expressions of primary antibody (1:100) and HRP-conjugated anti-rabbit sec- ¡ DDC miR-122-5p and DUSP4 were quantified by 2 method. ondary antibody (1:2000). Afterwards, immunoreactivity in the sections was detected using a horseradish peroxidase (3,3 -diami- nobenzidine substrate) kit (BioGenex, Fremont, CA, USA). The Western blot slides were then counterstained with hematoxylin (Beyotime, Shanghai, China), dehydrated and mounted. The sections were Total proteins were isolated by using 1 ml radioimmunopreci- evaluated by an optical microscopy. pitation assay buffer with 10 ml phenylmethanesulfonyl fluo- ride. Then protein concentration was examined by a bicinchoninic acid protein assay kit. Proteins were separated using 8% SDS-PAGE, then electro-transferred onto PVDF Hematoxylin-Eosin (HE) staining membranes, which were sealed with 5% non-fat milk for 2 h The tissues embedded in paraffin were first deparaffinized in and incubated in the primary antibodies rabbit polyclonal anti- xylene, and then soaked in alcohol with gradient concentration DUSP4 (ab72593, 1:500, Abcam Trading, Shanghai, China), (100%, 95%, 90%, 80%, 70%) for 10 min each. Hematoxylin rabbit polyclonal anti-MMP2 (ab37150, 1:1000, Abcam Trad- was instilled to stain the tissues for 15 min after washed with ing), rabbit polyclonal anti-MMP9 (1:1000, ab38898, Abcam distilled water for 2 min. The stained tissues were disposed in Trading), rabbit polyclonal anti-b-actin (ab8227, 1:1000, 1% hydrochloric acid ethanol to separate color for 20 s, and Abcam Trading) and rabbit polyclonal anti-GAPDH (ab9485, then soaked in warm water (50 C) for 5 min. The 0.25% eosin 1:2500, Abcam Trading). After rinsed by tris buffer saline con- dye solution was instilled for counterstaining for 2 min, and taining 20% Tween twice, the PVDF membrane was incubated the stained tissues were dehydrated by being soaked in gradient for 2 h at 37 C in the diluted HRP-labeled goat anti-rabbit IgG concentration of alcohol (70%, 80%, 90%, 95%, 100% ethyl H&L secondary antibodies (1:2000). Then the membranes were alcohol) for 10 min each. The samples were observed by using rinsed using TBST for 20 min and visualized using an ECL Plus an optical microscope (200 £). kit (Life Technology Inc., USA). GAPDH and b-actin were respectively considered as internal references for DUSP4 and MMP2/ MMP9. Wound healing assay GC cells BCG-823 were inoculated on 6-well plates, and the Immunohistochemistry germfree pipette was utilized to create a linear scratch in cell The primary and secondary antibodies used in this assay were monolayer when cell confluence was up to 80%. The cells were same as those in western blot. The tissues were fixed in 4% formal- then washed with PBS for 2»3 times and cultivated in the dehyde buffered with phosphate-buffered saline, then dehydrated, serum-free medium. Lastly, we observed the wound distance at embedded in paraffin and serial section cut (4 mm thick). DUSP4 0 h and 24 h by using image-Pro Plus 6.0 (Media Cybernetics, protein was detected after incubation with the rabbit polyclonal Inc., Rockville, MD, USA) and measure the cell mobility. CANCER BIOLOGY & THERAPY 431 Figure 4. MiR-122-5p inhibited gastric cancer cell migration and invasion ability by targeting DUSP4 (A) The transfection efficiency was confirmed by qRT-PCR. (B) The wound distance and cell mobility of the experimental groups at different time points were measured by wound healing assay. The migration ability of the cells in miR- 122-5p group or si-DUSP4 group was significantly lower than in NC group. The NC group and the Mix group could not be significantly distinguished (£ 200). (C) The num- ber of invasion cells in miR-122-5p mimics group and si-DUSP4 group was remarkably fewer compared with NC group, whereas that in miR-122-5p inhibitor group was considerably more than that in NC group detected