The Effects And Mechanisms Of Hsa-circ-0002755 In High Grade Serous Ovarian Cancer

Objective:Ovarian cancer is the second incidence of gynecological malignant tumors.Because the ovaries are located in the deep pelvic cavity,the symptoms are concealed,early detection is difficult,and the treatment is difficult,easy to recur and metastasize,the 5-year survival rate of Stage III-IV ovarian cancer is only 30%.Ovarian cancer is the leading cause of cancer-related mortality in the female reproductive system.CircRNA is a novel RNA with a closed loop structure.Because of its stable structure,good conservation in different species,tissue specificity and expression specificity at different developmental stages,circRNA has gradually become a research hotspot.So far,abnormal expression of circRNA has been confirmed in a variety of tumor studies.The expression of some circRNAs is related to TNM staging,differentiation,tumor size,metastasis,invasion and other factors.However,the current correlation between ovarian cancer and circRNA is rarely reported.The expression and mechanism of circRNAs in ovarian cancer are still largely unknown.Epithelial ovarian cancer(EOC)is the most important pathological subtype of ovarian cancer,accounting for more than 90%.Among the pathological subtypes of EOC,high-grade ovarian serous carcinoma(HGSOC)accounts for 60-70%,the highest proportion.HGSOC often indicates a worse clinical outcome.Existing studies have shown that HGSOC has a different pathogenesis from low-grade ovarian serous carcinoma.Despite the heterogeneity of HGSOC,the current treatment of clinical patients is not specific,further clarifying the pathogenesis of HGSOC and finding that potential tumor markers or therapeutic targets are essential for improving the 5-year survival rate of HGSOC patients.In this study,circRNA high-throughput sequencing was used to detect differentially expressed circRNAs in HGSOC tissues and normal ovarian tissues,and combined with fold change,P values,FDR,and fpkm based on differential expression,8 circRNAs were selected for qRT-PCR to verify the results of high-throughput sequencing.The circRNA with the highest fold change(hsa-circ-0002755)was selected for further verification in HGSOC tissues and ovarian cancer cell lines,and for clinical pathological biological characteristics analysis.Hsa-circ-0002755-siRNA plasmids and overexpression plasmids was transiently transfected into ovarian cancer cell line respectively,the biological function of hsa-circ-0002755 was determined by CCK-8 assay,Scratch test,Tranwell detection,flow cytometry and Western Blot.The bioinformatics software was used to predict the miRNA and target genes directly bound to hsa-circ-0002755.The targeted binding of hsa-circ-0002755 to hsa-miR-211-3p,hsa-miR-211-3p to CDKN1 B were verified by the dual luciferase assay.qRT-PCR was used to detected the expression of hsa-miR-211-3p in HGSOC and analysis of the correlation with hsa-circ-0002755.The hsa-circ-0002755-siRNA plasmids and overexpression plasmids were constructed to transfect ovarian cancer cells,and the expression changes of hsa-miR-211-3p and CDKN1 B were detected.The hsa-miR-211-3p-siRNA plasmids and overexpression plasmids were transfected to ovarian cancer cells,the expression of CDKN1 B was detected.The results clarified that hsa-circ-0002755 regulates the expression of target gene CDKN1 B by competitive inhibition of hsa-miR-211-3p,providing a new idea and evidence for the pathogenesis of HGSOC.Methods:1.CircRNA high-throughput sequencing was used to detect differentially expressed circRNAs in HGSOC tissues and normal ovarian tissues,and combined with fold change,P values,FDR,and fpkm based on differential expression,8 circRNAs were selected for qRT-PCR to verify the sequencing results high-throughput sequencing.The circRNA with the highest fold change(hsa-circ-0002755)was selected for further verification in HGSOC tissues and ovarian cancer cell lines,and for clinical pathological biological characteristics analysis.2.The hsa-circ-0002755-siRNA plasmids was transiently transfected to ovarian cancer cell line SKOV3,and the hsa-circ-0002755 overexpression plasmids was transiently transfected to ovarian cancer cell line OVCAR-3.The biological function of hsa-circ-0002755 was determined by CCK-8 assay,Scratch test,Tranwell detection,flow cytometry and Western Blot.3.The bioinformatics software was used to predict the miRNA and target genes directly bound to hsa-circ-0002755.The targeted binding of hsa-circ-0002755 to hsa-miR-211-3p,hsa-miR-211-3p to CDKN1 B were verified by the dual luciferase assay.The expression of hsa-circ-0002755,hsa-miR-211-3p and CDKN1 B in HGSOC tissues and ovarian cancer cell lines were detected by qRT-PCR and their correlations were analyzed.The effects of hsa-circ-0002755 expression changes