Role Of The LncRNA ABHD11-AS₁ In The Tumorigenesis And Progression Of Epithelial Ovarian Cancer Through Targeted Regulation Of RhoC

Objective:Ovarian cancer is one of the female genital malignancies,with the third highest incidence of gynecologic malignancies.However,its incidence is insidious,the prognosis is poor,the case fatality rate is high,and the overall survival rate of patients is not satisfied.Therefore,to explore the occurrence and development of ovarian cancer and find effective biomarkers may provide a new strategy for the clinical diagnosis and treatment of ovarian cancer.In recent years,long-chain non-coding RNAs（lncRNAs）play a role in tumorigenesis,invasion,metastasis and drug resistance.LncRNAs are non-coding RNA molecules consisting of more than 200 nucleotides that regulate gene expression and participate in several biological functions through a variety of pathways and molecular mechanisms.Aberrant expression and regulation of lncRNAs are closely linked to the development of many malignancies;therefore lncRNAs are thought to play an important role in cell proliferation,differentiation and apoptosis.However,more evidence is needed to confirm these findings.Studies have shown that the expression of Lnc RNA ABHD11-AS1（ABHD11 antisense RNA1）in gastric cancer tissue is significantly higher than that of normal gastric tissue,which means that lncRNA may serve as a cancer biomarker.Some studies have shown that gastric cancer patients gastric Lnc RNA ABHD11-AS1 expression was significantly higher than normal,but the mechanism of cancer-related lncRNA is unclear.In addition,there is no report on the role or mechanism of lncRNA ABHD11-AS1 in ovarian cancer.Methods:1.Ovarian cancer specimens:Fifty-one epithelial ovarian cancer tissue and thirteen normal ovarian tissue specimens were collected from patients who had undergone surgical resection at the Department of Gynecology of the First Affiliated Hospital of China Medical University（Shenyang,China）.Two pathologists confirmed the tumor specimens independently.Samples were frozen immediately in liquid nitrogen and stored at-80°C until use.None of the patients had undergone preoperative chemotherapy or radiotherapy.Informed consent was obtained from all the subjects.The China Medical University Ethics Committee approved of the study,and all the specimens were handled and anonymized according to ethical and legal standards（No:2014-27）.2.Cell culture and transfection:The human ovarian carcinoma cell lines A2780 and OVCAR3 were purchased from Jennio Biotech Co.Ltd.（GuangZhou,China）.The human cell lines A2780 cells were cultured in Dulbecco’s modified Eagle’s medium（DMEM;HyClone,Logan,UT,USA）and the OVCAR3 cells were cultured in RPMI1640（HyClone）supplemented with penicillin/streptomycin（100 U/mL）in 10%fetal bovine serum（FBS）.The cells were cultured in an incubator at 37°C in a 5%CO₂atmosphere.All transfection experiments were carried out using Lipofectamine 2000according to the manufacturer’s instructions（Invitrogen,Carlsbad,USA）.3.Tetrazolium assay:Cells were seeded in 96-well plates at a density of 3000 cells per well.At 0 h,24 h,48 h,and 72 h after seeding,the cells were incubated with 20μL of 5 mg/m L tetrazolium（MTT）solution（Solarbio,Beijing,China）at 37°C for 4 h.Then,the medium was removed,and the precipitated formazan was dissolved in 150μL dimethyl sulfoxide（DMSO）.After shaking for 10 min,the absorbance at 490 nm was determined using a microplate spectrophotometer（BioTek Instruments,Winooski,VT,USA）,and was used as an indicator of cell proliferation.4.Apoptosis assay:Cells were collected 48 h after transfection and washed twice with cold phosphate-buffered saline（PBS）.For the ABHD11-AS₁ plasmid transfection,cell apoptosis was quantified using 7AAD and PE-labeled Annexin V（BD Biosciences）with flow cytometry,according to the manufacturer’s protocol.For si RNA transfection,cell apoptosis was quantified using annexin V–FITC and PI（BD Biosciences）with flow cytometry.The apoptosis rate was determined by flow cytometry within 1 h.5.Wound healing assay:Cells were cultured to80%confluence in 6-well culture plates.Then,the monolayers were scratched with a200-μL pipette tip.The cells were washed with PBS and cultured in FBS-free medium with mitomycin C（20?g/ml）.Wounds were observed under a light microscope and photographed at 0 h,24 h,and 48 h.The wound areas were measured using the ImageJ software（National Institutes of Health,Bethesda,MD,USA）.The wound healing rate was determined as follows:（area of original wound-area of wound at different times）/area of original wound×100%.The wound healing rate was considered as an indicator of the migration ability of the cells.6.Invasion assay:We used Matrigel-coated Transwell cell culture chambers（BD Bioscience,San Jose,CA,USA）for the invasion assay.Filters were coated with 40μL of basement membrane Matrigel（1:10）.Cells（5×10⁴/L）resuspended in 200μL serum-free medium were layered in the top compartment of the Transwell inserts.The bottom chambers contained 600μL of complete medium that served as the chemoattractant.After 48 h of incubation at 37°C,cells on the upper surface of the filter were removed using a cotton swab.The cells that had invaded the bottom of the top chamber were fixed with formaldehyde,stained with crystal violet,and counted under an Olympus fluorescence microscope（Tokyo,Japan）.7.qRT-PCR:Total RNA was isolated from ovarian carcinoma cell lines and tissues with TRIzol reagent（Takara,Shiga,Japan）,and OD260/280 was measured with a spectrophotometer（Unico,Shanghai,China）.An OD260/280 value of 1.8–2.0 indicated that the RNA quality was good.Then,2μg of RNA was reverse-transcribed to complementary DNA（cDNA）using the avian