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LINC00035 Transcriptional Regulation of SLC16A3 via CEBPB Affects Glycolysis and Cell Apoptosis in Ovarian Cancer

LINC00035 Transcriptional Regulation of SLC16A3 via CEBPB Affects Glycolysis and Cell Apoptosis... Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2021, Article ID 5802082, 12 pages https://doi.org/10.1155/2021/5802082 Research Article LINC00035 Transcriptional Regulation of SLC16A3 via CEBPB Affects Glycolysis and Cell Apoptosis in Ovarian Cancer Shuyan Yang, Jing Wang, Rongjie Cheng, Bo Pang, and Pengcheng Sun Department of Obstetrics and Gynecology, e Fourth Affiliated Hospital of Harbin Medical University, No. 37, Yiyuan Street, Nangang District, Harbin 150001, Heilongjiang, China Correspondence should be addressed to Pengcheng Sun; [email protected] Received 8 September 2021; Accepted 25 September 2021; Published 11 October 2021 Academic Editor: Songwen Tan Copyright © 2021 Shuyan Yang et al. )is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. Ovarian cancer (OC) represents the most lethal gynecologic malignancy globally. Over the decades, lncRNAs have been considered as study focuses due to their genome-wide expression through multiple mechanisms in which regulation of target gene transcription through interaction with transcription factors or epigenetic proteins is proven. In the present work, we focus on the functional role of LINC00035 in OC and its regulation mechanism on gene expression. Methods. We collected OC tissues and adjacent tumor-free tissues surgically resected from 67 OC patients. Cultured human OC cell lines SKOV3 and A2780 were assayed for their viability, migration, invasion, apoptosis in vitro using CCK-8 assays, transwell assays, and flow cytometric analysis. OC cell tumorigenesis in vivo was evaluated by mouse xenograft experiments. Glycolysis was evaluated by glucose uptake, lactate release, and ATP production assays. Luciferase activity assay, RNA immunoprecipitation (RIP), and RNA pull-down were performed to confirm the interactions among LINC00035, CEBPB, and SLC16A3. Results. LINC00035 was upregulated in OC tissues. LINC00035 knockdown was shown to repress SKOV3 and A2780 cell viability, migration, invasion, induce their ap- optosis, and reduce glucose uptake, lactate release, and ATP production. LINC00035 could recruit CEBPB into the SLC16A3 promoter region, thus increasing the SLC16A3 transcription. SLC16A3 was upregulated in OC tissues. SLC16A3 knockdown exerted similar effects on SKOV3 and A2780 cells as LINC00035 knockdown. Rescue experiments found SLC16A3 overexpression resisting to LINC00035 knockdown on SKOV3 and A2780 cell viability, migration, invasion, apoptosis, glucose uptake, lactate release, and ATP production. Results also showed LINC00035 knockdown could inhibit OC cell tumorigenesis in vivo. Con- clusion. )e study reveals that LINC00035 promotes OC progression by regulating glycolysis and cell apoptosis through CEBPB- mediated transcriptional promotion of SLC16A3. diagnosed at stage III (51%) or IV (29%), with a high rate of 1. Introduction relapse and metastasis into the abdominal cavity [5]. A large- Ovarian cancer (OC) is the third most common gynecologic scale study of OC screening has shown that only using ultrasound or CA125 test for screening every year and, if malignancy and one of the leading causes of cancer death among women around the world [1]. OC is considered to be necessary, follow-up with ultrasound fails to decrease a heterogeneous malignant tumor with unique genomic mortality in the general population. Despite more early characteristics. Histologically, it can be divided into five diagnosis by screening, the increase was not sufficient to different subtypes at least [2]. )e report of Global Cancer significantly improve survival [6, 7]. Given that various Statistics 2020 estimated that the incidence and mortality of factors involving frequent recurrence, late stage during OC accounts for 1.6% and 2.1%, respectively [3]. Due to less initial diagnosis, the presence of resistance to drugs, and early clinical features and the presence of metastatic and adverse reaction to chemotherapeutic regimens occur in invasive cancer cells in most patients at the time of diagnosis, most of the patients, it has become imperative to extensively OC remains one of the most challenging malignancies to elucidate the underlying mechanisms behind OC develop- manage [4]. Most women with serous carcinomas are ment. Long noncoding RNAs (lncRNAs), a subgroup of 2 Evidence-Based Complementary and Alternative Medicine RNAs (>200 nucleotides in length), engage in OC initiation, lipofectamine 2000 reagent transfection (Invitrogen, USA). drug resistance, and progression [8, 9]. LINC00035, located Cell transfection was performed lasting for 24–48 h. on the human chromosome 7q11.23, with another known name ABHD11-AS1, has been widely confirmed as an on- 2.3. Quantitative Real-Time Polymerase Chain Reaction cogene, showing different power of gene control, including (qRT-PCR). After total RNA extractions from tissue and epigenetic, transcription, or posttranscriptional control in cells by using Trizol reagents (Invitrogen), cDNA was many types of human cancers, such as gastric cancer [10], generated according to the manuals of the PrimeScript RT pancreatic cancer [11], endometrial carcinoma [12], thyroid reagent Kit (Takara, Japan). )e SYBR Premix ExTaqTM II cancer [13], lung cancer [14], and breast cancer [15]. ® (RR820A, Takara) kit and the ABI PRISM 7300 System LncRNAs interact with a variety of transcription factors (ABI, USA) were applied to complete the qRT-PCR. Data to modulate gene expression and then participate in cell were relative to the expression of a reference gene GADPH growth and apoptosis [16]. )e transcription factor CCAAT/ −ΔΔCt and analyzed using the 2 method. )e primers of enhancer-binding protein beta (CEBPB) is required for LINC00035, SLC16A3, and GADPH were synthesized by maintenance of the tumor-initiating capacity and invasion RiboBio (Guangzhou, China). )e primer sequences of ability in several tumors, including hepatocellular carcinoma LINC00035 were 5′-TCCAGACAAGACTTGGTCGC-3’ [17], glioblastoma [18], and breast cancer [19]. SLC16A3 is a (forward) and 5′-CAGCTGGTTGTGTGGCTTTC-3’ (re- member of the SLC16 gene family that has fourteen verse), of SLC16A3 were 5′-CTTCCCGTCAGACGCCC-3’ members among which SLC16A1, SLC16A3, SLC16A7, and (forward) and 5′-GTGTTCAGTACCAGCCCTGT-3’ (re- SLC16A8 encode proton-coupled monocarboxylate trans- verse), and of GADPH were 5′-ATGGA- porters MCT1, MCT4, MCT2, and MCT3, respectively [20]. GAAGGCTGGGGCTC-3’ (forward) and 5′- SLC16A3 has the ability to catalyze the proton-linked AAGTTGTCATGGATGACCTTG-3’ (reverse). transport of monocarboxylates such as l-lactate, pyruvate, and ketone bodies across the plasma membrane, which was recognized as glycolysis-related gene signature in human 2.4. CCK-8 Assays. A2780 and SKOV3 cells were incubated cancers [21, 22]. )e LncMAP database (http://bio-bigdata. with 3 ×10 cells per well in the 96-well plate. Upon incu- hrbmu.edu.cn/LncMAP/) shows LINC00035 regulates bation at different time points, 0, 1, 2, and 3 d, each well transcription factor CEBPB and its downstream gene reacted with the addition of CCK-8 reaction solution SLC16A3 in OC. Based on the aforementioned data, we (Dojindo, Kumamoto, Japan). Another 2h incubation therefore speculate whether there is a promising correlation continued. )e optical value was obtained at 450 nm. among LINC00035, CEBPB, and SLC16A3 in OC. 2.5. Transwell Migration and Invasion Assays. A2780 and 2. Materials and Methods SKOV3 cells (6 ×10 cells for each) were suspended in 200 μL serum-free medium and supplemented into the 2.1. Human Tissue Specimen. We collected OC tissues and apical chamber of the transwell system (Corning-Costar, adjacent tumor-free tissues surgically resected from 67 OC Cambridge, MA) uncoated (for migration) or coated (for patients who were admitted into our hospital in the time invasion) with Matrigel (BD Biosciences, San Jose, CA). duration from January 2020 to December 2020. No patients Additional 700 μL DMEM with 10% FBS was added into the had received radiotherapy, chemotherapy, or immuno- basolateral chamber. Following 24-hour incubation, A2780 therapy before surgery. Signed informed consents were and SKOV3 cells were transferred from the apical chamber obtained from all participants. )e study was approved by into the basolateral one, followed by addition of methanol the Institutional Review Board of our hospital. Experiments and 0.1% crystal violet. )e number of migrating or invasive involving human beings were performed in strict accordance cells was photographed under an inverted microscope and with the Declaration of Helsinki. also calculated flow cytometric analysis. A2780 and SKOV3 cells were suspended in the DMEM to adjust the density into 1 × 10 cells/mL. )e cell sus- 2.2. Cell Culture and Transient Transfection. Under the pensions reacted with the addition of 5 μl Annexin V and 5 μl conditions of humidity of 5% CO and temperature of 37 C, propidium iodide (PI). Apoptotic A2780 and SKOV3 cells IOSE80 used as normal ovarian epithelial cells and Caov-3, were analyzed using a flow cytometer (FACScan ) equipped A2780, SKOV3, and CoC1 used as human OC