Prognostic Model Of Ovarian Cancer Based On Alternative Splicing Events And The Function Of LUC7L3 In Ovarian Cancer

Part 1 Establishment of ovarian cancer prognosis model based on 11-AS events and its relationship with immune microenvironmentBackground:Alternative splicing(AS)events play an important role in the occurrence and development of tumors.They are also closely related to the immune microenvironment.At the present moment,the role of AS events in ovarian cancer(OC)remains to be further explored.The purpose of this study was to explore the value of AS events in the prognosis of patients with ovarian cancer and its relationship with immune microenvironment.Methods:We downloaded the transcriptome sequencing data,gene expression data and clinical data of 544 patients from the Cancer Genome Atlas(TCGA)database.The percent-splice-in(PSI)of each AS event was obtained through TCGA Splice Seq software,which was matched with expression data and clinical data.Then Univariate and Multivariate Cox regression analysis was used to determine the AS events related to survival and establish the prognosis model.Based on the prognostic model,we analyzed the differences in the distribution of immune cells and their different responses to cytotoxic T lymphocyte associated antigen 4(CTLA-4)and programmed cell death protein 1(PD-1)blockers in low-risk and high-risk groups of OC patients.In addition,Spearman test was used to screen the splicing factor(SF)related to the expression of AS events,and bioinformatics tools were used to establish the regulatory network of SF-AS events.Finally,the functional enrichment of SF genes in the regulatory network was analyzed through gene ontology(GO)and Kyoto Encyclopedia of genes and genomes(KEGG).Results:We screened 1472 AS events related to OC survival,of which ES was the most important type(46.94%).Furthermore,11 AS events screened by lasso regression and multivariate regression are good prognostic indicators of OC,and a prognostic model based on these 11 AS events was constructed.The patients were divided into high-risk group and low-risk group,with 192 cases in each group.The median risk score was 0.9137.K-M survival analysis showed that the prognostic risk model had important value in distinguishing the prognosis of patients(P=9.485e-03),and the area under the curve(AUC)was calculated by drawing the ROC curve.The results showed that the AUC of 11 AS events was 0.733.Univariate and multivariate Cox regression analysis showed that age and risk score could predict the survival rate of OC and were independent prognostic factors.When studying the expression of immune cells in highrisk and low-risk,it was found that the expression of 10 immune cells in low-risk patients was up-regulated,and activated B cells,natural killer T cells,natural killer cells and regulatory T cells were related to the survival of OC.The response of patients in the low-risk group to CTLA 4 and PD-1 blockers was higher than that in the high-risk group.In addition,SF-AS related networks also revealed several central SF genes,including DDX39B、PNN、LUC7L3、ZC3H4、SRSF11,etc.GO analysis showed that "mRNA splicing through spliceosomes","regulation of RNA splicing","mRNA processing","RNA processing" and "regulation of selective mRNA splicing through spliceosomes" were the five most important biological processes;"Nucleoplasm","membrane","catalytic second step spliceosome" and "nuclear spot" are the four most important cellular components;In addition,"poly(a)RNA binding","nucleotide binding" and "ATP binding" are the three most important molecular functions.KEGG analysis revealed four significant enrichment pathways,including "spliceosome","RNA transport","mRNA monitoring pathway" and "RNA degradation".Conclusion:This study constructed a prognostic model based on 11 AS events,which has high predictive value for the prognosis of patients.The SF-AS regulatory network was established to analyze the relationship between the prognosis model and the immune microenvironment,which provided new ideas and potential therapeutic targets for exploring the potential mechanism of the occurrence and development of ovarian cancer.Part 2 Study on the function of LUC7L3 in ovarian cancerBackground:Some studies have shown that LUC7 like 3 pre-mRNA splicing factor(LUC7L3)plays an important role in some cancers and diseases,but its specific mechanism in ovarian cancer is not clear.LUC7L3 is one of the key SF genes found after the establishment of SF-AS regulatory network in OC.This study aims to explore the effect of abnormal expression of LUC7L3 on the biological function of ovarian cancer cells.Methods:We used RT-qPCR and Western blot to detect the differences of LUC7L3 mRNA and protein expression between ovarian cancer and normal ovarian epithelial cell lines.SKOV3 and OV8 cells were selected as further research objects.SKOV3 and OV8 cell lines were transfected with lentivirus LUC7L3 shRNA to knock down LUC7L3.The experiment was divided into three groups:control(Ctl),lentivirus LUC7L3 shRNAl and lentivirus LUC7L3 shRNA2.We used RT-qPCR and Western blot to confirm the knockdown efficiency of LUC7L3 shRNA.Then,the effect of LUC7L3 on the proliferation of OC cells was verified by MTT and plate cloning test,and we used scratch test and Transwell migration test to analyze the migration of OC cells;vivo experiments in mice were conducted to verify the effect of LUC7L3 on tumor growth.RESULTS:RT-qPCR and Western blot results showed that compared with normal ovarian epithelial cell line(IOSE),the expression of LUC7L3 was highly expressed in ovarian cancer cells(HEY,SKOV3,A2780,OV8)(P<0.05),and the expression of LUC7L3 was the highest in SKOV3 and OV8 cells.Therefore,SKOV3 and OV8 cells were selected as further research objects.We used RT-qPCR and Western blot to detect the expression level of LUC7L3 in cells transfected with lentivirus luc713 shRNA.The experimental results showed that compared with Ctl group,LUC7L3 shRNAl and LUC7L3 shRNA2 groups were significantly down regulated(P<0.05).The knockdown efficiency was 74%and 73%in SKOV3 cells and 79%and 64%in OV8 cells respectively.MTT results showed that compared with Ctl,the proliferation ability of LUC7L3 shRNAl and LUC7L3 shRNA2 groups decreased(P<0.05);Plate cloning experiment showed that the colony forming activity of LUC7L3 shRNA1 and LUC7L3 shRNA2 cells was inhibited(P<0.05);The results of cell scratch test showed that the relative migration area of cells in LUC7L3 shRNA1 and LUC7L3 shRNA2 groups decreased significantly(P<0.05);The results of Transwell experiment showed that the migration ability of ovarian cancer cells was significantly lower than that of Ctl group(P<0.05).In vivo experiments showed that the tumor growth of mice in LUC7L3 shRNA2 group was significantly slower than that of Ctl.At the same time,the down-regulation of LUC7L3 in vivo also reduced the tumor weight,and the difference was also statistically significant(P<0.05).Conclusion:In vitro and in vivo experiments on cells and animals have confirmed that LUC7L3 can promote the proliferation and migration of ovarian cancer cells,and LUC7L3 can play a role in the carcinogenesis and development of ovarian cancer,which provides new ideas and data support for the exploration of potential molecular mechanisms and targeted therapy of ovarian cancer.