on hsa-miR-211-3p and CDKN1 B were detected by qRT-PCR and analyzed.The effect of hsa-miR-211-3p expression changes on CDKN1 B was detected by qRT-PCR.The effects of hsa-circ-0002755 expression changes on CDKN1 B protein expression were detected by Western Blot.Results:1.High-throughput sequencing was used to detect differentially expressed circRNAs in HGSOC tissues and normal ovarian tissues.A total of 710 circRNAs differentially expressed in HGSOC tissues were found,of which 354 circRNAs were up-regulated and 356 circRNAS were down-regulated.The results of qRT-PCR showed circRNA385,circRNA2058,circRNA3336,circRNA2606,hsa-circ-0002755(circRNA1656),were all down-regulated,while circRNA1312 and circRNA7474 were up-regulated,and the P value was <0.05,which was statistically significant.Hsa-circ-0002755 is the circRNA with the highest fold change by qRT-PCR and consistent with high-throughput sequencing results.The down-regulation of hsa-circ-0002755 was confirmed in both HGSOC tissues and ovarian cancer cell lines,and correlated with tumor FIGO stage.2.The results of CCK-8 assay showed that the proliferative capacity of ovarian cancer cell hsa-circ-0002755-siRNA plasmids transfected enhanced,while decreased in the ovarian cancer cell hsa-circ-0002755 over-expressed transfected,indicating that hsa-circ-0002755 inhibited ovarian cancer cell proliferation.The results of scratch test showed that the migration ability of hsa-circ-0002755-siRNA transfected ovarian cancer cell enhanced,while decreased in the ovarian cancer cell hsa-circ-0002755 over-expressed transfected,indicating that hsa-circ-0002755 inhibited ovarian cancer cell migration.The results of Transwell assay showed that the invasive ability of ovarian cancer cell hsa-circ-0002755-siRNA plasmids transfected enhanced,while decreased in the ovarian cancer cell hsa-circ-0002755 over-expressed transfected;Western Blot assay was used to detect the expression of invasion-related proteins(E-cadherin,vimentin),the results showed that in the hsa-circ-0002755-siRNA transfected ovarian cancer cells,E-cadherin protein expression was decreased,vimentin protein expression was increased;while in the hsa-circ-0002755 overexpression transfected ovarian cancer cells,the expression of E-cadherin protein was up-regulated,and the expression of vimentin protein was down-regulated;the results of Transwell assay and western blot indicated that hsa-circ-0002755 inhibited the invasive ability of ovarian cancer cell.Flow cytometry detection of apoptosis showed that the apoptosis of hsa-circ-0002755-siRNA transfected ovarian cancer cell decreased,while increased in the ovarian cancer cell hsa-circ-0002755 over-expressed transfected;Western Blot assay was used to detect the expression of apoptosis-related protein(cleaved caspase3,cleaved caspase9,bcl-2 and bax),in hsa-circ-0002755-siRNA transfected ovarian cancer cells,cleaved caspase3,cleaved caspase9,bax protein expression was down-regulated,bcl-2 protein expression was up-regulated,while in the hsa-circ-0002755 overexpression transfected ovarian cancer cell,hsa-circ-0002755 expression was up-regulated,cleaved caspase3,cleaved caspase9,bax protein expression was up-regulated,bcl-2 protein expression was down-regulated,this results showed that hsa-circ-0002755 promoted ovarian cancer cell apoptosis,which was consistent with flow cytometry results,indicating that hsa-circ-0002755 promoted the ovarian cancer cells apoptosis.3.The target binding of hsa-circ-0002755 to hsa-miR-211-3p,hsa-miR-211-3p to CDKN1 B were verified by the dual luciferase assay.Hsa-miR-211-3p was high expressed in HGSOC tissues and negatively correlated with hsa-circ-0002755 expression in HGSOC tissues.In ovarian cancer cells,hsa-circ-0002755 is down-regulated,and hsa-miR-211-3p is up-regulated,while CDKN1 B is down-regulated.In the hsa-circ-0002755 overexpression transfected ovarian cancer cell,the expression of hsa-miR-211-3p decreased,the expression of CDKN1 B increased;In the hsa-miR-211-3p-siRNA transfected ovarian cancer cell,the expression of CDKN1 B increased;In the hsa-miR211-3p overexpression transfected ovarian cancer cell,the expression of CDKN1 B decreased.Conclusion:1.High-throughput sequencing results showed that 354 differential expression of circRNA was up-regulated and 356 circRNAs was down-regulated in HGSOC tissues.2.Hsa-circ-0002755 was down-regulated in both HGSOC tissues and ovarian cancer cell lines,and was significantly associated with FIGO stage.3.Hsa-circ-0002755 inhibits the proliferation,invasion and migration of ovarian cancer cells,promotes the apoptosis of ovarian cancer cells;hsa-circ-0002755 shows a tumor suppressing effect.4.Hsa-circ-0002755 competitively inhibits hsa-miR-211-3p and targets its downstream target gene CDKN1 B,and affects the biological function of HGSOC.