cell lines with Cell Quest software (BD Biosciences). (ATCC, USA) were grown in the RPMI1640 with 15% fetal bovine serum (FBS) (Gibco, USA). To construct OC cells with LINC00035 overexpression and knockdown, CEBPB 2.6. Immunoblotting Analysis. After 10% SDS-PAGE sepa- overexpression and knockdown, SLC16A3 overexpression ration, extracted total protein was wet-transferred to the and knockdown, siRNA targeting LINC00035, the expres- PVDF membrane (Sigma-Aldrich, USA). Immunoblots sion vector containing the full-fragments of LINC00035, were generated after addition of primary antibodies: anti- siRNA targeting CEBPB, the expression vector containing bcl-2 antibody (ab182858, Abcam, UK), anti-bax antibody the full-fragments of CEBPB, siRNA targeting SLC16A3 and (ab182734, Abcam), anti-CEBPB antibody (ab32358, the expression vector containing the full-fragments of Abcam), anti-SLC16A3 antibody (ab74109, Abcam), and SLC16A3 were delivered into selected OC cells by mean of anti-GAPDH antibody (ab171091, Abcam). Horseradish Evidence-Based Complementary and Alternative Medicine 3 2.11. Chromatin Immunoprecipitation (ChIP). To generate peroxidase-labeled IgG and enhanced chemiluminescence solution were added for visualization. With GAPDH as the DNA-protein cross-links, 10% formaldehyde was added to fix A2780 and SKOV3 cells for 10 min. Chromatin fragments loading control, immunoblots were photographed by the use of Bio-Rad image analysis system (BIO-RAD, Hercules, CA) was generated by sonication of the DNA-protein cross-links. and their densimetric analyses were performed using )ree parts with equal volume were divided from the Quantity One v4.6.2 software. chromatin fragments. Anti-CEBPB antibody (ab32358, Abcam) or normal mouse IgG was applied to generate immunoprecipitates. Protein agarose/sepharose beads were 2.7. Glucose Uptake, Lactate Release, and ATP Production used to precipitate the DNA-protein complexes. Cross-link Assays. A2780 and SKOV3 cells were seeded with 50,000 and elution were finished and qRT-PCR analysis was con- cells per well in 12-well plates and then underwent serum ducted to the DNA samples. Mouse xenograft experiments. starvation for 24 h. Afterwards, cells were stimulated with Animal studies consisted of 12 male and 12 female 100 nmol/L insulin for 30 min. Cells were treated with 10 μM BALB/c mice (Nanjing, Junke Biological Co., Ltd., Jiangsu, 2-NBDG for 1 h at 37 C. )e uptake of 2-NBDG was ana- China), with age of 4–6 weeks and weight of 18 to 22 g. lyzed with an FC500 Flow Cytometer (Beckman Coulter, SKOV3 cells transfected with si-LINC00035 plus empty Brea, CA, USA) to reflect glucose uptake. Lactate release and vector, si-SLC16A3, scramble siRNA plus empty vector, or ATP production were determined by Lactate Colorimetric/ si-LINC00035 plus SLC16A3 expression vector were ad- Fluorometric Assay Kit (BioVision, USA) and ATP Deter- justed into suspensions with 1 × 10 cells/mL, and 0.5 ml of mination Kit ()ermo Fisher Scientific, USA), respectively. them were subcutaneously implanted into the scapular re- gion of each mouse. )e growth of xenografted tumors in mice was monitored every 5 days. When the tumor was 2.8. Immunoprecipitation (RIP) Assays. A commercial kit visible by neck eyes, the length and width of the tumor were (Millipore, USA) was used to examine the combination of measured. After 6 weeks of implantation, the mice were LINC00035 and CEBPB following the manufacturer’s euthanized by exposure to prolonged inhalational anes- manual. A2780 and SKOV3 cell lysis were centrifuged thesia. All animal experiments were conducted under the (14000 × g, 10 min) and cell extracts for three parts with approval of Ethics Committee of our hospital and in ac- 100 μL each were collected. Immunoprecipitation was per- cordance with the Guide for the Care and Use of Laboratory formed in two cell extracts using protein A/G sepharose Animal. Tremendous efforts were done to minimize pain the beads equipped with either anti-CEBPB antibody (ab32358, included animals suffered from. Abcam) or normal mouse IgG. Immunoprecipitated RNA and total RNA from the input control were extracted for qRT-PCR analysis. 2.12. Data Analysis. SPSS 20.0 software (IBM Corp., Armonk, NY, USA) was used for statistical comparisons. A manner of mean± standard deviation was used to display all 2.9. RNA Pull-Down Assays. A2780 and SKOV3 cells were data. Paired t-test was used as a statistical tool to compare transfected with either biotin-labeled wild-type LINC00035 data between OC tissues and nontumor tissues. One-way or mutated LINC00035 probes for 48 h. Subsequently, cell analysis of variance (ANOVA) was used for multiple-group lysis was centrifuged (14000 × g, 10 min). Cell extracts were comparison, and repeated measurement ANOVA was used immunoprecipitated with M-280 streptavidin beads (S3762, upon different time points. A difference level less than 0.05 Sigma-Aldrich) precoated with RNase-free bovine serum was indicative of statistical significance. albumin (BSA) and yeast tRNA (TRNABAK-RO, Sigma- Aldrich). Immunoprecipitated protein and total protein 3. Results from the input control were extracted. Following 10% SDS- PAGE separation and membrane transfer, immunoblotting 3.1. LINC00035 Knockdown Attenuated Glycolysis and analysis of CEBPB was performed. Apoptosis in OC Cells. )e qRT-PCR demonstrated a re- markably regulated LINC00035 in OC tissues compared to that of adjacent tumor-free tissues (Figure 1(a)). Subse- 2.10. Promoter Luciferase Activity. )e oligonucleotides on quently, qRT-PCR was carried out to determine the ex- the 3′UTR of SLC16A3 mRNA and mutated ones were pression of LINC00035 in different OC cell lines. Similarly, inserted into commercially available luciferase reporter we found that LINC00035 in Caov-3, A2780, SKOV3, and vectors (pmirGLO, Promega, USA), named SLC16A3-wt CoC1 human OC cell lines was increased compared with and SLC16A3-mut. Treatment was done on A2780 and normal ovarian epithelial cells IOSE80, especially highly SKOV3 cells with SLC16A3-wt or SLC16A3-mut in the expressed in A2780 and SKOV3 cells (Figure 1(a)). Con- presence of CEBPB expression vector or si-CEBPB. Sub- sidering downregulated LINC00035 in OC tissues and cells, sequent to 48 h of transfection, the luciferase activity in the LINC00035 knockdown A2780 and SKOV3 cells, which cell lysis was determined by the dual-luciferase reporter gene were confirmed by the qRT-PCR (Figure 1(b)), were suc- assay (Promega). )e relative luciferase activity was calcu- cessfully constructed to present the function of LINC00035 lated using Firefly luciferase activity to ratio Renilla lucif- in OC. Next, LINC00035 knockdown A2780 and SKOV3 erase activity. cells were analyzed by CCK-8 assays, transwell assays, flow 4 Evidence-Based Complementary and Alternative Medicine A2780 SKOV3 6 * 5 * * 5 5 3 3 2 2 1 1 0 0 0 (a) (b) SKOV3 A2780 2.0 2.5 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 0 h 24 h 48 h 72 h 0 h 24 h48 h72 h scramble siRNA scramble siRNA si-LINC00035 si-LINC00035 (c) A2780 SKOV3 400 * 300 * A2780 50 μm 50 μm Bcl-2 25 KD si-LINC00035 si-LINC00035 Bax 21 KD 50 μm 50 μm GADPH 36 KD scramble siRNA scramble siRNA (d) A2780 SKOV3 SKOV3 50 μm 150 Bcl-2 25 KD 50 μm si-LINC00035 si-LINC00035 Bax 21 KD GADPH 36 KD 50 μm 50 μm 0 0 scramble siRNA scramble siRNA (e) (f) A2780 SKOV3 2.0 2.0 1.5 1.5 2.0 1.0 0.8 1.5 * 1.5 1.5 1.0 1.0 0.6 1.0 1.0 1.0 0.4 0.5 0.5 0.5 0.5 0.5 0.2 0.0 0.0 0.0 0.0 0.0 0.0 (g) Figure 1: LINC00035 knockdown attenuates glycolysis and apoptosis in OC cells. (a) Expression of LINC00035 in OC tissues (n � 67), adjacent tumor-free tissues (n � 67), ovarian epithelial cells, and OC cell lines determined by RT-qPCR. (b) Validation of LINC00035 knockdown A2780 and SKOV3 cells. (c) Reduced A2780 and SKOV3 cells following LINC00035 knockdown. (d, e) Fewer A2780 and SKOV3 cells transferring from the apical chamber of the transwell system uncoated or coated with Matrigel into the basolateral chamber following LINC00035 knockdown. (f) Immunoblots and quantification of bcl-2 and bax in A2780 and SKOV3 cells following LINC00035 knockdown. (g) Decreases in glucose uptake, lactate production, and ATP production in A2780 and SKOV3 cells following LINC00035 knockdown. p< 0.05. Glucose uptake LINC00035 expression normalized to GADPH si-LINC00035 OC tissues scramble siRNA Migrating cells Adjacent tissues Lactate production LINC00035 expression si-LINC00035 si-LINC00035 normalized to GADPH si-LINC00035 scramble scramble OD value siRNA siRNA IOSE80 scramble siRNA Caov-3 ATP level CoC1 A2780 si-LINC00035 Migrating cells SKOV3 scramble Invasive cells siRNA Glucose uptake si-LINC00035 OD value si-LINC00035 scramble scramble siRNA LINC00035 expression siRNA normalized to GADPH si-LINC00035 scramble siRNA Apotosis rate (%) si-LINC00035 Apotosis rate (%) Lactate production scramble si-LINC00035 si-LINC00035 siRNA si-LINC00035 scramble scramble LINC00035 expression siRNA siRNA scramble normalized to GADPH siRNA ATP level si-LINC00035 si-LINC00035 scramble siRNA scramble siRNA scramble scramble siRNA siRNA si-LINC00035 si-LINC00035 Evidence-Based Complementary and Alternative Medicine 5 siRNA plus empty vector, or si-LINC00035 plus SLC16A3 cytometric analysis, glucose uptake, lactate release, and ATP production assays. LINC00035 knockdown leading to re- expression