The Expression Of POLQ In Endometrial Carcinoma And Its Clinical Significance

BackgroundEndometrial Cancer(EC)is an epithelial malignancy that occurs in the endometrium.With an increasing incidence worldwide,it seriously endangers women’s reproductive health.The prognosis of advanced and recurrent endometrial cancer is poor,which is closely related to the insensitivity of advanced and recurrent endometrial cancer to conventional radiotherapy and chemotherapy.Through TCGA database,we found significant mutation of POLQ gene in EC patients,which was closely related to prognosis.POLQ is involved in DNA damage repair of higher eukaryotic cells and plays an important role in maintaining gene stability and preventing gene mutation.When the expression of POLQ is deficient,the ability of cell base excise and repair is reduced,which makes cells more sensitive to external physical and chemical DNA damage factors.This suggests that silencing the POLQ gene may enhance the killing effect of radiotherapy and chemotherapy on tumor cells.Research on POLQ gene in EC has rarely been reported.In this study,the expression of POLQ in EC and its relationship with the sensitivity of endometrial cancer cells to PARP1 inhibitor(olaparib)were analyzed,so as to provide a theoretical basis for the exploration of EC prognostic indicators and targeted therapy,thus helping to improve the prognosis of EC patients.ObjectiveTo investigate and verify the differential expression of POLQ in Endometrial Cancer(EC)and normal Endometrial tissues,and to analyze the relationship between POLQ and clinicopathological parameters of Endometrial Cancer and its prognostic value.To investigate whether silencing of POLQ protein expression in endometrial cancer cells affects their sensitivity to PARP1 inhibitor(olaparib)and cancer cell biological function.MethodsThis experiment is divided into three parts:1.Download POLQ gene expression profile and clinical data from TCGA database.The relationship between POLQ expression and clinicopathological factors in cancer and paracancerous tissues was compared by using R software.Kaplan-Meier method was used to analyze the correlation between POLQ expression and prognosis.GSEA enrichment analysis was used to predict the signaling pathways involved in POLQ in endometrial cancer.2.Immunohistochemical staining was performed on 127 cases of endometrial cancer patients and 22 cases of normal endometrial paraffin sections.The sections were double-blind read and scored by two experienced pathologists.Kaplan-Meier method and multivariate Cox method were used to analyze the correlation between POLQ expression and prognosis.3.Western blot was used to detect the content of target protein in endometrial cancer cells,and siRNA was transfected into endometrial cancer cells to establish a silenced POLQ cell model.Different concentrations of PARP1 inhibitors(olaparib)were applied to endometrial cancer cell lines,and CCK8 was used to detect cell viability after drug treatment.IC50 values of endometrial cancer cell lines after POLQ knockdown were compared.The proliferation,apoptosis and migration of endometrial cancer cells were detected by clone formation,flow cytometry and scratch assay.The effect of POLQ on DNA damage of endometrial cancer cells was detected by immunofluorescence assay.Results1.The expression of POLQ was significantly up-regulated in endometrial cancer tissue samples(P < 0.05);The expression of POLQ was correlated with grade,and the overall survival(OS)of patients with high POLQ expression was significantly lower than that of patients with low POLQ expression(P < 0.05).The enrichment analysis showed that POLQ gene was mainly concentrated in the base resection repair pathway,homologous recombination pathway,P53 pathway,etc.2.The expression of POLQ was significantly up-regulated in endometrial cancer tissue samples(P < 0.05);The expression of POLQ was correlated with stage and lymph node metastasis,and the disease-free survival(DFS)and overall survival(OS)of patients with high POLQ expression were significantly lower than those with low POLQ expression(P < 0.05).Multivariate Cox analysis suggested that POLQ could be an independent prognostic factor for EC.3.The expression levels of POLQ in HEC-1-A and AN3 CA cell lines were relatively high,while the protein levels of POLQ in RL95-2,KLE and Ishikawa cell lines were relatively low.CCK8 detected the cell viability of each cell line and olaparib treated cells and calculated the IC50.The IC50 values of HEC-1-A and AN3 CA cells were significantly decreased and theγ H2 AX level was significantly increased after the knockdown of POLQ with siRNA.The results of clone formation experiment showed that the number of bacteria decreased significantly after POLQ knockdown,the flow detection results showed that the level of cell apoptosis increased after POLQ knockdown,and the scratch test results showed that the scratch healing rate decreased after POLQ knockdown.ConclusionPOLQ is highly expressed in endometrial cancer cells and is closely related to prognostic factors such as grade,stage,lymphatic metastasis,etc.POLQ can be used as an independent factor to predict poor prognosis of endometrial cancer patients.POLQ knockdown increased the sensitivity of endometrial cancer cells to olaparib,aggravated DNA damage of endometrial cancer cells,significantly inhibited the proliferation and migration of endometrial cancer cells after olaparib treatment,and significantly promoted the apoptosis of endometrial cancer cells.