The Effects And Mechanisms Of MicroRNA-10a-5p Targeting GATA6 In Ovarian Cancer,and Its Impact On Shikonin-Induced Apoptosis Of Ovarian Cancer

Chapter 1:The Impact of GATA6 Regulated by miRNA on the Prognosis of Ovarian CancerObjective:To investigate the expression of transcription factor GATA binding protein 6(GATA6)in ovarian cancer and its impact on the prognosis of ovarian cancer,as well as to clarify the microRN A regulating GATA6.Methods:(1)The expression levels of GATA6 protein and mRNA in patient tissues were detected by Western blot(WB)and quantitative real-time PCR(qRT-PCR),respectively;(2)The Cancer Genome Atlas Program(TCGA)database was used to analyze the expression levels of GATA6 and its impact on overall survival(OS)and progression-free survival(PFS)of ovarian cancer patients;(3)Target miRNAs regulating GATA6 were predicted using online databases and confirmed by dual-luciferase assay.Results:(1)The expression levels of GATA6 protein and mRNA in ovarian cancer tissues were lower than those in normal ovarian tissues;(2)TCGA database further verified the lower expression levels of GATA6 in ovarian cancer than in normal ovarian tissues;(3)Multivariate regression analysis suggested that GATA6 and tumor stage were independent risk factors for the prognosis of ovarian cancer;(4)The Kaplan-Meier plotter database revealed that low expression of GATA6 was associated with better prognosis in ovarian cancer patients,and was independent of tumor stage,tumor differentiation,histological type,and p53 mutation status;(5)The database predicted that miR-10a-5p regulated GATA6,and the dual-luciferase assay further confirmed the targeting relationship between them.Conclusion:(1)The expression level of GATA6 is lower in ovarian cancer than in normal ovarian tissues;(2)GATA6 and tumor stage are independent risk factors for ovarian cancer patients,and patients with high expression of GATA6 have a poor prognosis;(3)miR-10a-5p targets GATA6.Chapter 2:The Impact and Mechanism of miR-10a-5p Targeting GATA6 on the Malignant Phenotype of Ovarian Cancer Cells Mediated by LentivirusObjective:To investigate the impact of miR-10a-5p targeting regulation of GATA6 on the phenotype of ovarian cancer cells,and to further clarify the related mechanisms.Methods:(1)Lentiviral vectors overexpressing miR-10a-5p and GATA6 were constructed to regulate the expression levels of miR-10a-5p and GATA6 in ovarian cancer cells SKOV3 and OVCAR3;(2)qRT-PCR was used to detect the expression levels of miR10a-5p and GATA6 in ovarian cancer cells,and Western blot was used to detect the expression levels of GATA6,N-Cadherin,E-Cadherin,p-AktS473,Akt,p53,GAPDH,and MUC1 proteins;(3)The impact of GATA6 on tumor cell proliferation,tumor formation ability,migration,and invasion was studied using CCK-8,colony formation,scratch assay,and transwell assay;(4)A nude mouse xenograft experiment was performed using the constructed SKOV3-related cell lines to detect the impact of miR-10a-5p silencing GATA6 on tumor growth;(5)The morphology of tumor cells was observed by HE staining,and the expression levels of GATA6,Ki-67,N-Cadherin,E-Cadherin,and p-AktS473 in the xenograft tumor were detected by immunohistochemistry,while the expression levels of GATA6,N-Cadherin,E-Cadherin,pAktS473,Akt,p53,and GAPDH were detected by Western blot.Results:(1)The expression level of miR-10a-5p was upregulated,and the mRNA expression level of GATA6 was downregulated in ovarian cancer cells after lentivirus transfection;(2)Upregulation of miR-10a-5p inhibited ovarian cancer cell proliferation,tumor formation ability,migration,and invasion through targeting GATA6,while restoration of GATA6 expression increased cell proliferation,tumor formation ability,migration,and invasion.However,there was no statistically significant difference in migration and invasion ability in OVCAR3 cells.(3)miR-10a-5p downregulated the expression level of GATA6,decreased the expression levels of Akt and MUC1 in SKOV3 cells,and inhibited epithelialmesenchymal transition(EMT).In OVCAR3 cells,the Akt pathway was also inhibited,but EMT was activated,and the expression level of p53 protein slightly decreased.After restoration of GATA6 expression,the Akt pathway and EMT were restored in both cell lines.(4)In vivo experiments showed that the growth of tumors in mice with upregulated miR-1Oa5p expression was slow,and the expression levels of related pathway proteins were consistent with those in SKOV3 cells in vitro.Conclusion:In vitro and in vivo experiments demonstrated that the downregulation of GATA6 mediated by miR-10a-5p inhibited the activation of the Akt pathway and the expression of MUC1 in ovarian cancer cells with loss of p53,which may inhibit EMT.However,in p53-mutated cell lines,although the Akt pathway was also inhibited,the performance in EMT was opposite.Chapter 3:The Role of miR-10a-5p in Shikonin-Induced Apoptosis of Ovarian Cancer CellsObjective:This study aims to elucidate the role of miR-10a-5p in shikonin resistance in ovanan cancer.Methods:(1)Analysis of the relationship between GATA6 expression levels in ovarian cancer and the sensitivity of ovarian cancer to shikonin based on databases;(2)Use of CCK8 assay to detect the half-maximal inhibitory concentration(IC50)of shikonin in SKOV3 cells;(3)Flow cytometry to detect the apoptotic ability of ovarian cancer cells;(4)Western blot to detect the expression levels of apoptotic proteins in ovarian cancer cells.Results:(1)Database analysis revealed that the decrease in GATA6 expression can increase the sensitivity of ovarian cancer to shikonin;(2)Upregulation of miR-10a-5p can promote apoptosis of ovarian cancer cells,significantly increasing the sensitivity of SKOV3 cells to shikonin;(3)The combination of shikonin and miR-10a-5p can significantly increase the expression levels of apoptotic proteins in ovarian cancer cells.Conclusion:MiR-10a-5p increases the sensitivity of ovarian cancer cells to shikonin and promotes apoptosis by inhibiting GATA6 expression in ovarian cancer.