vector into A2780 and SKOV3 cells (Figure 3(c)). )e qRT-PCR and immunoblotting analysis both demon- duced viability (Figure 1(c)), migration (Figure 1(d)), and invasion (Figure 1(e)) in A2780 and SKOV3 cells were strated declined SLC16A3 mRNA and protein expressions in noted. In addition, flow cytometric analysis demonstrated A2780 and SKOV3 cells following LINC00035 knockdown. more apoptotic A2780 and SKOV3 cells upon LINC00035 As shown in Figures 4(a)–4(c), A2780 and SKOV3 cells knockdown (Figure 1(f)). Furthermore, immunoblotting exhibited reduced viability, migration and invasion fol- analysis found declined bcl-2 expression concomitant with lowing SLC16A3 knockdown. We also found more apo- elevated bax expression in A2780 and SKOV3 cells with ptotic A2780 and SKOV3 cells, a lower bcl-2 protein LINC00035 knockdown (Figure 1(f)). )e LINC00035 expression and a higher bax upon SLC16A3 knockdown knockdown A2780 and SKOV3 cells showed decreases in (Figure 4(d)). It was revealed that A2780 and SKOV3 cells with SLC16A3 knockdown presented decreases in glucose glucose uptake, lactate production and ATP production as compared with the scramble siRNA cells (Figure 1(g)). )ese uptake, lactate production, and ATP production as com- pared to the scramble siRNA cells (Figure 4(e)). In A2780 results thus suggested that LINC00035 knockdown atten- uated glycolysis and apoptosis in OC cells. and SKOV3 cells concurrently treated by si-LINC00035 and SLC16A3 expression vector, we found SLC16A3 over- expression negated the effects of LINC00035 knockdown 3.2. LINC00035 Increased the SLC16A3 Transcription by concerning no significant differences in A2780 and SKOV3 Recruiting CEBPB in OC Cells. Next, we retrieved the cell viability, migration, invasion, apoptosis, glucose uptake, LncMAP database (http://bio-bigdata.hrbmu.edu.cn/ lactate production, and ATP production between scramble LncMAP/) by entering LINC00035 and found LINC00035 siRNA plus empty vector and si-LINC00035 plus SLC16A3 regulates transcription factor CEBPB and its downstream expression vector. )ese results thus suggested that gene SLC16A3 (Figure 2(a)). )en, we conducted RIP assays LINC00035 promoted glycolysis and cell apoptosis through in A2780 and SKOV3 cells. LINC00035 enrichments were CEBPB-mediated transcriptional promotion of SLC16A3. observed in that the CEBPB immunoprecipitates in A2780 and SKOV3 cells and more LINC00035 enrichments were presented after transfection with LINC00035 expression 3.4. e LINC00035/CEBPB/SLC16A3 Pathway Affected the vector (Figure 2(b)). RNA pull-down followed by immu- Tumorigenesis of OC Cells In Vivo. Finally, the effect of noblotting showed CEBPB could be pulled down by biotin- LINC00035 on the tumorigenesis of OC cells in vivo was labeled LINC00035 probes (Figure 2(c)). Accordingly, we analyzed after transfected SKOV3 cells were xenografted mapped CEBPB and SLC16A3 into the starBase database into nude mice. )e data revealed that LINC00035 and found CEBPB and SLC16A3 were coexpressed in OC knockdown and SLC16A3 knockdown were associated with (r � 0.405; p< 0.001, Figure 2(d)). It was found elevated inhibition of xenotransplanted tumors in nude mice luciferase activity of the SLC16A3-wt in A2780 and SKOV3 (Figure 5(a)). Six weeks after implantation, the mice were cells with CEBPB overexpression and declined luciferase euthanized by exposure to prolonged inhalational anes- activity upon CEBPB knockdown (Figure 2(e)). )e ChIP thesia, with tumor bodies excised. )e data showed that assays demonstrated SLC16A3 in CEBPB immunoprecipi- LINC00035 knockdown and SLC16A3 knockdown could tates using anti-CEBPB rather than normal mouse IgG. increase the weight and volume of xenotransplanted tumors Furthermore, we found more SLC16A3 was detected in in nude mice (Figures 5(b) and 5(c)). No significant dif- CEBPB immunoprecipitates from A2780 and SKOV3 cells ference between scramble siRNA plus empty vector and si- upon LINC00035 expression vector transfection and less LINC00035 plus SLC16A3 expression vector, suggesting that detection of SLC16A3 in CEBPB immunoprecipitates upon SLC16A3 overexpression negated the effects of LINC00035 si-LINC00035 transfection (Figure 2(f)). )ese results thus knockdown on the tumorigenesis of SKOV3 cells in vivo. suggested that LINC00035 could recruit CEBPB into the SLC16A3 promoter region, thus increasing the SLC16A3 4. Discussion transcription. Recent evidence indicates that high propensity of OC cells to 3.3. e LINC00035/CEBPB/SLC16A3 Pathway Affected consume and metabolize glucose by glycolysis can fuel Glycolysis and Apoptosis in OC Cells. Next, the investigation uncontrolled growth of abnormal cells, finally leading to focused on the role of SLC16A3 in OC cells. At the be- disease progression and chemoresistance [23]. In the case of ginning, we mapped SLC16A3 into the GEPIA database and OC, dysregulation of lncRNAs has been reported to engage found SLC16A3 was highly expressed in OC tissue in tumor growth and metastasis [24]. )e obtained data (Figure 3(a)). Results of qRT-PCR and immunoblotting revealed LINC00035 knockdown led to reduced viability analysis showed SLC16A3 mRNA and protein expressions migration and invasion of OC cells concurrent with an were upregulated in OC tissues compared to adjacent tu- increased apoptosis, leading to decreases in glucose uptake, mor-free tissue, in four human OC cell lines compared with lactate release, and ATP production. )e OC mouse model normal ovarian epithelial cells (Figure 3(b)). Subsequently, confirmed anti-tumor effect of LINC00035 knockdown on we performed rescue experiments by successfully delivering tumor growth as well. More specifically, another investi- si-LINC00035 plus empty vector, si-SLC16A3, scramble gation in this study uncovered the detailed mechanism 6 Evidence-Based Complementary and Alternative Medicine (a) A2780 SKOV3 1.0 1.5 1.0 1.5 0.8 0.8 1.0 1.0 0.6 0.6 0.4 0.4 0.5 0.5 0.2 0.2 0.0 0.0 0.0 0.0 scramble siRNA scramble siRNA si-LINC00035 si-LINC00035 (b) A2780 CEBPB vs. SLC16A3, 379 samples (OV) SKOV3 Data Source: starBase v3.0 project * 5 4 3 -1 CEBPB, Expression level: log2 (FPKM+0.01) Regression (y = 0.6665x - 1.8482) r = 0.405, p-value = 2.17e-16 (c) (d) A2780 2.5 * 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 SLC16A3-wt SLC16A3-mut SLC16A3-wt SLC16A3-mut Empty vector scramble siRNA CEBPB expression vector si-CEBPB SKOV3 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 SLC16A3-wt SLC16A3-mut SLC16A3-wt SLC16A3-mut Empty vector scramble siRNA CEBPB expression vector si-CEBPB (e) Figure 2: Continued. Bio-NC Bio-NC LINC00035 enrichment Luciferase activity Luciferase activity Input anti-CEBPB IgG Bio-LINC00035-mut LINC00035 enrichment CEBPB enrichment anti-CEBPB IgG Luciferase activity Luciferase activity Bio-LINC00035-mut LINC00035 enrichment SLC16A3, Expression level: log2 [FPKM+0.01)] Input anti-CEBPB IgG LINC00035 enrichment anti-CEBPB IgG Bio-LINC00035-wt Bio-LINC00035-wt Evidence-Based Complementary and Alternative Medicine 7 A2780 IgG anti-CEBPB SKOV3 IgG anti-CEBPB (f) Figure 2: LINC00035 increases the SLC16A3 transcription by recruiting CEBPB in OC cells. (a) LINC00035 regulation of SLC16A3 via transcription factor CEBPB is predicted in the LncMAP database. (b) RIP-qPCR examined the LINC00035 in CEBPB immunoprecipitates from A2780 and SKOV3 cells with or without LINC00035 overexpression. (c) RNA pull-down followed by immunoblotting analysis examined the CEBPB pulled down by biotin-labeled LINC00035 probes. (d) Coexpression between CEBPB and SLC16A3 in the starBase database. (e) Dual-luciferase reporter gene assays to validate SLC16A3 binding with CEBPB in A2780 and SKOV3 cells. (f) ChIP assays followed by immunoblotting analysis examined the SLC16A3 in CEBPB immunoprecipitates from A2780 and SKOV3 cells with or without LINC00035 knockdown p< 0.05. * * 2.5 2.0 38-42 KD 1.5 36 KD 1.0 2 2 0.5 0 0.0 OC (num (T)=426; num (N)=88) 2.0 1.5 1.0 38-42 KD 0.5 36 KD SKOV3 Caov-3 CoC1 A2780 IOSE80 0.0 SKOV3 Caov-3 CoC1 A2780 IOSE80 SKOV3 Caov-3 CoC1 A2780 IOSE80 (a) (b) Figure 3: Continued. SLC16A3 mRNA expression normalized to GADPH Expression-log2 (TPM+1) SLC16A3 mRNA expression normalized to GADPH SLC16A3 enrichment SLC16A3 enrichment Adjacent tissues OC tissues Adjacent tissues OC tissues SLC16A3 protein expression normalized to GADPH SLC16A3 protein expression normalized to GADPH Adjacent tissues OC tissues 8 Evidence-Based Complementary and Alternative Medicine A2780 SKOV3 A2780 8 8 38-42 KD 36 KD SKOV3 38-42 KD 2 2 36 KD 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 (c) Figure 3: LINC00035 increases SLC16A3 expression in OC cells. (a) SLC16A3 was highly expressed in OC tissue in the GEPIA database. (b) SLC16A3 expressions in OC tissues and cells, measured by qRT-PCR and immunoblotting analysis. (c) si-LINC00035 plus empty vector, si-SLC16A3, scramble siRNA plus empty vector, or si-LINC00035 plus SLC16A3 expression vector were delivered into A2780 and SKOV3 cells and qRT-PCR, and immunoblotting analysis were used to determine SLC16A3 mRNA and protein expressions p< 0.05. A2780 SKOV3 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h si-SLC16A3 si-SLC16A3 si-LINC00035+oe-SLC16A3 si-LINC00035+oe-SLC16A3 scramble siRNA+empty vector scramble siRNA+empty vector (a) A2780 SKOV3 50 μm 50 μm 50 μm 50 μm 50 μm 50 μm 0 