Construction And Research Of Cervical Cell Line Model And Cervical Cancer Prognostic Model Based On Folic Acid Metabolism

Objective:1.To construct a cervical cell line model of folic acid deficiency and folic acid metabolism disorder,and to explore whether folic acid deficiency and folic acid metabolism disorder can induce cervical cell carcinogenesis.2.To explore the differential expression and correlation of folic acid metabolismrelated genes in cervical cancer,and to screen the genes related to prognosis to construct a prognosis model of cervical cancer patients,so as to provide reference for the prognosis prediction of cervical cancer patients and subsequent molecular biology experiments.Methods:1.Selected cervical epithelial cell line(Hcer Epic)to build a folate deficiency(FD)cell model with folic acid-free 1640 medium,and used the key enzyme inhibitors of folic acid metabolism(methotrexate,raltitrexed 5-azacytidine,5-fluorouracil)to build a cell model of folic acid metabolism disorder.ELISA assay was used to detect intracellular folic acid levels and related enzymatic indicators to confirm the successful modeling,and the following methods were used to explore the functional biological behavior changes of cell models: colony formation assay to detect cell proliferation ability,wound healing assay to detect migration ability,Transwell assay to detect invasion ability,Western blot,immunofluorescence Changes in the expression of cancer markers P16 and Ki67 were detected.2.Collected cervical cancer-related data from the public dataset,the cancer genome atlas database(TCGA),including RNA sequencing,clinical and survival data,and used R Project to analyze nine folate metabolism regulator genes(DHFR,MTHFD1,TYMS,SHMT1,MTR,MTHFR,MTRR,NNMT,SLC19A1)for differential expression analysis,correlation analysis,and survival analysis.These genes were incorporated into the least absolute shrinkage and selection(LASSO)Cox regression model to construct a cervical cancer prognostic model and displayed with a nomogram.The sensitivity and specificity of the prognostic model were assessed by receiver operating characteristic(ROC)and area under the ROC curves(AUC).Boot resampling method was used for internal validation to assess the accuracy of the prognostic model.Results:1.Cell models of the normal control group(NC group),folic acid deficiency group(FD group),and folic acid metabolism disorder group(MTX group,RTX group,5-N group,5-FU group)constructed in the previous preliminary experiments were used for cell modeling.The detection of intracellular folic acid concentration and related enzyme indicators showed that the intracellular folic acid levels in the FD group was significantly lower than that in the NC group,and enzyme indicators in MTX group,RTX group,5-N group,and 5-FU group(dihydrofolate reductase,thymidylate synthase,DNA methyltransferase and thymidine phosphorylase,respectively)were significantly lower than those in the NC group(P<0.001),proving the above cell model has been constructed successfully.Colony formation experiments showed that the number of colonies in the FD group,MTX group,RTX group,5-N group,and 5-FU group was significantly higher than that in the NC group(P<0.05).The wound healing experiment found that compared with the NC group,the wound healing ability of the FD group,the MTX group,the RTX group,the 5-N group,and the 5-FU group was significantly increased after 24 hours(all P<0.001).Western blot and immunofluorescence assays confirmed that compared with the NC group,the expressions of P16 and Ki67 in the FD group,MTX group,RTX group,5-N group,and 5-FU group were all enhanced.2.Compared with normal tissues,the mRNA levels of DHFR,MTHFD1,TYMS,SHMT1 in cervical cancer tissues were significantly increased,while MTR,MTHFR,MTRR,NNMT were significantly decreased,and no significant difference was found in SLC19A1.Nine folate metabolism-regulating genes were related to each other.TYMS and MTHFD1 were significantly correlated(r=0.44,P<0.01).The LASSO algorithm was used to screen the prognostic characteristics of 9 folate metabolismregulating genes in cervical cancer,and 4 folate metabolism-regulating genes were finally included,namely MTR,MTHFR,NNMT,SLC19A1,and the following formula was used to calculate the risk score of each cervical cancer patient: risk score=0.332895*MTR expression value+0.007054*MTHFR expression value+0.119373*NNMT expression value+0.221179*SLC19A1 expression value.Cervical cancer patients in TCGA were divided into low-risk and high-risk groups according to the median risk score.Compared with the low-risk group,the high-risk group had worse survival(HR=2.33,P=0.001).Univariate and multivariate Cox regression analyses were performed to match risk scores of genes regulating folate metabolism with clinical data for cervical cancer in TCGA.Univariate analysis showed that BMI(HR=0.951,95%CI=0.910-0.997,P=0.024),stage Ⅲ/Ⅳ(HR=1.818,95%CI=1.068-3.093,P=0.028),and risk score(HR=4.298,95%CI=2.262-8.166,P<0.001)was significantly correlated with cervical cancer survival rate.The multivariate Cox regression model showed that age(HR=1.029,95%CI=1.008-1.051,P=0.007)and risk score(HR=5.336,95%CI=2.459-11.578,P<0.001)were independent of cervical cancer prognostic factors.Finally,a prognostic model based on folate metabolism-regulated genes was constructed,which could predict the prognosis of cervical cancer patients at 1,3,and 5 years,and displayed it as a nomogram.The internal validation results showed that the model prediction results were in good agreement with the actual results.The C-index(Concordance,C-index)was 0.726(0.688-0.763),indicating that the model is reliable.Conclusion:1.In this study,an in vitro model of cervical epithelial cells with folic acid deficiency and folic acid metabolism disorder was constructed,and it was confirmed that folic acid deficiency and metabolic disorder can enhance the proliferation,migration,and invasion of cervical epithelial cells,which may lead to malignant transformation of cervical epithelial cells.2.The abnormal expression of MTR,NNMT and SLC19A1 in folic acid metabolism-related genes is significantly correlated with the survival and prognosis of cervical cancer patients.The prognostic model constructed based on genes related to folic acid metabolism may have important significance for predicting the prognosis of cervical cancer patients.