by transwell invasion assay. Little difference could be found between NC group and the Mix group. P < 0.05, com- pared with NC group. Transwell assay including luciferase genes to construct pMIR-DUSP4-wt plasmids after recovery, purification and enzyme digestion. The transfected cells were seeded onto the upper chamber Likewise, the pMIR-DUSP4-mut plasmids were also con- along with serum-free medium. The medium containing 10% structed based on this method. The recombinant plasmids FBS was instilled into the lower chamber and incubated for were identified through PCR, double-restrict-enzyme diges- 24 h. The cells on the upper chamber were gently removed by tion of EarI (CTCTTC) and PspOMI (GGGCCC) and DNA cotton swabs. The migrating or invading cells in the lower sequence. The HEK-293T cells were first carefully seeded chamber were fixed with alcohol for 15 min and stained using into 24-well plates. Following transfection, RIPA lysis buffer crystal violet for 10 min. Lastly, the cells were detected under (Thermo Fisher Scientific) was added into the 24-well plates an optical microscope (200 £). (100 mL / well). Then the mixture was centrifuged and the supernatant was collected into a 96-well plate. Renilla lucif- erase activity was properly normalized to fireflyluciferase Dual luciferase reporter assay activity. Finally, the relative luciferase activities were was determined following the instructions of Dual-Luciferase Thesequence3 UTR of DUSP4 was cloned into pMIR Reporter Assay kit (Promega). eukaryotic expression vectors (Promega, Madison, USA) 432 X. XU ET AL. Figure 5. MiR-122-5p influenced the expression of MMP2 and MMP9 proteins in gastric cancer by regulating DUSP4 (A-B) The protein expressions of MMP2 and MMP9 were observantly down-regulated in BGC-823 cells transfected with miR-122-5p mimics or si-DUSP detected by western blot and immunohistochemistry. Conversely, the expressions of MMP2 and MMP9 were significantly up-regulated in BGC-823 cells transfected with miR-122-5p inhibitor. The expressions of MMP2 and MMP9 in MIX group were equivalent to those in NC group. P < 0.05, compared with NC group. Murine pulmonary metastasis models procedures were approved by Animal Ethics Committee in C57BL/6nudemice(4-5weeksold,18–20 g, male and Jingjiang People’sHospital. 12 C57BL/6 micewereran- female) were bought from Shanghai Experimental Animal domly divided into two groups and labeled. 5 £ 10 trans- Center (Shanghai, China) and maintained in specificpatho- fected cells in 100 ml of PBS were first injected into the tail th gen free (SPF) level environment. All the experimental vein of the mice. All mice were sacrificed at 24 day after Figure 6. MiR-122-5p inhibited pulmonary tumor metastasis by repressing DUSP4 in vivo (A) The number of murine pulmonary nodules determined by dissecting micro- scope. The number of mice lung metastasis nodules in miR-122-5p mimics group or si-DUSP4 group remarkably decreased, while that in miR-122-5p inhibitor group con- spicuously increased. The number of mice lung metastasis nodules in Mix group was equivalent to that in NC group. (B) HE staining was conducted to detect the number of lung metastatic nodules after tail vein injection. The lungs of mice injected with transfected cells in miR-122-5p mimics group or si-DUSP4 group had almost no visible metastatic nodules, while that of mice in miR-122-5p inhibitor group was fully covered with nodules. (C) The lung weight of mice in miR-122-5p mimics group or si- DUSP4 group decreased significantly, whereas that of mice in miR-122-5p inhibitor increased sharply. There was no significant difference between Mix group and NC group in terms of lung weight. P < 0.05, P < 0.01, compared with NC group. CANCER BIOLOGY & THERAPY 433 injection. Next, the lung, liver and brain tissues were iso- DUSP4 was a potential target of miR-122-5p lated