0 si-SLC16A3 si-LINC00035+ scramble siRNA+ si-LINC00035+ scramble siRNA+ si-SLC16A3 oe-SLC16A3 empty vector oe-SLC16A3 empty vector (b) Figure 4: Continued. SLC16A3 mRNA expression normalized to GADPH si-LINC00035 +empty vector si-SLC16A3 SLC16A3 protein expression normalized to GADPH scramble siRNA +empty vector OD value si-LINC00035 si-LINC00035 +oe-SLC16A3 +empty vector Migrating cells si-SLC16A3 SLC16A3 mRNA expression normalized to GADPH si-SLC16A3 scramble siRNA +empty vector si-LINC00035+ oe-SLC16A3 si-LINC00035 si-LINC00035 scramble siRNA+ +empty vector empty vector +oe-SLC16A3 si-SLC16A3 OD value SLC16A3 protein expression normalized to GADPH scramble siRNA +empty vector si-LINC00035 si-LINC00035 +oe-SLC16A3 +empty vector si-SLC16A3 scramble siRNA si-LINC00035 +empty vector +empty vector si-LINC00035 si-SLC16A3 +oe-SLC16A3 scramble siRNA +empty vector Migrating cells si-LINC00035 +oe-SLC16A3 si-SLC16A3 si-LINC00035+ oe-SLC16A3 scramble siRNA+ empty vector Evidence-Based Complementary and Alternative Medicine 9 300 200 SKOV3 A2780 50 μm 50 μm 50 μm 50 μm 50 μm 50 μm 0 0 si-SLC16A3 si-LINC00035+ scramble siRNA+ si-SLC16A3 si-LINC00035+ scramble siRNA+ oe-SLC16A3 empty vector oe-SLC16A3 empty vector (c) A2780 SKOV3 Bcl-2 20 25 KD Bcl-2 25 KD Bax 21 KD Bax 21 KD GADPH 36 KD 36 KD 10 GADPH (d) SKOV3 A2780 2.0 2.0 1.5 2.0 1.5 2.0 * * * 1.5 1.5 * 1.5 1.5 1.0 1.0 1.0 1.0 1.0 1.0 0.5 0.5 0.5 0.5 0.5 0.5 0.0 0.0 0.0 0.0 0.0 0.0 (e) Figure 4: )e LINC00035/CEBPB/SLC16A3 pathway affects glycolysis and apoptosis in OC cells. (a) CCK-8 assays examined A2780 and SKOV3 cells upon SLC16A3 knockdown or LINC00035 knockdown and SLC16A3 overexpression concurrently. (b, c) Representative view and statistics of A2780 and SKOV3 cells transferring from the apical chamber of the transwell system uncoated or coated with Matrigel into the basolateral chamber upon SLC16A3 knockdown or LINC00035 knockdown and SLC16A3 overexpression concurrently. (d) Im- munoblots and quantification of bcl-2 and bax in A2780 and SKOV3 cells upon SLC16A3 knockdown or LINC00035 knockdown and SLC16A3 overexpression concurrently. (e) Glucose uptake, lactate production, and ATP production measurements in A2780 and SKOV3 cells upon SLC16A3 knockdown or LINC00035 knockdown and SLC16A3 overexpression concurrently p< 0.05. knockdown could notably repress the viability, migration, behind the action of LINC00035 in OC, namely, a regulatory network consisting of LINC00035, the transcription factor and invasion of OC, induce the apoptosis, and reduce CEBPB, and downstream gene SLC16A3. glycolysis. Accordingly, in vivo experiments further vali- Increasing studies have begun to focus on the effects of dated that tumor growth inhibition and tumor size reduc- lncRNAs, such as LINC00494, GClnc1, and SNHG17 in OC tion were related to LINC00035 knockdown. Taken together, by gene regulation from aspect of transcription regulation the oncogenic functions of LINC00035 in OC was uncovered [25–27]. Aberrant expression of LINC00035 has been re- in our study. ported to regulate the development and progression of LncRNA regulation of transcription factor to modulate various tumors, including OC, and its high expression may the transcription of downstream genes is a novel mechanism contribute to tumor growth and invasion. For example, in tumor growth and metastasis. CEBPB is one of four Zhang et al. demonstrated LINC00035 promoter mediated known members of the C/EBP family of basic region-leucine zipper transcription factors (CEBPA, CEBPB, CEBPD, and by EZH2 modulates OC progression by targeting miR-133a- 3p [28]. In the study of Zeng et al., they found LINC00035 CEBPE) [31]. )e transcriptional regulation of CEBPB on governed by the EGFR pathway promotes the tumorigenesis downstream genes is regulated by lncRNA in human dis- of OC via epigenetically suppressing TIMP2 [29]. A previous eases. For example, Wu et al. demonstrated LINC00160 study conducted by Wu and his team found LINC00035 mediated acquired resistance of chemotherapeutic regimes regulation of RhoC conferred tumor-promoting effects in of paclitaxel and doxorubicin drugs in breast cancer cells by OC [30]. Further functional studies verified that LINC00035 regulating TFF3 via transcription factor C/EBPβ [32]. Gao Apotosis rate (%) Glucose uptake si-SLC16A3 si-SLC16A3 si-LINC00035+ oe-SLC16A3 si-LINC00035+ oe-SLC16A3 scramble siRNA+ empty vector scramble siRNA+ empty vector Lactate production si-SLC16A3 si-LINC00035+ oe-SLC16A3 scramble siRNA+ si-SLC16A3 empty vector Invasive cells ATP level si-LINC00035+ oe-SLC16A3 si-SLC16A3 si-SLC16A3 scramble siRNA+ si-LINC00035+ empty vector oe-SLC16A3 si-LINC00035+ oe-SLC16A3 scramble siRNA+ scramble siRNA+ empty vector empty vector Glucose uptake Apotosis rate (%) si-SLC16A3 si-SLC16A3 si-LINC00035+ oe-SLC16A3 scramble siRNA+ si-LINC00035+ empty vector oe-SLC16A3 Lactate production scramble siRNA+ empty vector si-SLC16A3 si-LINC00035+ oe-SLC16A3 scramble siRNA+ empty vector Invasive cells ATP level si-SLC16A3 si-LINC00035+ oe-SLC16A3 si-SLC16A3 si-SLC16A3 si-LINC00035+ scramble siRNA+ si-LINC00035+ oe-SLC16A3 empty vector oe-SLC16A3 scramble siRNA+ scramble siRNA+ empty vector empty vector 10 Evidence-Based Complementary and Alternative Medicine 2.0 800 1.5 1.0 0.5 1 25 3 4 6 0.0 Time (week) scramble si-LINC00035 siRNA+ +empty vector empty vector si-LINC00035 si-SLC16A3 +oe-SLC16A3 (a) (b) (c) Figure 5: )e LINC00035/CEBPB/SLC16A3 pathway affects the tumorigenesis of OC cells in vivo. (a) Subcutaneously xenografted tumors. (b) Tumor weight. (c) Tumor growth p< 0.05. LINC00035 LINC00035 Glycolysis CEBPB Apoptosis CEBPB Promoter SLC16A3 Figure 6: A schematic diagram showing how the LINC00035/CEBPB/SLC16A3 pathway regulates the development of OC. LINC00035 was triggered in OC and could recruit the transcription factor CEBPB into the SLC16A3 promoter region, thus increasing the SLC16A3 transcription. Abundance of SLC16A3 inhibited OC cell viability, migration, invasion, and glycolysis and induced the apoptosis. et al. found that silencing of lncRNA LEF1-AS1 reduced knockdown exerted similar effects on SKOV3 and EZH2 expression to repress hepatocellular carcinoma pro- A2780 cells as LINC00035 knockdown, especially attenu- gression by disruption of CEBPB interaction with CDCA7 ating glucose uptake, lactate production, and ATP pro- [33]. Only a previous study investigates the role of CEBPB in duction. In a gliomas report, Reuss et al. indicated that the function SLC16A3 is lactate and proton exporter. )e the case of OC that CEBPB expression was not significantly changed in OC tissues and cells, which was partially con- expression of SLC16A3 was negatively correlated with sistent with our results that CEBPB was just responsible for extracellular pH and proportional to extracellular lactate the regulation of LINC00035 on the SLC16A3 expression in concentrations [35]. Yu and his team performed integrated OC. bioinformatics that comprehensively analyzed the SLC16A SLC16A3 is greatly involved in tumor pH modulation gene family in pancreatic cancer, showing SLC16A as a lactate transporter. In a mouse tumor model, lactate exhibited biomarker potential for prognosis of pancreatic modulation and anti-tumor activity were evaluated cancer [36]. Concurring with our data concerning through the direct target interaction of the optimized SLC16A3 expression in OC, SLC16A3 was remarkably compound 18n with the cytosolic domain of SLC16A3 increased in liver cancer [37]. suggesting pharmacokinetic inhibition of SLC16A3 by 18n In summary, the study mainly highlights a new mechanism by regulating the transcription factor CEBPB is a useful outcome measure for anti-tumor effects on tumor biology [34]. Our following investigations demon- and the downstream genes SLC16A3 to confirm the strated SLC16A3 was upregulated in OC tissues. SLC16A3 function of LINC00035 in OC. LINC00035 acts as an si-LINC00035 +empty vector si-SLC16A3 scramble siRNA +empty vector si-LINC00035 +oe-SLC16A3 Tumor weight (g) si-LINC00035 +empty vector si-SLC16A3 scramble siRNA +empty vector si-LINC00035 +oe-SLC16A3 Cytoplasm Evidence-Based Complementary and Alternative Medicine 11 endometrial carcinoma by targeting cyclin D1,” Journal of oncogenic lncRNA in OC tumorigenesis. )e finding Cellular and Molecular Medicine, vol. 22, pp. 3955–3964, 2018. revealed in this study may confer valuable view to develop [13] X. Zhuang, H. Tong, Y. Ding et al., “Long noncoding RNA new therapeutic targets for OC (Figure 6). )e interaction ABHD11-AS1 functions as a competing endogenous RNA to between LINC00035, CEBPB, and SLC16A3 needs to be regulate papillary thyroid cancer progression by miR-199a- further studied to deeply understand and clarify the specific 5p/SLC1A5 axis,” Cell Death & Disease, vol. 10, no. 8, p. 620, mechanisms related to antiglycolysis action of SLC16A3 in OC, in order to verify its application value from the per- [14] L. Xue, J. Li, Y. Lin et al., “m(6) A transferase METTL3-induced spective of clinical practice. lncRNA ABHD11-AS1 promotes the Warburg effect of non- small-cell lung cancer,” Journal of Cellular Physiology, vol. 236, Data Availability no. 4, pp. 2649–2658, 2021. [15] S. Mehrpour Layeghi, M. Arabpour, A. 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LINC00035 Transcriptional Regulation of SLC16A3 via CEBPB Affects Glycolysis and Cell Apoptosis in Ovarian Cancer

