| Ovarian cancer is the leading cause of death among gynecologic malignances.Ovarian cancer is susceptible to invasion and metastasis in the early stages,and its distant metastasis is more common in the peritoneum and liver.Due to its lack of typical clinical symptoms at the early stages and no specific diagnostic indicators,75% of patients with ovarian cancer advanced late stages(III~IV)at the time of diagnosis.Despite advances in chemotherapy,the five-year survival rate of advanced ovarian cancer patients with peritoneal metastasis remains approximately 30%.In the United States have 22,240 new ovarian cancer cases and 14,070 ovarian cancer deaths occured in 2018.Hence,understanding of the molecular alterations associated with ovarian cancer metastasis could lead to the identification of targets for novel therapeutic interventions.Metastasis is the dissemination of cancer cells from primary tumors to other distant organs and represents a major barrier in the treatment of cancer.The epithelial-mesenchymal transition(EMT)is an important biological process in which cancer cells lose their epithelial characteristics and acquire mesenchymal properties,thus promoting cancer cell migration,invasion and metastasis.The transition from epithelial to mesenchymal allows cancer cells to break away from its original tissue and diffuse throughout the body.The majority of the signalling pathways,while alone are capable of EMT induction,often converge and compose a complex network of EMT signaling.The EMT pathway is of increasing interest as a novel therapeutic avenue in the treatment of cancer,and could be targeted to prevent tumor cell dissemination in early stage of patients or to eradicate existing metastatic cells in advanced stages.Therefore,it is important to understand the molecular mechanisms underlying EMT regulation which help us determine valuable biomarkers for the prognosis of patients and even provides novel approaches for a more effective therapeutics against cancers.MiRNA are a class of highly-conserved,small endogenous non-coding RNAs of 18 to 25 nucleotides in length function as indispensable and negative regulators of gene expression at post-transcription level by either degrading m RNAs or blocking protein translation by binding to the 3?-untranslated region(3?-UTR)Mounting evidences indicate that Dysregulation of mi RNAs is associated with cancer development.Mi RNAs contribute to the malignant tumor progression and metastasis process,such as cell proliferation,invasion,apoptosis,angiogenesis.Mi R-203 located at the chromosome 14q32.33,which inhibits tumor cell growth,proliferation,and metastasis.Many studies have demonstrated that mi R-203 as a tumor suppressor in multiple malignancies,including oral,lung,glioma,bladder,gastric,and colorectal cancers,and breast cancers.However,there are very few studies showing that mi R-203 plays a tumor suppressor role in ovarian cancer.Survivin also called BIRC5,is a member of the apoptosis inhibitor protein family and as an apoptosis inhibitor plays an important role in this cellular event.Survivin expression is limited in a variety of normal tissues but highly expressed in various cancer cells and its expression correlates with aggressiveness and poor survival,which suggests the direct role of survivin in tumorigenesis.However,little is known about the role of survivin in ovarian cancer.YM155,a small molecule inhibitor of survivin,specifically inhibit the expression of survivin in a dose-dependent manner.A number of studies have reported that YM155 is able to effectively inhibit survivin expression and induce the apoptosis of human cancer cells.Furthermore,YM155 has been evaluated in phase II clinical trials for melanoma and breast cancer.We previously demonstrated that mi R-203 is a tumor suppressor in ovarian cancer,and also showed that survivin was highly expressed in ovarian cancer but not in the normal ovaries tissues.Survivin was reported to be a target gene of mi R-203 in leukemia and hepatocellular carcinoma.Based on the literature and our previous data,we hypothesize that mi R-203 may regulate ovarian tumor metastasis by targeting survivin in ovarian cancer.We tested our hypothesis that mi R-203 inhibits ovarian tumor metastasis by suppressing EMT through targeting survivin using an orthotopic ovarian cancer mouse model.We elucidated the underlying mechanism of mi R-203 in inhibiting EMT by targeting BIRC5 in ovarian cancer cells and enhancing the efficacy of survivin inhibitor YM155 in cell migration and invasion.Mi R-203 expression inhibits ovarian tumor metastasis by targeting survivin and attenuating the TGF? pathway in an orthotopic ovarian cancer mouse model.mi R-203 could be potential novel molecular markers for predicting the risk of recurrence and prognosis of ovarian cancer.This helps us further learn the mechanism of tumorigenesis of ovarian cancer and provides molecular foundation for the early detection and therapy of ovarian cancer.Objective(1)Overexpression of mi R-203 in ovarian cancer cells,survivin and EMT marker gene expression was examined in both mi R-203-expressing and control ovarian cancer cells confirming that mi R-203 inhibits EMT by targeting survivin in ovarian cancer cells.