and the number of lung metastatic nodules was The candidate target gene of miR-122-5p was predicted by counted under anatomical microscope. At last, 4% parafor- bioinformatics software, of which the results suggested that maldehyde was used to fix lung, liver and brain tissues for there was a potential binding site of miR-122-5p on the 3'- HE staining and weighting. UTR of DUSP4 (Fig. 3A). Meanwhile, dual luciferase reporter assay also illustrated that after transfected with pMIR-DUSP4-wild type plasmids, the luciferase activity of Statistical analysis BGC-823 cells decreased by about 50% in miR-122-5p Statistical software SPSS version 19.0 (SPSS Inc., USA) and mimics group compared with NC group (P < 0.05, Graphpad 6.0 (GraphPad Software, Inc., USA) were applied to Fig. 3B). However, no significant difference was found data analysis. Results were presented in form of mean § stan- between miR-122-5p mimics group and NC group in terms dard deviation. The differences between two groups were com- of luciferase activity of the cells after transfection with pared by Student’s t-test, while that among three or more pMIR-DUSP4-mutated type plasmids (P > 0.05), suggesting groups was analyzed using one-way ANOVA method. All that miR-122-5p could directly bind to 3'-UTR of DUSP4 experiments were conducted in triplicate and P < 0.05 was and hinder the transcriptional activity of DUSP4.Further- considered as an indicator of statistical significance. more, the results of qRT-PCR indicated that the relative expression of DUSP4 mRNA was negatively correlated to that of miR-122-5p both in HEK-293T cells (P < 0.05, Results Fig. 3C) and BCG-823 cells (P < 0.05, Fig. 3D), which fur- MiR-122-5p was low-expressed in gastric cancer tissues ther validated the above results. and cells We took fold change over 2 and P < 0.05 as the filtration MiR-122-5p suppressed gastric cancer cell mobility and standard to screen out abnormally expressed miRNAs invasiveness by targeting DUSP4 (Fig. 1A) and found that there were 111 up-regulated miR- The mRNA expressions of miR-122-5p and DUSP4 after cell NAs and 76 down-regulated miRNAs in GC tissues in com- transfection were also examined by qRT-PCR. As shown in parison with the adjacent tissues. Among these miRNAs, Fig. 4A, the expression of miR-122-5p was dramatically up-reg- miR-122-5p was found to be down-regulated and miR-122-5p ulated in miR-122-5p mimics group, whereas that in miR-122- expression in GC tissues was averagely 2.99 times lower than 5p inhibitor group was remarkably down-regulated (both that in the adjacent tissues (P D 0.001249428). The top 10 P < 0.05). Conversely, DUSP4 expression levels in miR-122-5p highly expressed miRNAs and the first 10 lowly expressed mimics group and si-DUSP4 group drastically decreased, while miRNAs were selected for the heat map (Fig. 1B). Further- those in miR-122-5p inhibitor group observantly increased (all more, qRT-PCR results exhibited that miR-122-5p expression P < 0.05). There was no conspicuous distinction between Mix was observantly lower in GC tissues than in adjacent tissues group and NC group in terms of the mRNA expressions of (P < 0.01, Fig. 1C). Beyond that, the expressions of miR-122- miR-122-5p and DUSP4 (both P > 0.05). Additionally, wound 5p in human gastric cancer cell lines (BGC-823, SGC-7901, healing assay results exhibited that the cell mobility ability was MGC-803 and HGC-27) were conspicuously lower compared conspicuously attenuated after transfection with miR-122-5p with human normal gastric mucosal cell line GES1 (P < 0.05, mimics or DUSP4 siRNA, while the migration ability of the Fig. 1D). BCG-823 cell line was chosen for subsequent experi- cells transfected with miR-122-5p inhibitor was significantly ments as presented the most significant difference of expres- sion in contrast to GES1 cell line (P < 0.01). enhanced (all P < 0.05, Fig. 4B). No significant difference of cell mobility was detected between Mix group and NC group (P > 0.05). Furthermore, the GC cell invasion was determined DUSP4 was high-expressed in gastric cancer tissues and by transwell invasion assay, of which the results suggested that cells the number of invasion cells in miR-122-5p mimics group and si-DUSP4 group was remarkably fewer compared with NC Likewise, we screened out aberrantly expressed mRNAs based group, whereas that in miR-122-5p inhibitor group was consid- on fold change over 2 and P < 0.05 (Fig. 2A), finding that 245 erably more than that in NC group (all P < 0.05, Fig. 4C). Little mRNAs were up-regulated and 336 mRNAs were down-regu- difference could be observed between NC group and Mix group lated in GC tissues. DUSP4 was found in the up-regulated (P > 0.05). Overall, miR-122-5p inhibited GC cell mobility and mRNAs, and the DUSP4 expression level was averagely invasiveness by down-regulating DUSP4. 1.01 times higher in gastric carcinoma tissues than in noncan- cerous tissues (P D 0.003334112). The top 10 highly or lowly expressed mRNAs were selected for the heat map (Fig. 2B). In MiR-122-5p influenced the expression of MMP2 and MMP9 addition, qRT-PCR also exhibited that DUSP4 expression in proteins by regulating DUSP4 GC tissues was dramatically higher compared with adjacent tissues (P < 0.01, Fig. 2C). In addition, the results from Western blot and immunohistochemistry were both immunohistochemistry (Fig. 2D) and western blot (Fig. 2E) employed to identify the expressions of MMP2 and MMP9 manifested that the expression of DUSP4 protein displayed a proteins related to cell metastasis. Results from western blot considerable increase in GC tissues (P < 0.01). (Fig. 5A) immunohistochemistry (Fig. 5B)bothindicated 434 X. XU ET AL. that the expressions of MMP2 and MMP9 proteins were For instance, miR-122-5p could play key roles in diagnosis and 29, 30 observantly down-regulated in BGC-823 cells transfected specific early prognosis of acute myocardial infarction. In with miR-122-5p mimics or si-DUSP (P < 0.05). the present research, we substantiated that miR-122-5p could Conversely, the expressions of MMP2 and MMP9 were sig- exerted inhibitory influence in GC cell metastasis through nificantly up-regulated in BGC-823 cells transfected with down-regulating DUSP4, suggesting that miR-122-5p could miR-122-5p inhibitor (P < 0.05). The expressions of MMP2 function as a tumor inhibitor in GC. and MMP9 in MIX group were equivalent to those in NC Additionally, the regulation function of DUSP4 was reported 31–33 group (P > 0.05), suggesting that miR-122-5p affected the in many previous study reports, which indicated that expression of MMP2 and MMP9 proteins related to cell DUSP4 had been thought to be a critical signaling regulator of migration and invasiveness by regulating DUSP4. multiple cellular processes, such as cell invasion, migration, dif- ferentiation and so forth. Zhang et al. found that sanguinarine exerted a inhibitive influence on GC cell growth and invasion MiR-122-5p inhibited pulmonary tumor metastasis by via regulation of the DUSP4/ERK pathway. Lai et al. reported repressing DUSP4 in vivo that the increased phosphorylation of ERK, correlated with To further confirm the effects of miR-122-5p and DUSP4 on decreased abundance of the phosphatases DUSP4 and DUSP6, GC cell metastasis, murine pulmonary metastasis model assay might counteract resistance to c-Met inhibitors in GC. Our was performed, from which the results displayed that the num- study verified that DUSP4 could be repressed by miR-122-5p, ber of mice lung metastasis nodules in miR-122-5p mimics hence impeding the progression of GC. Besides, a few signaling group or si-DUSP4 group remarkably decreased, while that in pathways have been found to be involved in DUSP-mediated 35 36 miR-122-5p