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Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2021, Article ID 5802082, 12 pages https://doi.org/10.1155/2021/5802082 Research Article LINC00035 Transcriptional Regulation of SLC16A3 via CEBPB Affects Glycolysis and Cell Apoptosis in Ovarian Cancer Shuyan Yang, Jing Wang, Rongjie Cheng, Bo Pang, and Pengcheng Sun Department of Obstetrics and Gynecology, e Fourth Affiliated Hospital of Harbin Medical University, No. 37, Yiyuan Street, Nangang District, Harbin 150001, Heilongjiang, China Correspondence should be addressed to Pengcheng Sun; [email protected] Received 8 September 2021; Accepted 25 September 2021; Published 11 October 2021 Academic Editor: Songwen Tan Copyright © 2021 Shuyan Yang et al. )is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. Ovarian cancer (OC) represents the most lethal gynecologic malignancy globally. Over the decades, lncRNAs have been considered as study focuses due to their genome-wide expression through multiple mechanisms in which regulation of target gene transcription through interaction with transcription factors or epigenetic proteins is proven. In the present work, we focus on the functional role of LINC00035 in OC and its regulation mechanism on gene expression. Methods. We collected OC tissues and adjacent tumor-free tissues surgically resected from 67 OC patients. Cultured human OC cell lines SKOV3 and A2780 were assayed for their viability, migration, invasion, apoptosis in vitro using CCK-8 assays, transwell assays, and flow cytometric analysis. OC cell tumorigenesis in vivo was evaluated by mouse xenograft experiments. Glycolysis was evaluated by glucose uptake, lactate release, and ATP production assays. Luciferase activity assay, RNA immunoprecipitation (RIP), and RNA pull-down were performed to confirm the interactions among LINC00035, CEBPB, and SLC16A3. Results. LINC00035 was upregulated in OC tissues. LINC00035 knockdown was shown to repress SKOV3 and A2780 cell viability, migration, invasion, induce their ap- optosis, and reduce glucose uptake, lactate release, and ATP production. LINC00035 could recruit CEBPB into the SLC16A3 promoter region, thus increasing the SLC16A3 transcription. SLC16A3 was upregulated in OC tissues. SLC16A3 knockdown exerted similar effects on SKOV3 and A2780 cells as LINC00035 knockdown. Rescue experiments found SLC16A3 overexpression resisting to LINC00035 knockdown on SKOV3 and A2780 cell viability, migration, invasion, apoptosis, glucose uptake, lactate release, and ATP production. Results also showed LINC00035 knockdown could inhibit OC cell tumorigenesis in vivo. Con- clusion. )e study reveals that LINC00035 promotes OC progression by regulating glycolysis and cell apoptosis through CEBPB- mediated transcriptional promotion of SLC16A3. diagnosed at stage III (51%) or IV (29%), with a high rate of 1. Introduction relapse and metastasis into the abdominal cavity [5]. A large- Ovarian cancer (OC) is the third most common gynecologic scale study of OC screening has shown that only using ultrasound or CA125 test for screening every year and, if malignancy and one of the leading causes of cancer death among women around the world [1]. OC is considered to be necessary, follow-up with ultrasound fails to decrease a heterogeneous malignant tumor with unique genomic mortality in the general population. Despite more early characteristics. Histologically, it can be divided into five diagnosis by screening, the increase was not sufficient to different subtypes at least [2]. )e report of Global Cancer significantly improve survival [6, 7]. Given that various Statistics 2020 estimated that the incidence and mortality of factors involving frequent recurrence, late stage during OC accounts for 1.6% and 2.1%, respectively [3]. Due to less initial diagnosis, the presence of resistance to drugs, and early clinical features and the presence of metastatic and adverse reaction to chemotherapeutic regimens occur in invasive cancer cells in most patients at the time of diagnosis, most of the patients, it has become imperative to extensively OC remains one of the most challenging malignancies to elucidate the underlying mechanisms behind OC develop- manage [4]. Most women with serous carcinomas are ment. Long noncoding RNAs (lncRNAs), a subgroup of 2 Evidence-Based Complementary and Alternative Medicine RNAs (>200 nucleotides in length), engage in OC initiation, lipofectamine 2000 reagent transfection (Invitrogen, USA). drug resistance, and progression [8, 9]. LINC00035, located Cell transfection was performed lasting for 24–48 h. on the human chromosome 7q11.23, with another known name ABHD11-AS1, has been widely confirmed as an on- 2.3. Quantitative Real-Time Polymerase Chain Reaction cogene, showing different power of gene control, including (qRT-PCR). After total RNA extractions from tissue and epigenetic, transcription, or posttranscriptional control in cells by using Trizol reagents (Invitrogen), cDNA was many types of human cancers, such as gastric cancer [10], generated according to the manuals of the PrimeScript RT pancreatic cancer [11], endometrial carcinoma [12], thyroid reagent Kit (Takara, Japan). )e SYBR Premix ExTaqTM II cancer [13], lung cancer [14], and breast cancer [15]. ® (RR820A, Takara) kit and the ABI PRISM 7300 System LncRNAs interact with a variety of transcription factors (ABI, USA) were applied to complete the qRT-PCR. Data to modulate gene expression and then participate in cell were relative to the expression of a reference gene GADPH growth and apoptosis [16]. )e transcription factor CCAAT/ −ΔΔCt and analyzed using the 2 method. )e primers of enhancer-binding protein beta (CEBPB) is required for LINC00035, SLC16A3, and GADPH were synthesized by maintenance of the tumor-initiating capacity and invasion RiboBio (Guangzhou, China). )e primer sequences of ability in several tumors, including hepatocellular carcinoma LINC00035 were 5′-TCCAGACAAGACTTGGTCGC-3’ [17], glioblastoma [18], and breast cancer [19]. SLC16A3 is a (forward) and 5′-CAGCTGGTTGTGTGGCTTTC-3’ (re- member of the SLC16 gene family that has fourteen verse), of SLC16A3 were 5′-CTTCCCGTCAGACGCCC-3’ members among which SLC16A1, SLC16A3, SLC16A7, and (forward) and 5′-GTGTTCAGTACCAGCCCTGT-3’ (re- SLC16A8 encode proton-coupled monocarboxylate trans- verse), and of GADPH were 5′-ATGGA- porters MCT1, MCT4, MCT2, and MCT3, respectively [20]. GAAGGCTGGGGCTC-3’ (forward) and 5′- SLC16A3 has the ability to catalyze the proton-linked AAGTTGTCATGGATGACCTTG-3’ (reverse). transport of monocarboxylates such as l-lactate, pyruvate, and ketone bodies across the plasma membrane, which was recognized as glycolysis-related gene signature in human 2.4. CCK-8 Assays. A2780 and SKOV3 cells were incubated cancers [21, 22]. )e LncMAP database (http://bio-bigdata. with 3 ×10 cells per well in the 96-well plate. Upon incu- hrbmu.edu.cn/LncMAP/) shows LINC00035 regulates bation at different time points, 0, 1, 2, and 3 d, each well transcription factor CEBPB and its downstream gene reacted with the addition of CCK-8 reaction solution SLC16A3 in OC. Based on the aforementioned data, we (Dojindo, Kumamoto, Japan). Another 2h incubation therefore speculate whether there is a promising correlation continued. )e optical value was obtained at 450 nm. among LINC00035, CEBPB, and SLC16A3 in OC. 2.5. Transwell Migration and Invasion Assays. A2780 and 2. Materials and Methods SKOV3 cells (6 ×10 cells for each) were suspended in 200 μL serum-free medium and supplemented into the 2.1. Human Tissue Specimen. We collected OC tissues and apical chamber of the transwell system (Corning-Costar, adjacent tumor-free tissues surgically resected from 67 OC Cambridge, MA) uncoated (for migration) or coated (for patients who were admitted into our hospital in the time invasion) with Matrigel (BD Biosciences, San Jose, CA). duration from January 2020 to December 2020. No patients Additional 700 μL DMEM with 10% FBS was added into the had received radiotherapy, chemotherapy, or immuno- basolateral chamber. Following 24-hour incubation, A2780 therapy before surgery. Signed informed consents were and SKOV3 cells were transferred from the apical chamber obtained from all participants. )e study was approved by into the basolateral one, followed by addition of methanol the Institutional Review Board of our hospital. Experiments and 0.1% crystal violet. )e number of migrating or invasive involving human beings were performed in strict accordance cells was photographed under an inverted microscope and with the Declaration of Helsinki. also calculated flow cytometric analysis. A2780 and SKOV3 cells were suspended in the DMEM to adjust the density into 1 × 10 cells/mL. )e cell sus- 2.2. Cell Culture and Transient Transfection. Under the pensions reacted with the addition of 5 μl Annexin V and 5 μl conditions of humidity of 5% CO and temperature of 37 C, propidium iodide (PI). Apoptotic A2780 and SKOV3 cells IOSE80 used as normal ovarian epithelial cells and Caov-3, were analyzed using a flow cytometer (FACScan ) equipped A2780, SKOV3, and CoC1 used as human OC cell lines with Cell Quest software (BD Biosciences). (ATCC, USA) were grown in the RPMI1640 with 15% fetal bovine serum (FBS) (Gibco, USA). To construct OC cells with