(2)Survivin inhibitor YM155 was used to treat ovarian cancer cells,and the effects of treatment on the biological behavior of ovarian cancer cells were observed to explore the effect of mi R-203 on the efficacy of YM155.(3)To observe the effect of mi R-203 and YM155 on TGF-? signaling pathway.(4)Generate an orthotopic ovarian cancer mouse model,observe the effect of mi R-203 on tumor growth and dissemination and TGF-? signaling pathway.Material and methods1.Object of studySKOV3 and OVCAR3 ovarian cancer cells,HEK293 FT cells and Cg-Prkdcscid Il2rgtm1Wjl/Sz J(NSG)female immunodeficient mice.2.Methods(1)Cell culture: Both SKOV3 and OVCAR3 cells were cultured in DMEM(Dulbecco's Modified Eagle Medium)supplemented with 10% FBS(Hyclone;Logan,UT),100 U/ml penicillin,and 100 ?g/ml streptomycin.HEK293 FT cells were cultured in DMEM media with 10% FBS,100 U/ml penicillin,100 ?g/ml streptomycin,1% glutamine,1% nonessential amino acid,and geneticin with a final concentration of 1 ?g/ml.(2)Lentiviral packaging,purification and transfection: HEK293 FT cells are used to package lentivirus,the plasmid containing the gene of interest is introduced,the virus is purified,and the purified lentivirus was transduced into the ovarian cancer cells to construct a control vector and mi R-203 overexpressing cells.(3)Western blot:Cellular protein was extracted to detect the expression of survivin and EMT-related proteins.The mouse tumor tissue protein was extracted,and the survivin,phosphos-SMAD2 and EMT related proteins were detected by the same method.(4)Cellular immunofluorescence experiments: The expression of survivin and EMT-related proteins in cells was observed.(5)Transwell experiment: The cells in the control group and the mi R-203 overexpression group were added with or without YM155 to observe the changes of cell migration and invasion ability.(6)Cell scratch assay: mi R-203 overexpression on cell migration was tested using cell scratch.(7)Animal experiment: Establish a mouse model of orthotopic ovarian cancer xenografts.Tumor size and weight were measured,tumors and organs were collected,and the effect of mi R-203 overexpression on ovarian tumor growth and metastasis was observed.(8)Immunofluorescence and HE staining: observe the metastasis of visceral tumor in mice.3.Statistical analysesThe experimental data were analyzed by SPSS23.0.The two groups were analyzed by independent t test.Difference between multiple sets using single factor analysis of variance,two comparison use of LSD-t,?=0.05 was used as the test level,P < 0.05 was statistically significant.Results(1)Western blot and cellular immunofluorescence showed that compared with the control,survivin was significantly downregulated in mi R-203-expressing cells compared to controls,the EMT related proteins including Snail,vimentin and ?-catenin were downregulated,while the epithelial marker cytokeratin-7 was up-regulated.(2)The results of Transwell experiment showed that the perforating cells of the mi R-203 overexpressing group and the YM155 treated group were lower than the control group,and the number of transduced cells in the overexpressed mi R-203 combined with YM155 group was significantly decreased.(3)The results of the scratch test were consistent with the transwell experiment.(4)Proteins were extracted at different time points after treatment with TGF-?.Western blot analysis showed that mi R-203 expression significantly attenuated phospho-SMAD2,but did not affect the total SMAD2 in SKOV3 and OVCAR3 cell lines.(5)Xenogen imaging system was used to image tumors in mouse ovary and metastatic organs.The results showed that the volume and weight of tumors in the mi R-203 overexpressing group were smaller than those in the control group,and no distant metastasis occurred.(6)Tumors in the ovary were verified in section HE staining.The results showed that liver and spleen metastasis occurred in the control group,but no metastasis was observed in the mi R-203 overexpression group.(7)Tumor tissue proteins were extracted and the survivin and EMT marker genes were detected by Western blot.The results were consistent with those obtained from the cell lines.(8)In ovarian tumor sections were immunostained using survivin,vimentin,and cytokeratin-7.In the mi R-203 overexpressing group,Survivin and vimentin staining was attenuated,while cytokeratin-7 staining was enhanced.Conclusions(1)Mi R-203 inhibits EMT by targeting survivin and attenuating the TGF-? pathway and enhances the efficacy of survivin inhibitor YM155 on inhibiting ovarian cell migration and invasion.(2)Mi R-203 inhibits primary tumor growth and metastasis by targeting survivin to attenuate the TGF-? pathway.in an orthotopic ovarian cancer mouse model.(3)As a biomarker for ovarian cancer,mi R-203 is expected to become a new clinical diagnosis and treatment index for ovarian cancer. |
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