inhibitor group conspicuously increased (Fig. 6A, regulation like ERK1/2/JNK pathway and MAPK pathways. P < 0.05). The number of mice lung metastasis nodules in Mix However, a lack of study on signaling pathway was one of the group was equivalent to that in NC group (P > 0.05). HE stain- limitations in this study. Apart from it, the small number of tis- ing assay also indicated that the lungs of mice injected with sue samples might make our results not completely convincing. transfected cells in miR-122-5p mimics group or si-DUSP4 In conclusion, over-expression of miR-122-5p could inhibit group had almost no visible metastatic nodules, while that of the cell mobility and invasiveness in GC through targeting mice in miR-122-5p inhibitor group was fully covered with DUSP4. The finding not only laid a foundation for an intensive nodules (Fig. 6B, P < 0.05). The number of mice lung nodules study on GC, but also provided two promising therapeutic tar- in Mix group was equivalent to that in NC group (P > 0.05). gets in GC treatment. Additionally, the lung weight of mice in miR-122-5p mimics group or si-DUSP4 group decreased significantly (P < 0.05), while that of mice in miR-122-5p inhibitor group increased Conflict of interest sharply (Fig. 6C, P < 0.01). There was no significant difference The authors declare no potential conflicts of interest. between Mix group and NC group in terms of lung weight (P > 0.05). Taken together, miR-122-5p restrained pulmonary metastasis by repressing DUSP4 in vivo. Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed. Discussion The current experiments have already identified that miR-122- Funding 5p was low-expressed, while DUSP4 was high-expressed in GC tissues and cell lines. Over-expression of miR-122-5p or knock- None. down of DUSP4 could inhibit the metastasis of GC cells. We disclosed that miR-122-5p played a tumor-suppressive role in cell mobility and invasiveness by targeting DUSP4 in GC cells. References Meanwhile, we also validated that miR-122-5p impeded pul- 1. Van Cutsem E, Sagaert X, Topal B, Haustermans K, Prenen H. Gastric monary tumor metastasis by repressing DUSP4 in vivo. cancer. Lancet. 2016;388:2654–64. doi:10.1016/S0140-6736(16)30354-3. The molecular mechanism of GC was a process involving 2. Li L, Ying XJ, Sun TT, Yi K, Tian HL, Sun R, Tian JH, Yang KH. Over- multiple factor, including epigenetic factors and genetic factors. view of methodological quality of systematic reviews about gastric Up to now, considerable researches have substantiated that cancer risk and protective factors. Asian Pac J Cancer Prev: APJCP. 2012;13:2069–79. doi:10.7314/APJCP.2012.13.5.2069. miRNAs play a crucial function in the gene regulation in multi- 3. Kato M, Asaka M. Recent knowledge of the relationship between Heli- ple cancers. Zhang et al. unraveled that miR-200c suppressed cobacter pylori and gastric cancer and recent progress of gastroendo- GC cell reproduction and metastasis through directly targeting scopic diagnosis and treatment for gastric cancer. Jpn J Clin Oncol. FN1. Wang et al. demonstrated that miR-190b enhanced the 2010;40:828–37. doi:10.1093/jjco/hyq119. radio-sensitivity of GC cells through negatively regulated Bcl- 4. Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J. Cancer statistics in China, 2015. CA: a cancer journal for 2. Ding et al. revealed that miR-27a stimulated the tumorgen- clinicians. 