LINC00035 overexpression and knockdown, CEBPB 2.6. Immunoblotting Analysis. After 10% SDS-PAGE sepa- overexpression and knockdown, SLC16A3 overexpression ration, extracted total protein was wet-transferred to the and knockdown, siRNA targeting LINC00035, the expres- PVDF membrane (Sigma-Aldrich, USA). Immunoblots sion vector containing the full-fragments of LINC00035, were generated after addition of primary antibodies: anti- siRNA targeting CEBPB, the expression vector containing bcl-2 antibody (ab182858, Abcam, UK), anti-bax antibody the full-fragments of CEBPB, siRNA targeting SLC16A3 and (ab182734, Abcam), anti-CEBPB antibody (ab32358, the expression vector containing the full-fragments of Abcam), anti-SLC16A3 antibody (ab74109, Abcam), and SLC16A3 were delivered into selected OC cells by mean of anti-GAPDH antibody (ab171091, Abcam). Horseradish Evidence-Based Complementary and Alternative Medicine 3 2.11. Chromatin Immunoprecipitation (ChIP). To generate peroxidase-labeled IgG and enhanced chemiluminescence solution were added for visualization. With GAPDH as the DNA-protein cross-links, 10% formaldehyde was added to fix A2780 and SKOV3 cells for 10 min. Chromatin fragments loading control, immunoblots were photographed by the use of Bio-Rad image analysis system (BIO-RAD, Hercules, CA) was generated by sonication of the DNA-protein cross-links. and their densimetric analyses were performed using )ree parts with equal volume were divided from the Quantity One v4.6.2 software. chromatin fragments. Anti-CEBPB antibody (ab32358, Abcam) or normal mouse IgG was applied to generate immunoprecipitates. Protein agarose/sepharose beads were 2.7. Glucose Uptake, Lactate Release, and ATP Production used to precipitate the DNA-protein complexes. Cross-link Assays. A2780 and SKOV3 cells were seeded with 50,000 and elution were finished and qRT-PCR analysis was con- cells per well in 12-well plates and then underwent serum ducted to the DNA samples. Mouse xenograft experiments. starvation for 24 h. Afterwards, cells were stimulated with Animal studies consisted of 12 male and 12 female 100 nmol/L insulin for 30 min. Cells were treated with 10 μM BALB/c mice (Nanjing, Junke Biological Co., Ltd., Jiangsu, 2-NBDG for 1 h at 37 C. )e uptake of 2-NBDG was ana- China), with age of 4–6 weeks and weight of 18 to 22 g. lyzed with an FC500 Flow Cytometer (Beckman Coulter, SKOV3 cells transfected with si-LINC00035 plus empty Brea, CA, USA) to reflect glucose uptake. Lactate release and vector, si-SLC16A3, scramble siRNA plus empty vector, or ATP production were determined by Lactate Colorimetric/ si-LINC00035 plus SLC16A3 expression vector were ad- Fluorometric Assay Kit (BioVision, USA) and ATP Deter- justed into suspensions with 1 × 10 cells/mL, and 0.5 ml of mination Kit ()ermo Fisher Scientific, USA), respectively. them were subcutaneously implanted into the scapular re- gion of each mouse. )e growth of xenografted tumors in mice was monitored every 5 days. When the tumor was 2.8. Immunoprecipitation (RIP) Assays. A commercial kit visible by neck eyes, the length and width of the tumor were (Millipore, USA) was used to examine the combination of measured. After 6 weeks of implantation, the mice were LINC00035 and CEBPB following the manufacturer’s euthanized by exposure to prolonged inhalational anes- manual. A2780 and SKOV3 cell lysis were centrifuged thesia. All animal experiments were conducted under the (14000 × g, 10 min) and cell extracts for three parts with approval of Ethics Committee of our hospital and in ac- 100 μL each were collected. Immunoprecipitation was per- cordance with the Guide for the Care and Use of Laboratory formed in two cell extracts using protein A/G sepharose Animal. Tremendous efforts were done to minimize pain the beads equipped with either anti-CEBPB antibody (ab32358, included animals suffered from. Abcam) or normal mouse IgG. Immunoprecipitated RNA and total RNA from the input control were extracted for qRT-PCR analysis. 2.12. Data Analysis. SPSS 20.0 software (IBM Corp., Armonk, NY, USA) was used for statistical comparisons. A manner of mean± standard deviation was used to display all 2.9. RNA Pull-Down Assays. A2780 and SKOV3 cells were data. Paired t-test was used as a statistical tool to compare transfected with either biotin-labeled wild-type LINC00035 data between OC tissues and nontumor tissues. One-way or mutated LINC00035 probes for 48 h. Subsequently, cell analysis of variance (ANOVA) was used for multiple-group lysis was centrifuged (14000 × g, 10 min). Cell extracts were comparison, and repeated measurement ANOVA was used immunoprecipitated with M-280 streptavidin beads (S3762, upon different time points. A difference level less than 0.05 Sigma-Aldrich) precoated with RNase-free bovine serum was indicative of statistical significance. albumin (BSA) and yeast tRNA (TRNABAK-RO, Sigma- Aldrich). Immunoprecipitated protein and total protein 3. Results from the input control were extracted. Following 10% SDS- PAGE separation and membrane transfer, immunoblotting 3.1. LINC00035 Knockdown Attenuated Glycolysis and analysis of CEBPB was performed. Apoptosis in OC Cells. )e qRT-PCR demonstrated a re- markably regulated LINC00035 in OC tissues compared to that of adjacent tumor-free tissues (Figure 1(a)). Subse- 2.10. Promoter Luciferase Activity. )e oligonucleotides on quently, qRT-PCR was carried out to determine the ex- the 3′UTR of SLC16A3 mRNA and mutated ones were pression of LINC00035 in different OC cell lines. Similarly, inserted into commercially available luciferase reporter we found that LINC00035 in Caov-3, A2780, SKOV3, and vectors (pmirGLO, Promega, USA), named SLC16A3-wt CoC1 human OC cell lines was increased compared with and SLC16A3-mut. Treatment was done on A2780 and normal ovarian epithelial cells IOSE80, especially highly SKOV3 cells with SLC16A3-wt or SLC16A3-mut in the expressed in A2780 and SKOV3 cells (Figure 1(a)). Con- presence of CEBPB expression vector or si-CEBPB. Sub- sidering downregulated LINC00035 in OC tissues and cells, sequent to 48 h of transfection, the luciferase activity in the LINC00035 knockdown A2780 and SKOV3 cells, which cell lysis was determined by the dual-luciferase reporter gene were confirmed by the qRT-PCR (Figure 1(b)), were suc- assay (Promega). )e relative luciferase activity was calcu- cessfully constructed to present the function of LINC00035 lated using Firefly luciferase activity to ratio Renilla lucif- in OC. Next, LINC00035 knockdown A2780 and SKOV3 erase activity. cells were analyzed by CCK-8 assays, transwell assays, flow 4 Evidence-Based Complementary and Alternative Medicine A2780 SKOV3 6 * 5 * * 5 5 3 3 2 2 1 1 0 0 0 (a) (b) SKOV3 A2780 2.0 2.5 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 0 h 24 h 48 h 72 h 0 h 24 h48 h72 h scramble siRNA scramble siRNA si-LINC00035 si-LINC00035 (c) A2780 SKOV3 400 * 300 * A2780 50 μm 50 μm Bcl-2 25 KD si-LINC00035 si-LINC00035 Bax 21 KD 50 μm 50 μm GADPH 36 KD scramble siRNA scramble siRNA (d) A2780 SKOV3 SKOV3 50 μm 150 Bcl-2 25 KD 50 μm si-LINC00035 si-LINC00035 Bax 21 KD GADPH 36 KD 50 μm 50 μm 0 0 scramble siRNA scramble siRNA (e) (f) A2780 SKOV3 2.0 2.0 1.5 1.5 2.0 1.0 0.8 1.5 * 1.5 1.5 1.0 1.0 0.6 1.0 1.0 1.0 0.4 0.5 0.5 0.5 0.5 0.5 0.2 0.0 0.0 0.0 0.0 0.0 0.0 (g) Figure 1: LINC00035 knockdown attenuates glycolysis and apoptosis in OC cells. (a) Expression of LINC00035 in OC tissues (n � 67), adjacent tumor-free tissues (n � 67), ovarian epithelial cells, and OC cell lines determined by RT-qPCR. (b) Validation of LINC00035 knockdown A2780 and SKOV3 cells. (c) Reduced A2780 and SKOV3 cells following LINC00035 knockdown. (d, e) Fewer A2780 and SKOV3 cells transferring from the apical chamber of the transwell system uncoated or coated with Matrigel into the basolateral chamber following LINC00035 knockdown. (f) Immunoblots and quantification of bcl-2 and bax in A2780 and SKOV3 cells following LINC00035 knockdown. (g) Decreases in glucose uptake, lactate production, and ATP production in A2780 and SKOV3 cells following LINC00035 knockdown. p< 0.05. Glucose uptake LINC00035 expression normalized to GADPH si-LINC00035 OC tissues scramble siRNA Migrating cells Adjacent tissues Lactate production LINC00035 expression si-LINC00035 si-LINC00035 normalized to GADPH si-LINC00035 scramble scramble OD value siRNA siRNA IOSE80 scramble siRNA Caov-3 ATP level CoC1 A2780 si-LINC00035 Migrating cells SKOV3 scramble Invasive cells siRNA Glucose uptake si-LINC00035 OD value si-LINC00035 scramble scramble siRNA LINC00035 expression siRNA normalized to GADPH si-LINC00035 scramble siRNA Apotosis rate (%) si-LINC00035 Apotosis rate (%) Lactate production scramble si-LINC00035 si-LINC00035 siRNA si-LINC00035 scramble scramble LINC00035 expression siRNA siRNA scramble normalized to GADPH siRNA ATP level si-LINC00035 si-LINC00035 scramble siRNA scramble siRNA scramble scramble siRNA siRNA si-LINC00035 si-LINC00035 Evidence-Based Complementary and Alternative Medicine 5 siRNA plus empty vector, or si-LINC00035 plus SLC16A3 cytometric analysis, glucose uptake, lactate release, and ATP production assays. LINC00035 knockdown leading to re- expression vector into A2780 and SKOV3 cells (Figure 3(c)). )e qRT-PCR and immunoblotting analysis both demon- duced viability (Figure 1(c)), migration (Figure 1(d)), and