myeloblastosis virus transcriptase and random primers（Takara）according to the manufacturer’s protocol.Then,the cDNA was amplified by real-time quantitative PCR with the SYBR Premix Ex Taq?II kit（Takara,Shiga,Japan）.The relative expression of the target genes was determined by comparing the threshold cycle（Ct）of the target genes to that of 18S rRNA（18 s）using the 2^-ΔΔCtΔΔCt method（GenePharma）.8.Western blotting:The complete ovarian carcinoma proteome was extracted in radio-immunoprecipitation assay buffer,which prevented protease-mediated sample degradation.The protein concentration was determined for each sample.Then,40μg of the denatured proteome was resolved by 10%or 12%sodium dodecyl sulfate（SDS）-polyacrylamide gel electrophoresis and then electrotransferred to Hybond membranes（Amersham,Munich,Germany）.After the membranes were blocked with 5%fat-free milk at room temperature for 2 h,they were incubated with primary antibodies against RhoC,P70S6K,MMP2,and Bcl-x L（1:1000;Proteintech,Proteintech Group,USA）at 4°C overnight.The membranes were washed three times with Tris-buffered saline containing Tween-20（TBST）,and then anti-rabbit secondary antibodies（1:5000）were added.After 2 h of incubation at room temperature,the protein bands were visualized by enhanced chemiluminescence according to the manufacturer’s instructions（Santa Cruz Biotechnology,Santa Cruz,CA,USA）.β-actin（1:3000;Proteintech,Proteintech Group,USA）served as the loading control.9.RNA pull-down assay:OVCAR3 normal and ABHD11-AS₁-overexpressing cells were collected and lysed using a protein lysis buffer.Streptavidin magnetic beads were used to capture the biotin-labeled ABHD11-AS₁ probe（Beijing Dingguo Changsheng biotech CO.China）according to the manufacturer’s protocol.Then,the biotinylated nucleic acid compounds were incubated with the protein extract of cells in a 42°C water bath for 40 min.After elution of the magnetic beads,the protein samples were detected by western blotting analysis,with the extracted protein as the positive control and the antisense RNA as the negative control.10.In vivo tumorigenesis model:An in vivo model of ovarian cancer was established by subcutaneously and intraperitoneally injecting 5-week-old female BALB/c nude mice with 1×10⁷A2780 cells transfected with lncRNA ABHD11-AS₁（or mock transfected）suspended in PBS（8 mice per group）.The tumor volumes of the mice were measured every 3 days.Tumor volume was assessed by measuring the length（L）and width（W）of the tumor with calipers（tumor volume[mm³]=0.5×L×W²）.After 4 weeks,the mice were euthanized,and the subcutaneous implanted tumors as well as the intraperitoneal metastatic lesions were excised,measured and photographed.All mice were obtained from Vital River Laboratories（Beijing,China）and housed in a specific pathogen-free environment.This experiment was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the China Medical University Animal Care and Use Committee.11.Immunohistochemistry:Consecutive tissue sections were deparaffinized with xylene,rehydrated with alcohol,and subjected to antigen retrieval by heating in a target retrieval solution（Dako）for 15min in a microwave oven（Oriental Rotor）.The sections were quenched with 3%hydrogen peroxide for 20 min to block endogenous peroxidase activity.Nonspecific binding was prevented by adding 5%bovine serum albumin for 5 min.The sections were incubated for 15 min with the RhoC antibody and then incubated with HRP-conjugated anti-rabbit antibodies（Dako）for 15 min in a microwave oven.After each treatment,the slides were washed three times with TBST for 5 min,and the binding sites were visualized with 3,3′-diaminobenzidine.After the sections were counterstained with Mayer’s hematoxylin,they were dehydrated,cleared,and mounted.Negative controls were prepared by omitting the primary antibody.12.Statistical analysis:Data are presented as the mean±SD values,and were analyzed using the SPSS 18.0 software（SPSS Inc.,Chicago,IL,USA）.The unpaired two-tailed Student’s t-test,Mann–Whitney U-test,and Spearman’s correlation test were used to compare the two groups.All p-values are two-sided;p<0.05 is indicative of statistical significance.Results:The expression of lncRNA ABHD11-AS1 in epithelial ovarian cancer was higher than that in normal ovarian tissue.It was positively correlated with tumor stage（stage I/II and stage III/IV）,and was lower in the well-differentiated group than in the low/medium stage.Overexpression of ABHD11-AS1 in ovarian cancer cell lines A2780and OVCAR3 promoted the proliferation,invasion and migration of ovarian cancer cells and inhibited apoptosis.Silencing ABHD11-AS1 has the opposite effect.Subcutaneous injection of tumor cells in nude mice showed that ABHD11-AS1 significantly promoted tumor growth.In addition,overexpression of ABHD11-AS1 upregulated the expression of RhoC and its downstream molecules P70s6k,MMP2 and BCL-xL.Silencing ABHD11-AS1 has the opposite effect.RNA pull-down assays showed that ABHD11-AS1 binds directly to RhoC.RhoC silencing was found to inhibit the oncogenic effect of lncRNA ABHD11-AS1.In addition,intraperitoneal injection of tumor cells in nude mice results in an increased ability of the tumor to metastasize.Therefore,it appears that RhoC is the primary target for lncRNA ABHD11-AS1.Conclusion:This study demonstrated that lncRNA ABHD11-AS1 can promote the proliferation,invasion and metastasis of ovarian cancer cells and inhibit the apoptosis of ovarian cancer cells by targeting RhoC and its downstream molecules.This discovery reveals the molecular mechanism of the development of malignant ovarian tumors.In addition,lncRNA ABHD11-AS1 is expected to be a potential new target for the treatment of ovarian cancer.