2016;66:115–32. esis of GC by activating the AKT/GSK3b pathway. Mi et al. 5. Zheng L, Jiao W, Song H, Qu H, Li D, Mei H, Chen Y, Yang F, Li H, validated that miR-181a-5p facilitated the development of GC Huang K, et al. miRNA-558 promotes gastric cancer progression via RASSF6-modulated MAPK signaling pathway. Recently, through attenuating Smad4-mediated repression of heparanase miR-122-5p has been identified as a potential novel biomarker. expression. Cell Death Dis. 2016;7:e2382. doi:10.1038/cddis.2016.293. CANCER BIOLOGY & THERAPY 435 6. Chia NY, Tan P. Molecular classification of gastric cancer. Ann Oncol: 23. Wei X, Tang C, Lu X, Liu R, Zhou M, He D, Zheng D, Sun C, Wu Z. official journal of the European Society for Medical Oncology. MiR-101 targets DUSP1 to regulate the TGF-beta secretion in sorafe- 2016;27:763–9. doi:10.1093/annonc/mdw040. nib inhibits macrophage-induced growth of hepatocarcinoma. Onco- 7. Tsai MM, Wang CS, Tsai CY, Huang HW, Chi HC, Lin YH, Lu PH, target. 2015;6:18389–405. doi:10.18632/oncotarget.4089. Lin KH. Potential Diagnostic, Prognostic and Therapeutic Targets of 24. Samsonov R, Burdakov V, Shtam T, Radzhabovsmall a CZ, Vasilyev MicroRNAs in Human Gastric Cancer. Int J Mol Sci. 2016;17. D, Tsyrlina E, Titov S, Ivanov M, Berstein L, Filatov M, et al. Plasma doi:10.3390/ijms17060945. exosomal miR-21 and miR-181a differentiates follicular from papillary 8. Sundaram P, Hultine S, Smith LM, Dews M, Fox JL, Biyashev D, thyroid cancer. Tumour Biol: the journal of the International Society Schelter JM, Huang Q, Cleary MA, Volpert OV, et al. p53-responsive for Oncodevelopmental Biology and Medicine. 2016;37:12011–21. miR-194 inhibits thrombospondin-1 and promotes angiogenesis in doi:10.1007/s13277-016-5065-3. colon cancers. Cancer Res. 2011;71:7490–501. doi:10.1158/0008-5472. 25. Zhang H, Sun Z, Li Y, Fan D, Jiang H. MicroRNA-200c binding to CAN-11-1124. FN1 suppresses the proliferation, migration and invasion of gastric 9. Bar-Eli M. Searching for the ‘melano-miRs’: miR-214 drives melanoma cancer cells. Biomed Pharmacother D Biomedecine & pharmacothera- metastasis. EMBO J. 2011;30:1880–1. doi:10.1038/emboj.2011.132. pie. 2017;88:285–92. 10. An Y, Zhang Z, Shang Y, Jiang X, Dong J, Yu P, Nie Y, Zhao Q. miR- 26. Wang C, Qiao C. MicroRNA-190b confers radio-sensitivity through 23b-3p regulates the chemoresistance of gastric cancer cells by target- negative regulation of Bcl-2 in gastric cancer cells. Biotechnol Lett. ing ATG12 and HMGB2. Cell Death Dis. 2015;6:e1766. doi:10.1038/ 2017;39:485–90. doi:10.1007/s10529-016-2273-2. cddis.2015.123. 27. Ding L, ZhangS,XuM,Zhang R, SuiP,YangQ.MicroRNA-27a 11. Zhang X, Zeng J, Zhou M, Li B, Zhang Y, Huang T, Wang L, Jia J, contributes to the malignant behavior of gastric cancer cells by Chen C. The tumor suppressive role of miRNA-370 by targeting directly targeting PH domain and leucine-rich repeat protein FoxM1 in acute myeloid leukemia. Mol Cancer. 2012;11:56. phosphatase 2. J Exp Clin Cancer Res: CR. 2017;36:45. doi:10.1186/1476-4598-11-56. doi:10.1186/s13046-017-0516-2. 12. Zhu M, Zhang N, He S, Lui Y, Lu G, Zhao L. MicroRNA-106a targets 28. Mi Y, Zhang D, Jiang W, Weng J, Zhou C, Huang K, Tang H, Yu Y, TIMP2 to regulate invasion and metastasis of gastric cancer. FEBS Liu X, Cui W, et al. miR-181a-5p promotes the progression of gastric Lett. 2014;588:600–7. doi:10.1016/j.febslet.2013.12.028. cancer via RASSF6-mediated MAPK signalling activation. Cancer 13. Miko E, Margitai Z, Czimmerer Z, Varkonyi I, Dezso B, Lanyi A, Lett. 2017;389:11–22. doi:10.1016/j.canlet.2016.12.033. Bacso Z, Scholtz B. miR-126 inhibits proliferation of small cell lung 29. Cortez-Dias N, Costa MC, Carrilho-Ferreira P, Silva D, Jorge C, cancer cells by targeting SLC7A5. FEBS Lett. 2011;585:1191–6. Calisto C, Pessoa T, Robalo Martins S, de Sousa JC, da Silva PC, et al. doi:10.1016/j.febslet.2011.03.039. Circulating miR-122-5p/miR-133b Ratio Is a Specific Early Prognostic 14. Chen Q, Ge X, Zhang Y, Xia H, Yuan D, Tang Q, Chen L, Pang X, Biomarker in Acute Myocardial Infarction. Circ J: official journal of Leng W, Bi F. Plasma miR-122 and miR-192 as potential novel bio- the Japanese Circulation Society. 2016;80:2183–91. doi:10.1253/circj. markers for the early detection of distant metastasis of gastric cancer. CJ-16-0568. Oncol Rep. 2014;31:1863–70. doi:10.3892/or.2014.3004. 