invasion (Figure 1(e)) in A2780 and SKOV3 cells were strated declined SLC16A3 mRNA and protein expressions in noted. In addition, flow cytometric analysis demonstrated A2780 and SKOV3 cells following LINC00035 knockdown. more apoptotic A2780 and SKOV3 cells upon LINC00035 As shown in Figures 4(a)–4(c), A2780 and SKOV3 cells knockdown (Figure 1(f)). Furthermore, immunoblotting exhibited reduced viability, migration and invasion fol- analysis found declined bcl-2 expression concomitant with lowing SLC16A3 knockdown. We also found more apo- elevated bax expression in A2780 and SKOV3 cells with ptotic A2780 and SKOV3 cells, a lower bcl-2 protein LINC00035 knockdown (Figure 1(f)). )e LINC00035 expression and a higher bax upon SLC16A3 knockdown knockdown A2780 and SKOV3 cells showed decreases in (Figure 4(d)). It was revealed that A2780 and SKOV3 cells with SLC16A3 knockdown presented decreases in glucose glucose uptake, lactate production and ATP production as compared with the scramble siRNA cells (Figure 1(g)). )ese uptake, lactate production, and ATP production as com- pared to the scramble siRNA cells (Figure 4(e)). In A2780 results thus suggested that LINC00035 knockdown atten- uated glycolysis and apoptosis in OC cells. and SKOV3 cells concurrently treated by si-LINC00035 and SLC16A3 expression vector, we found SLC16A3 over- expression negated the effects of LINC00035 knockdown 3.2. LINC00035 Increased the SLC16A3 Transcription by concerning no significant differences in A2780 and SKOV3 Recruiting CEBPB in OC Cells. Next, we retrieved the cell viability, migration, invasion, apoptosis, glucose uptake, LncMAP database (http://bio-bigdata.hrbmu.edu.cn/ lactate production, and ATP production between scramble LncMAP/) by entering LINC00035 and found LINC00035 siRNA plus empty vector and si-LINC00035 plus SLC16A3 regulates transcription factor CEBPB and its downstream expression vector. )ese results thus suggested that gene SLC16A3 (Figure 2(a)). )en, we conducted RIP assays LINC00035 promoted glycolysis and cell apoptosis through in A2780 and SKOV3 cells. LINC00035 enrichments were CEBPB-mediated transcriptional promotion of SLC16A3. observed in that the CEBPB immunoprecipitates in A2780 and SKOV3 cells and more LINC00035 enrichments were presented after transfection with LINC00035 expression 3.4. e LINC00035/CEBPB/SLC16A3 Pathway Affected the vector (Figure 2(b)). RNA pull-down followed by immu- Tumorigenesis of OC Cells In Vivo. Finally, the effect of noblotting showed CEBPB could be pulled down by biotin- LINC00035 on the tumorigenesis of OC cells in vivo was labeled LINC00035 probes (Figure 2(c)). Accordingly, we analyzed after transfected SKOV3 cells were xenografted mapped CEBPB and SLC16A3 into the starBase database into nude mice. )e data revealed that LINC00035 and found CEBPB and SLC16A3 were coexpressed in OC knockdown and SLC16A3 knockdown were associated with (r � 0.405; p< 0.001, Figure 2(d)). It was found elevated inhibition of xenotransplanted tumors in nude mice luciferase activity of the SLC16A3-wt in A2780 and SKOV3 (Figure 5(a)). Six weeks after implantation, the mice were cells with CEBPB overexpression and declined luciferase euthanized by exposure to prolonged inhalational anes- activity upon CEBPB knockdown (Figure 2(e)). )e ChIP thesia, with tumor bodies excised. )e data showed that assays demonstrated SLC16A3 in CEBPB immunoprecipi- LINC00035 knockdown and SLC16A3 knockdown could tates using anti-CEBPB rather than normal mouse IgG. increase the weight and volume of xenotransplanted tumors Furthermore, we found more SLC16A3 was detected in in nude mice (Figures 5(b) and 5(c)). No significant dif- CEBPB immunoprecipitates from A2780 and SKOV3 cells ference between scramble siRNA plus empty vector and si- upon LINC00035 expression vector transfection and less LINC00035 plus SLC16A3 expression vector, suggesting that detection of SLC16A3 in CEBPB immunoprecipitates upon SLC16A3 overexpression negated the effects of LINC00035 si-LINC00035 transfection (Figure 2(f)). )ese results thus knockdown on the tumorigenesis of SKOV3 cells in vivo. suggested that LINC00035 could recruit CEBPB into the SLC16A3 promoter region, thus increasing the SLC16A3 4. Discussion transcription. Recent evidence indicates that high propensity of OC cells to 3.3. e LINC00035/CEBPB/SLC16A3 Pathway Affected consume and metabolize glucose by glycolysis can fuel Glycolysis and Apoptosis in OC Cells. Next, the investigation uncontrolled growth of abnormal cells, finally leading to focused on the role of SLC16A3 in OC cells. At the be- disease progression and chemoresistance [23]. In the case of ginning, we mapped SLC16A3 into the GEPIA database and OC, dysregulation of lncRNAs has been reported to engage found SLC16A3 was highly expressed in OC tissue in tumor growth and metastasis [24]. )e obtained data (Figure 3(a)). Results of qRT-PCR and immunoblotting revealed LINC00035 knockdown led to reduced viability analysis showed SLC16A3 mRNA and protein expressions migration and invasion of OC cells concurrent with an were upregulated in OC tissues compared to adjacent tu- increased apoptosis, leading to decreases in glucose uptake, mor-free tissue, in four human OC cell lines compared with lactate release, and ATP production. )e OC mouse model normal ovarian epithelial cells (Figure 3(b)). Subsequently, confirmed anti-tumor effect of LINC00035 knockdown on we performed rescue experiments by successfully delivering tumor growth as well. More specifically, another investi- si-LINC00035 plus empty vector, si-SLC16A3, scramble gation in this study uncovered the detailed mechanism 6 Evidence-Based Complementary and Alternative Medicine (a) A2780 SKOV3 1.0 1.5 1.0 1.5 0.8 0.8 1.0 1.0 0.6 0.6 0.4 0.4 0.5 0.5 0.2 0.2 0.0 0.0 0.0 0.0 scramble siRNA scramble siRNA si-LINC00035 si-LINC00035 (b) A2780 CEBPB vs. SLC16A3, 379 samples (OV) SKOV3 Data Source: starBase v3.0 project * 5 4 3 -1 CEBPB, Expression level: log2 (FPKM+0.01) Regression (y = 0.6665x - 1.8482) r = 0.405, p-value = 2.17e-16 (c) (d) A2780 2.5 * 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 SLC16A3-wt SLC16A3-mut SLC16A3-wt SLC16A3-mut Empty vector scramble siRNA CEBPB expression vector si-CEBPB SKOV3 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 SLC16A3-wt SLC16A3-mut SLC16A3-wt SLC16A3-mut Empty vector scramble siRNA CEBPB expression vector si-CEBPB (e) Figure 2: Continued. Bio-NC Bio-NC LINC00035 enrichment Luciferase activity Luciferase activity Input anti-CEBPB IgG Bio-LINC00035-mut LINC00035 enrichment CEBPB enrichment anti-CEBPB IgG Luciferase activity Luciferase activity Bio-LINC00035-mut LINC00035 enrichment SLC16A3, Expression level: log2 [FPKM+0.01)] Input anti-CEBPB IgG LINC00035 enrichment anti-CEBPB IgG Bio-LINC00035-wt Bio-LINC00035-wt Evidence-Based Complementary and Alternative Medicine 7 A2780 IgG anti-CEBPB SKOV3 IgG anti-CEBPB (f) Figure 2: LINC00035 increases the SLC16A3 transcription by recruiting CEBPB in OC cells. (a) LINC00035 regulation of SLC16A3 via transcription factor CEBPB is predicted in the LncMAP database. (b) RIP-qPCR examined the LINC00035 in CEBPB immunoprecipitates from A2780 and SKOV3 cells with or without LINC00035 overexpression. (c) RNA pull-down followed by immunoblotting analysis examined the CEBPB pulled down by biotin-labeled LINC00035 probes. (d) Coexpression between CEBPB and SLC16A3 in the starBase database. (e) Dual-luciferase reporter gene assays to validate SLC16A3 binding with CEBPB in A2780 and SKOV3 cells. (f) ChIP assays followed by immunoblotting analysis examined the SLC16A3 in CEBPB immunoprecipitates from A2780 and SKOV3 cells with or without LINC00035 knockdown p< 0.05. * * 2.5 2.0 38-42 KD 1.5 36 KD 1.0 2 2 0.5 0 0.0 OC (num (T)=426; num (N)=88) 2.0 1.5 1.0 38-42 KD 0.5 36 KD SKOV3 Caov-3 CoC1 A2780 IOSE80 0.0 SKOV3 Caov-3 CoC1 A2780 IOSE80 SKOV3 Caov-3 CoC1 A2780 IOSE80 (a) (b) Figure 3: Continued. SLC16A3 mRNA expression normalized to GADPH Expression-log2 (TPM+1) SLC16A3 mRNA expression normalized to GADPH SLC16A3 enrichment SLC16A3 enrichment Adjacent tissues OC tissues Adjacent tissues OC tissues SLC16A3 protein expression normalized to GADPH SLC16A3 protein expression normalized to GADPH Adjacent tissues OC tissues 8 Evidence-Based Complementary and Alternative Medicine A2780 SKOV3 A2780 8 8 38-42 KD 36 KD SKOV3 38-42 KD 2 2 36 KD 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 (c) Figure 3: LINC00035 increases SLC16A3 expression in OC cells. (a) SLC16A3 was highly expressed in OC tissue in the GEPIA database. (b) SLC16A3 expressions in OC tissues and cells, measured by qRT-PCR and immunoblotting analysis. (c) si-LINC00035 plus empty vector, si-SLC16A3, scramble siRNA plus empty vector, or si-LINC00035 plus SLC16A3 expression vector were delivered into A2780 and SKOV3 cells and qRT-PCR, and immunoblotting analysis were used to determine SLC16A3 mRNA and protein expressions p< 0.05. A2780 SKOV3 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h si-SLC16A3 si-SLC16A3 si-LINC00035+oe-SLC16A3 si-LINC00035+oe-SLC16A3 scramble siRNA+empty vector scramble siRNA+empty vector (a) A2780 SKOV3 50 μm 50 μm 50 μm 50 μm 50 μm 50 μm 0 0 si-SLC16A3 si-LINC00035+ scramble siRNA+ si-LINC00035+ scramble siRNA+ si-SLC16A3 oe-SLC16A3 empty vector oe-SLC16A3 empty vector (b) Figure 4: Continued. SLC16A3 mRNA