The Role And Mechanism Of TFCP2L1 In Promoting Ovarian Cancer Progression And Reducing Cisplatin Sensitivity Through FEN1

BackgroundOvarian cancer is a common cause that threatens women’s health.Due to its insidious onset,ovarian cancer patients are diagnosed in the advanced stage.The causes of ovarian cancer are diverse and not fully understood,involving multiple factors such as genetics,environment,and lifestyle.In recent years,research has revealed significant heterogeneity in ovarian cancer,including differences in its molecular characteristics,cell origin,and clinical manifestations,which poses challenges for the diagnosis and treatment of the disease.In the face of these problems,the development of targeted therapy,immunotherapy,and personalized medical strategies has provided patients with more precise and effective treatment choices.The comprehensive application of multi omics research and advanced bioinformatics technologies is driving a deeper understanding of the heterogeneity of ovarian cancer,which is expected to guide the optimization of future treatment plans and bring hope to patients.TFCP2L1(transcription factor CP2 Like 1)is a transcription factor that plays an important role in cell development and differentiation.In recent years,with the deepening of molecular biology and oncology research,the role of TFCP2L1 in tumor occurrence and progression has begun to receive widespread attention.Research has found that TFCP2L1 can affect the proliferation,migration and invasion of tumor cells by regulating the expression of specific genes,and is closely related to the development of bladder cancer,renal medullary carcinoma,renal clear cell carcinoma and other malignant tumors.It suggests that it may serve as a potential biomarker and therapeutic target for malignant tumors.At present,there is no research to elucidate the role and specific mechanism of TFCP2L1 in ovarian cancer.Therefore,in this study,we determined through joint analysis of TCGA and GTEx databases that TFCP2L1 is highly expressed in ovarian cancer and is associated with poor prognosis in ovarian cancer patients.Subsequently,the specific effects and possible mechanisms of action on ovarian cancer will be explored through the detection of clinically collected ovarian cancer samples and a series of in vitro and in vivo experiments.Methods(1)Through the joint analysis of TCGA and GTEx databases,the differential expression of TFCP2L1 in ovarian cancer tissue and normal ovarian tissue was screened.The expression of TFCP2L1 in ovarian cancer was validated using the GEO database ovarian cancer sample dataset.Analyze the impact of TFCP2L1 expression on the prognosis of ovarian cancer patients based on clinical information from the TCGA database.Analyze the expression of TFCP2L1 in different types of tumors and validation of its impact on the prognosis of ovarian cancer patients through GEPIA database.(2)Western blot and immunohistochemical staining techniques were used to detect the expression of TFCP2L1 in ovarian cancer cell lines and clinical samples collected from ovarian cancer patients.The correlation between TFCP2L1 expression level and clinical pathological characteristics of ovarian cancer was determined through chi square analysis based on patient clinical information.The relationship between TFCP2L1 expression level and prognosis of ovarian cancer patients was determined through COX univariate and multivariate analysis.(3)Construct ovarian cancer A2780 and SKOV3 cells with stable knockdown/overexpression of TFCP2L1,and detect the effect of TFCP2L1knockdown/overexpression on ovarian cancer cell proliferation through CCK-8 and clone formation experiments.Western blot was used to detect the effect of TFCP2L1knockdown/overexpression on the expression of apoptosis related proteins Bax and Bcl2.(4)Transwell assays explore the effect of TFCP2L1 knockdown/overexpression on the migration and invasion ability of ovarian cancer cells.Western blot was used to detect the effects of TFCP2L1 knockdown/overexpression on the transfer related proteins E-cadherin and N-cadherin.(5)Explore the effects of TFCP2L1 expression on the proliferation and migration of ovarian cancer by constructing a mice subcutaneous transplant tumor model of A2780 cell with TFCP2L1 knockdown.Detect the proliferation related protein Ki67 and metastasis related proteins E-cadherin and N-cadherin expression in tumor tissues in vivo through immunohistochemical staining(6)Analyze the expression of TFCP2L1 in cisplatin resistant ovarian cancer cells datasets through the GEO database.Detect the effect of TFCP2L1knockdown/overexpression on cisplatin sensitivity in ovarian cancer cells through CCK-8 and flow cytometry.(7)By conducting transcriptome sequencing and pathway analysis on SKOV3 cells with TFCP2L1 knockdown,the pathway and target of TFCP2L1 affecting malignant behavior of ovarian cancer were identified.GEPIA database searches for the expression of potential downstream targets of TFCP2L1 in ovarian cancer.Correlation analysis of TCGA database to determine the expression correlation between TFCP2L1 and its potential downstream targets in ovarian cancer tissue.(8)Western blot and immunofluorescence detection to explore the effect of TFCP2L1 on FEN1 and DNA damage marker γ-H2 Ax expression.Immunofluorescence detection of DNA damage marker γ-H2 Ax levels in TFCP2L1knockdown/overexpression ovarian cancer cells treated with cisplatin.Immunohistochemical staining was used to detect DNA damage levels in TFCP2L1 knockdown tumor tissues.