Proliferation And Invasion Effect And Mechanism Of Long Non-Coding RNA CCAT1 In Ovarian Cancer Cells

Objection: To explore the expression of CCAT1(colon-related transcription factor-1)in epithelial ovarian cancer tissues and cells,and the biological function of CCAT1 in ovarian cancer cells,to investigate the regulatory mechanisms of CCAT1 on the proliferation,invasion,migration,in order to provide a theoretical basis for the clinical diagnosis and treatment of ovarian cancer.Method: Part 1: Study lncRNA CCAT1 expression in epithelial ovarian cancer and its relationship with clinical features: 1)collected ovarian epithelial carcinoma tissue samples and normal ovarian tissue specimens of 40 cases,who have the surgeries in the Second Hospital of Tianjin Medical University.The expression of CCAT1 in epithelial ovarian cancer tissue and normal ovarian tissue was detected by qRT-PCR.According to the average expression level of CCAT1 in ovarian cancer,the ≥ 2.5-fold average level was defined as high expression,and <2.5 times was defined as low expression,then analyze the results of the clinical and pathological correlation with the patient.2)A2780,SKOV3,HO8910 and ES-2 ovarian cancer cell lines were cultured.The expression of lncRNA CCAT1 in each cell line was detected by Realtime PCR,and the cell line with the highest expression was screened out.Part2:Determined the effect of CCAT1 on ovarian cancer cells.We screened out the cell lines with the highest level of CCAT1 expression and targeted the expression of CCAT1 in the silenced cell line by siRNA interference design.We designed 3 siRNA fragments that could specifically inhibit the expression of CCAT1,and detected the expression of CCAT1 by RT-PCR.The cell proliferation was detected by CCK8 assay.The effect of CCAT1 down-regulation on the clonality of ovarian cancer cells was detected by plate clone formation assay.The migration and invasion ability of si-CATT1 and its negative control cells were detected by Transwell assay.Apoptosis level of si-CATT1 and its negative control cells were detected by flow cytometry.Part3: To investigate the regulatory mechanism of CCAT1 on the proliferation,invasion and migration of ovarian cancer cells: The expression of let-7,miR-218-5p,Bmi1,c-myc,E-cadherin,N-cadherin in CCAT1 siRNA knockdown cells was detected by Realtime PCR and Western blotting.Result: Part 1: The expression of lncRNA CCAT1 in epithelial ovarian cancer tissues was significantly increased,which was significantly correlated with the clinicopathological features of patients.The overexpression of lncRNA CCAT1 was positively correlated with the FIGO staging,histological grade and distant metastasis of ovarian cancer.The expression of lncRNA CCAT1 in ovarian cancer cell lines A2780,SKOV3,HO8910,ES-2 was increased,and the highest expression is in SKOV3.Part2: Three CCAT1-siRNAs were successfully constructed and transfected into SKOV3 cell line respectively to screen out the most down-regulated si-RNA.The CCK8 and plate cloning experiments confirmed that the CCAT1 expression was down-regulated by si-RNA and the SKOV3 cell’s proliferation and cloning ability was reduced.Transwell experiments confirmed that after down-regulated the expression of CCAT1 by si-RNA,SKOV3 cell’s migration and invasion were decreased;Flow cytometry determined that CCAT1 inhibite the apoptosis of SKOV3 cell.The above results show that CCAT1 can increase ovarian cancer cell proliferation by inhibiting apoptosis in ovarian cancer cells.Part3: The qRT-PCR results showed that compared with the negative control group,the expression levels of let-7 and miRNA-218-5p were significantly increased in si-CCAT1 group;in the si-CCAT1 group,both qRT-PCR and Western blot showed that the expression levels of c-myc and Bim1 were significantly reduced,E-cadherin levels were elevated and N-cadherin levels were significantly reduced(all P <0.05).Conclusion: Our study showed that the expression of lncRNA CCAT1 was increased in epithelial ovarian cancer tissues and cell lines,and has a clear correlation with the clinical pathological stage of patients;CCAT1 can promote ovarian cancer cell proliferation,migration and invasion,and inhibit ovarian cancer cell apoptosis;the mechanism may be through inhibiting the expression of let-7 and miR-218-5p,up-regulating c-myc and Bim1,regulating the levels of E-cadherin and N-cadherin,and promoting EMT.To further investigate the regulatory mechanism of CCAT1 in ovarian cancer will help elucidate the pathogenesis of ovarian cancer and provide a new target for the treatment of ovarian cancer.