The Study On The Effect And Mechanism Of SRD5A3 Promoting The Malignant Behavior Of High Serous Ovarian Carcinoma

Background and objectiveOvarian cancer lacks effective early diagnosis and treatment,and the prognosis of most patients are poor.Recently,although significant progression has been made in the research and treatment of HGSOC,the potential molecular pathogenesis of HGSOC still needs to be further explored.It is of great significance to explore the key molecules involved in the tumorigenesis and progression of ovarian cancer for the early screening and treatment.Steroid-5a reductase 3(SRD5A3)is a member of the steroid-5a reductase family,NADPH-dependent microsomal enzyme,which is involved in the metabolism of steroid compounds.SRD5A3 plays a role in the occurrence and development of human malignant tumors,and its up-regulation can promote the growth of tumors.However,the role of SRD5A3 in HGSOC have not been fully elucidated.This study aims to detect the expression of SRD5A3 in HGSOC tissues and ovarian cancer cell lines,and to explore the biological characteristics and molecular mechanism of SRD5A3 in the tumorigenesis and development of HGSOC,so as to provide a possible new target for therapy of HGSOC.Materials and methodsReal-time quantitative PCR(RT-qPCR)and western blot(WB)were used to elucidate the expression of SRD5A3 in fimbriae tube(FT)and HGSOC tissues.The expression of SRD5A3 in FT and HGSOC and the relationship between the expression level of SRD5A3 and the clinicopathological characteristics of patients were detected by immunohistochemistry(IHC).The upregulating plasmid and interfering plasmid of SRD5A3 were constructed,and the SRD5A3 overexpressing and interfering stable cell lines were established by lentivirus packaging system.MTT and colony formation assay were used to investigate the effect of SRD5A3 on the proliferation and clonogenic ability of ovarian cancer cells,and flow cytometry was used to analyse the effect of SRD5A3 on the cycle distribution of ovarian cancer cells..Transwell assay was used to explore the effect of SRD5A3 on the migration and invasion ability of ovarian cancer.Cisplatin and SRD5A3 small molecule inhibitor were applied to ovarian cancer cells with a certain concentration gradient,which were used to observe the effect of SRD5A3 on cisplatin chemotherapy on ovarian cancer cells.MTT was used to detect the effect of SRD5A3 small molecule inhibitor combined with cisplatin.WB was used to detect whether SRD5A3 affects the biological behavior of high-level serous ovarian cancer by regulating P13K/AKT/mTOR pathway.ResultsRT-PCR and WB showed that the expression level of SRD5A3 in HGSOC tissues and ovarian cancer cells was higher.Immunohistochemical staining showed that the expression level of SRD5A3 was associated with poor prognosis,lymph node metastasis and resistance to platinum chemotherapy.After overexpression of SRD5A3,MTT assay showed that the cell growth rate were increased,and clonoy formation assay showed that the ability of cell clonogenesis were increased.After interference with SRD5A3 expression,the results were reversed.The result of flow cytometry showed that the up-regulation of SRD5A3 promoted the transformation of ovarian cancer cell cycle from G1 to S stage,while knockdown of SRD5A3 induced the cell cycle arrest in Gl/S stage.And the mRNA levels of c-MYC,CDK2,CCNE1,CDK4,CDK6,CCND1 in A2780 cell lines were increased,while the levels of P21 and P27 were decreased with SRD5A3 overexpression.Moreover the protein expression of cycle-related molecular were detected corresponding changes.Transwell assay showed thatSRD5A3 overexpression promoted ovarian cancer cells enter into the lower chamber.After SRD5A3 overexpression,the mRNA level and protein of E-cadherin levels of epithelial marker molecules decreased,while N-cadherin,vimentin,ZEB1 and p-cateninn levels of interstitial marker molecules increased.and the results were reversed after the interference of SRD5A3.RT-PCR showed that with the increase of cisplatin concentration,the mRNA level of SRD5A3 gradually increased.Western blot showed that the protein level of SRD5A3 increased gradually with the increase of cisplatin time.And with the up-regulation of SRD5A3,the expression of Atg12 and LC3 ?/? were increased,while the levels of P62 were decreased.The results were reversed after SRD5A3 interference;In the drug MTT experiment,with the increase of the concentration and time of small molecule inhibitors of SRD5A3,the growth inhibition rate of ovarian cancer cells increased.In the drug-resistant MTT assay,with the increase of cisplatin concentration gradient,the inhibitory rate of SRD5A3 interfering ovarian cancer cell lines increased than that of the control group.The combination of cisplatin and small molecule inhibitor showed stronger inhibitory effect on ovarian cancer cells than cisplatin or small molecule inhibitor alone.After the overexpression of SRD5A3,Western blot showed that the phosphorylated P13K,AKT and mTOR were up-regulated,while the phosphorylated P13K,AKT,mTOR were down-regulated after the interference of SRD5A3.Conclusion1.SRD5A3 is highly expressed in HGSOC,and its expression level is related to poor prognosis of HGSOC patients,platinum chemotherapy resistance and lymph node metastasis.2.SRD5A3 affects the growth and proliferation of ovarian cancer by regulating the distribution of G1 to S phase of cell cycle.3.SRD5A3 induces cell EMT by regulating the changes of EMT-related molecules,and ultimately affects cell invasion and migration ability.4.SRD5A3 confers chemotherapy resistance by promoting autophagy of ovarian cancer cells.5.Knockdown SRD5A3 expression or using small molecule inhibitors of SRD5A3 can enhance the sensitivity of cisplatin chemotherapy.6.SRD5A3 plays a biological role in ovarian cancer accompanied by P13K/AKT/mTOR pathway activation.