expression normalized to GADPH si-LINC00035 +empty vector si-SLC16A3 SLC16A3 protein expression normalized to GADPH scramble siRNA +empty vector OD value si-LINC00035 si-LINC00035 +oe-SLC16A3 +empty vector Migrating cells si-SLC16A3 SLC16A3 mRNA expression normalized to GADPH si-SLC16A3 scramble siRNA +empty vector si-LINC00035+ oe-SLC16A3 si-LINC00035 si-LINC00035 scramble siRNA+ +empty vector empty vector +oe-SLC16A3 si-SLC16A3 OD value SLC16A3 protein expression normalized to GADPH scramble siRNA +empty vector si-LINC00035 si-LINC00035 +oe-SLC16A3 +empty vector si-SLC16A3 scramble siRNA si-LINC00035 +empty vector +empty vector si-LINC00035 si-SLC16A3 +oe-SLC16A3 scramble siRNA +empty vector Migrating cells si-LINC00035 +oe-SLC16A3 si-SLC16A3 si-LINC00035+ oe-SLC16A3 scramble siRNA+ empty vector Evidence-Based Complementary and Alternative Medicine 9 300 200 SKOV3 A2780 50 μm 50 μm 50 μm 50 μm 50 μm 50 μm 0 0 si-SLC16A3 si-LINC00035+ scramble siRNA+ si-SLC16A3 si-LINC00035+ scramble siRNA+ oe-SLC16A3 empty vector oe-SLC16A3 empty vector (c) A2780 SKOV3 Bcl-2 20 25 KD Bcl-2 25 KD Bax 21 KD Bax 21 KD GADPH 36 KD 36 KD 10 GADPH (d) SKOV3 A2780 2.0 2.0 1.5 2.0 1.5 2.0 * * * 1.5 1.5 * 1.5 1.5 1.0 1.0 1.0 1.0 1.0 1.0 0.5 0.5 0.5 0.5 0.5 0.5 0.0 0.0 0.0 0.0 0.0 0.0 (e) Figure 4: )e LINC00035/CEBPB/SLC16A3 pathway affects glycolysis and apoptosis in OC cells. (a) CCK-8 assays examined A2780 and SKOV3 cells upon SLC16A3 knockdown or LINC00035 knockdown and SLC16A3 overexpression concurrently. (b, c) Representative view and statistics of A2780 and SKOV3 cells transferring from the apical chamber of the transwell system uncoated or coated with Matrigel into the basolateral chamber upon SLC16A3 knockdown or LINC00035 knockdown and SLC16A3 overexpression concurrently. (d) Im- munoblots and quantification of bcl-2 and bax in A2780 and SKOV3 cells upon SLC16A3 knockdown or LINC00035 knockdown and SLC16A3 overexpression concurrently. (e) Glucose uptake, lactate production, and ATP production measurements in A2780 and SKOV3 cells upon SLC16A3 knockdown or LINC00035 knockdown and SLC16A3 overexpression concurrently p< 0.05. knockdown could notably repress the viability, migration, behind the action of LINC00035 in OC, namely, a regulatory network consisting of LINC00035, the transcription factor and invasion of OC, induce the apoptosis, and reduce CEBPB, and downstream gene SLC16A3. glycolysis. Accordingly, in vivo experiments further vali- Increasing studies have begun to focus on the effects of dated that tumor growth inhibition and tumor size reduc- lncRNAs, such as LINC00494, GClnc1, and SNHG17 in OC tion were related to LINC00035 knockdown. Taken together, by gene regulation from aspect of transcription regulation the oncogenic functions of LINC00035 in OC was uncovered [25–27]. Aberrant expression of LINC00035 has been re- in our study. ported to regulate the development and progression of LncRNA regulation of transcription factor to modulate various tumors, including OC, and its high expression may the transcription of downstream genes is a novel mechanism contribute to tumor growth and invasion. For example, in tumor growth and metastasis. CEBPB is one of four Zhang et al. demonstrated LINC00035 promoter mediated known members of the C/EBP family of basic region-leucine zipper transcription factors (CEBPA, CEBPB, CEBPD, and by EZH2 modulates OC progression by targeting miR-133a- 3p [28]. In the study of Zeng et al., they found LINC00035 CEBPE) [31]. )e transcriptional regulation of CEBPB on governed by the EGFR pathway promotes the tumorigenesis downstream genes is regulated by lncRNA in human dis- of OC via epigenetically suppressing TIMP2 [29]. A previous eases. For example, Wu et al. demonstrated LINC00160 study conducted by Wu and his team found LINC00035 mediated acquired resistance of chemotherapeutic regimes regulation of RhoC conferred tumor-promoting effects in of paclitaxel and doxorubicin drugs in breast cancer cells by OC [30]. Further functional studies verified that LINC00035 regulating TFF3 via transcription factor C/EBPβ [32]. Gao Apotosis rate (%) Glucose uptake si-SLC16A3 si-SLC16A3 si-LINC00035+ oe-SLC16A3 si-LINC00035+ oe-SLC16A3 scramble siRNA+ empty vector scramble siRNA+ empty vector Lactate production si-SLC16A3 si-LINC00035+ oe-SLC16A3 scramble siRNA+ si-SLC16A3 empty vector Invasive cells ATP level si-LINC00035+ oe-SLC16A3 si-SLC16A3 si-SLC16A3 scramble siRNA+ si-LINC00035+ empty vector oe-SLC16A3 si-LINC00035+ oe-SLC16A3 scramble siRNA+ scramble siRNA+ empty vector empty vector Glucose uptake Apotosis rate (%) si-SLC16A3 si-SLC16A3 si-LINC00035+ oe-SLC16A3 scramble siRNA+ si-LINC00035+ empty vector oe-SLC16A3 Lactate production scramble siRNA+ empty vector si-SLC16A3 si-LINC00035+ oe-SLC16A3 scramble siRNA+ empty vector Invasive cells ATP level si-SLC16A3 si-LINC00035+ oe-SLC16A3 si-SLC16A3 si-SLC16A3 si-LINC00035+ scramble siRNA+ si-LINC00035+ oe-SLC16A3 empty vector oe-SLC16A3 scramble siRNA+ scramble siRNA+ empty vector empty vector 10 Evidence-Based Complementary and Alternative Medicine 2.0 800 1.5 1.0 0.5 1 25 3 4 6 0.0 Time (week) scramble si-LINC00035 siRNA+ +empty vector empty vector si-LINC00035 si-SLC16A3 +oe-SLC16A3 (a) (b) (c) Figure 5: )e LINC00035/CEBPB/SLC16A3 pathway affects the tumorigenesis of OC cells in vivo. (a) Subcutaneously xenografted tumors. (b) Tumor weight. (c) Tumor growth p< 0.05. LINC00035 LINC00035 Glycolysis CEBPB Apoptosis CEBPB Promoter SLC16A3 Figure 6: A schematic diagram showing how the LINC00035/CEBPB/SLC16A3 pathway regulates the development of OC. LINC00035 was triggered in OC and could recruit the transcription factor CEBPB into the SLC16A3 promoter region, thus increasing the SLC16A3 transcription. Abundance of SLC16A3 inhibited OC cell viability, migration, invasion, and glycolysis and induced the apoptosis. et al. found that silencing of lncRNA LEF1-AS1 reduced knockdown exerted similar effects on SKOV3 and EZH2 expression to repress hepatocellular carcinoma pro- A2780 cells as LINC00035 knockdown, especially attenu- gression by disruption of CEBPB interaction with CDCA7 ating glucose uptake, lactate production, and ATP pro- [33]. Only a previous study investigates the role of CEBPB in duction. In a gliomas report, Reuss et al. indicated that the function SLC16A3 is lactate and proton exporter. )e the case of OC that CEBPB expression was not significantly changed in OC tissues and cells, which was partially con- expression of SLC16A3 was negatively correlated with sistent with our results that CEBPB was just responsible for extracellular pH and proportional to extracellular lactate the regulation of LINC00035 on the SLC16A3 expression in concentrations [35]. Yu and his team performed integrated OC. bioinformatics that comprehensively analyzed the SLC16A SLC16A3 is greatly involved in tumor pH modulation gene family in pancreatic cancer, showing SLC16A as a lactate transporter. In a mouse tumor model, lactate exhibited biomarker potential for prognosis of pancreatic modulation and anti-tumor activity were evaluated cancer [36]. Concurring with our data concerning through the direct target interaction of the optimized SLC16A3 expression in OC, SLC16A3 was remarkably compound 18n with the cytosolic domain of SLC16A3 increased in liver cancer [37]. suggesting pharmacokinetic inhibition of SLC16A3 by 18n In summary, the study mainly highlights a new mechanism by regulating the transcription factor CEBPB is a useful outcome measure for anti-tumor effects on tumor biology [34]. Our following investigations demon- and the downstream genes SLC16A3 to confirm the strated SLC16A3 was upregulated in OC tissues. SLC16A3 function of LINC00035 in OC. LINC00035 acts as an si-LINC00035 +empty vector si-SLC16A3 scramble siRNA +empty vector si-LINC00035 +oe-SLC16A3 Tumor weight (g) si-LINC00035 +empty vector si-SLC16A3 scramble siRNA +empty vector si-LINC00035 +oe-SLC16A3 Cytoplasm Evidence-Based Complementary and Alternative Medicine 11 endometrial carcinoma by targeting cyclin D1,” Journal of oncogenic lncRNA in OC tumorigenesis. )e finding Cellular and Molecular Medicine, vol. 22, pp. 3955–3964, 2018. revealed in this study may confer valuable view to develop [13] X. Zhuang, H. Tong, Y. 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Shakoori et al., )e data used for this study were included in the article. “Expression profiles and functional prediction of long non- coding RNAs LINC01133, ZEB1-AS1 and ABHD11-AS1 in the luminal subtype of breast cancer,” Journal of Translational Conflicts of Interest Medicine, vol. 19, no. 1, p. 364, 2021. [16] D. Fu, Y. Shi, J.-B. Liu et al., “Targeting long non-coding RNA )e authors declare that they have no conflicts of interest. to therapeutically regulate gene expression in cancer,” Mo- lecular erapy—Nucleic Acids, vol. 21, pp. 712–724, 2020. Acknowledgments [17] G. G. Jinesh, M. Napoli, H. D. Ackerman et al., “Regulation of MYO18B mRNA by a network of C19MC miRNA-520G, )is study was supported by the Heilongjiang Postdoctoral IFN-c, CEBPB, p53 and bFGF in hepatocellular carcinoma,” Fund (LBH-Z18190) and the Wutong Tree Foundation of Scientific Reports, vol. 10, no. 1, Article ID 12371, 2020. the Forth Affiliated Hospital of Harbin Medical University [18] Y. Gao, B. Liu, L. 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