30. Yao XL, Lu XL, Yan CY, Wan QL, Cheng GC, Li YM. Circulating 15. Lu W, Zhang Y, Zhou L, Wang X, Mu J, Jiang L, Hu Y, Dong P, Liu Y. miR-122-5p as a potential novel biomarker for diagnosis of acute miR-122 inhibits cancer cell malignancy by targeting PKM2 in gall- myocardial infarction. Int J Clin Exp Pathol. 2015;8:16014–9. bladder carcinoma. Tumour Biol. 2015. doi:10.1007/s13277-015- 31. Barajas-Espinosa A, Basye A, Angelos MG, Chen CA. Modulation of 4308-z. p38 kinase by DUSP4 is important in regulating cardiovascular func- 16. Wang Y, Xing QF, Liu XQ, Guo ZJ, Li CY, Sun G. MiR-122 targets tion under oxidative stress. Free Radic Biol Med. 2015;89:170–81. VEGFC in bladder cancer to inhibit tumor growth and angiogenesis. doi:10.1016/j.freeradbiomed.2015.07.013. Am J Transl Res. 2016;8:3056–66. 32. Schmid CA, Robinson MD, Scheifinger NA, Muller S, Cogliatti S, 17. Ergun S, Ulasli M, Igci YZ, Igci M, Kirkbes S, Borazan E, Balik A, Tzankov A, Muller € A. DUSP4 deficiency caused by promoter hyper- Yumrutas ¸ O, Camci C, Cakmak EA, et al. The association of the expres- methylation drives JNK signaling and tumor cell survival in diffuse sion of miR-122-5p and its target ADAM10 with human breast cancer. large B cell lymphoma. J Exp Med. 2015;212:775–92. doi:10.1084/ Mol Biol Rep. 2015;42:497–505. doi:10.1007/s11033-014-3793-2. jem.20141957. 18. Bai S, Nasser MW, Wang B, Hsu SH, Datta J, Kutay H, Yadav A, Nuovo 33. Kim SY, Han YM, Oh M, Kim WK, Oh KJ, Lee SC, Bae KH, Han BS. G, Kumar P, Ghoshal K, et al. MicroRNA-122 inhibits tumorigenic prop- DUSP4 regulates neuronal differentiation and calcium homeostasis by erties of hepatocellular carcinoma cells and sensitizes these cells to sorafe- modulating ERK1/2 phosphorylation. Stem Cells Dev. 2015;24:686– nib. J Biol Chem. 2009;284:32015–27. doi:10.1074/jbc.M109.016774. 700. doi:10.1089/scd.2014.0434. 19. Patterson KI, Brummer T, O’Brien PM, Daly RJ. Dual-specificity 34. Lai AZ, Cory S, Zhao H, Gigoux M, Monast A, Guiot MC, Huang S, phosphatases: critical regulators with diverse cellular targets. Biochem Tofigh A, Thompson C, Naujokas M, et al. Dynamic reprogramming J. 2009;418:475–89. doi:10.1042/BJ20082234. of signaling upon met inhibition reveals a mechanism of drug resis- 20. Huang CY, Tan TH. DUSPs, to MAP kinases and beyond. Cell Biosci. tance in gastric cancer. Sci Signal. 2014;7:ra38. doi:10.1126/ 2012;2:24. doi:10.1186/2045-3701-2-24. scisignal.2004839. 21. Zhang R, Wang G, Zhang PF, Zhang J, Huang YX, Lu YM, Da W, Sun 35. Xu T, Wu X, Chen Q, Zhu S, Liu Y, Pan D, Chen X, Li D. The anti- Q, Zhu JS. Sanguinarine inhibits growth and invasion of gastric cancer apoptotic and cardioprotective effects of salvianolic acid a on rat car- cells via regulation of the DUSP4/ERK pathway. J Cell Mol Med. diomyocytes following ischemia/reperfusion by DUSP-mediated regu- 2017;21:1117–27. doi:10.1111/jcmm.13043. lation of the ERK1/2/JNK pathway. PloS One. 2014;9:e102292. 22. De Vriendt V, De Roock W, Di Narzo AF, Tian S, Biesmans B, Jacobs doi:10.1371/journal.pone.0102292. B, Budinska E, Sagaert X, Rossi S, D’Ario G, et al. DUSP 4 expression 36. Balko JM, Schwarz LJ, Bhola NE, Kurupi R, Owens P, Miller TW, identifies a subset of colorectal cancer tumors that differ in MAPK Gomez H, Cook RS, Arteaga CL. Activation of MAPK pathways due activation, regardless of the genotype. Biomarkers: biochemical indica- to DUSP4 loss promotes cancer stem cell-like phenotypes in basal-like tors of exposure, response, and susceptibility to chemicals. breast cancer. Cancer Res. 2013;73:6346–58. doi:10.1158/0008-5472. 2013;18:516–24. doi:10.3109/1354750X.2013.819038. CAN-13-1385.

Journal

Cancer Biology & Therapy – Taylor & Francis

Published: May 4, 2018

Keywords: DUSP4; Gastric cancer; invasion; migration; miR-122-5p

There are no references for this article.