(9)Exploring the effects of overexpression of FEN1 on ovarian cancer proliferation,migration,invasion,and cisplatin sensitivity in TFCP2L1 knockdown ovarian cancer cells through cloning,transwell,flow cytometry,immunofluorescence,and western blot analysis.Results(1)The joint analysis of TCGA and GTEx databases showed that TFCP2L1 was highly expressed in ovarian cancer tissue,and the GEO ovarian cancer tissue dataset confirmed the upregulation of TFCP2L1 expression in ovarian cancer tissue.The TCGA survival analysis results showed that high expression of TFCP2L1 was associated with poor prognosis in ovarian cancer patients,and the ROC curve results showed that TFCP2L1 may become a potential biomarker for predicting ovarian cancer.GEPIA database search results showed that TFCP2L1 was highly expressed in ovarian cancer,colon cancer,pancreatic cancer,rectal adenocarcinoma,testicular germ cell tumor and endometrial cancer,while it was low expressed in invasive breast cancer,renal clear cell carcinoma,renal papillary cell carcinoma,prostate adenocarcinoma,skin melanoma,gastric adenocarcinoma and thyroid cancer.GEPIA prognostic analysis results also showed that high expression of TFCP2L1 was associated with poor prognosis of ovarian cancer patients.(2)The western blot results of normal ovarian cells and ovarian cancer cell lines,as well as normal ovarian tissue and ovarian cancer tissue,showed that TFCP2L1 was highly expressed in ovarian cancer cell lines and ovarian cancer tissues.The immunohistochemical staining results of normal ovarian tissue and ovarian cancer tissue sections collected clinically showed that TFCP2L1 was highly expressed in ovarian cancer tissue.The chi square analysis combined with the clinical and pathological characteristics of ovarian cancer patients showed that the expression level of TFCP2L1 was associated with FIGO stage and platinum resistance in ovarian cancer patients.The results of COX univariate and multivariate analysis showed that high expression of TFCP2L1,high FIGO stage,and cisplatin resistance were independent risk factors affecting the prognosis of ovarian cancer patients.(3)Knocking down TFCP2L1 can effectively inhibit the proliferation of ovarian cancer cells,upregulate the expression of pro-apoptotic protein Bax and downregulate the expression of anti-apoptotic protein Bcl2 in ovarian cancer cells,suggesting that knocking down TFCP2L1 has a pro-apoptotic effect on ovarian cancer cells.Overexpression of TFCP2L1 can promote the proliferation of ovarian cancer cells,downregulate the expression of pro-apoptotic protein Bax and upregulate the expression of anti-apoptotic protein Bcl2 in ovarian cancer cells,suggesting that overexpression of TFCP2L1 has an anti-apoptotic effect on ovarian cancer cells.(4)Knocking down TFCP2L1 can inhibit the migration and invasion of ovarian cancer cells,upregulate the expression of anti-metastatic protein E-cadherin and downregulate the expression of pro-metastatic protein N-cadherin in ovarian cancer cells.On the contrary,overexpression of TFCP2L1 can promote the metastasis and invasion of ovarian cancer cells,downregulate the expression of anti-metastatic protein E-cadherin and upregulate the expression of pro-metastatic protein N-cadherin in ovarian cancer cells.(5)Tumor formation experiments in mice have shown that knocking down TFCP2L1 slows down the growth rate of ovarian cancer cells and reduces tumor weight.The immunohistochemical staining results of tumor tissue showed that the TFCP2L1 knockdown group showed a decrease in Ki67 protein expression,an increase in E-cadherin protein expression,and a decrease in N-cadherin protein levels in tumor tissue.This also proves that TFCP2L1 knockdown can effectively inhibit tumor cell proliferation and migration in vivo.(6)The analysis results of the GEO dataset showed that TFCP2L1 was highly expressed in A2780,SKOV3,and PEA ovarian cancer cisplatin resistant cells.Inhibiting TFCP2L1 can enhance the sensitivity of ovarian cancer cells to cisplatin,while overexpression of TFCP2L1 can reduce the sensitivity of ovarian cancer cells to cisplatin.(7)The transcriptome sequencing results of SKOV3 cells with TFCP2L1 knockdown showed that the differentially expressed genes induced by TFCP2L1 knockdown were significantly enriched in pathways such as cell stress,cancer,cell growth and death,DNA repair,and DNA replication.Ultimately,it was determined that TFCP2L1 may participate in DNA damage repair pathways by regulating the FEN1 gene.The GEPIA database retrieval results showed that FEN1 is also highly expressed in ovarian cancer tissues.The gene expression correlation analysis based on the TCGA database showed a positive correlation between TFCP2L1 and FEN1 expression in ovarian cancer.(8)Western blot analysis showed that the expression of FEN1 decreased and DNA damage marker γ-H2 Ax increased in ovarian cancer cells with TFCP2L1 knockdown,while the expression of FEN1 increased and γ-H2 Ax protein levels decreased in ovarian cancer cells with TFCP2L1 overexpression.The results of immunofluorescence and immunohistochemistry showed that ovarian cancer cells with TFCP2L1 knockdown had higher levels of DNA damage,and the DNA damage level in cells treated with cisplatin is also much higher than that in the control group.The DNA damage level in ovarian cancer cells overexpressing TFCP2L1 is lower,and the DNA damage levels in cells treated with cisplatin is also much lower than that in the control group.(9)Overexpression of FEN1 in TFCP2L1 knockdown ovarian cancer cells can reverse the inhibitory effects of TFCP2L1 knockdown on proliferation,migration and invasion of ovarian cancer cells,reverse the cisplatin sensitization effect caused by TFCP2L1 knockdown.At the same time,overexpression of FEN1 can reduce the levels of DNA damage in TFCP2L1 knockdown cells and inhibit cisplatin induced DNA damage.Conclusion(1)TFCP2L1 is highly expressed in ovarian cancer and is associated with poor prognosis in ovarian cancer patients.The expression of TFCP2L1 in ovarian cancer patients is associated with FIGO staging and cisplatin resistance.High expression of TFCP2L1,high FIGO staging,and cisplatin resistance are independent risk factors affecting the prognosis of ovarian cancer.(2)TFCP2L can promote the proliferation,metastasis,and invasion of ovarian cancer cells,and reduce their sensitivity to cisplatin.(3)TFCP2L1 can participate in the DNA damage repair pathway by inducing the expression of FEN1,inducing proliferation,metastasis,invasion,and reducing the sensitivity of ovarian cancer cells to cisplatin.