Preliminary Study On The Role Of M6A Modification And Its Methyltransferase METTL14 In Ovarian Cancer

Ovarian cancer is one of the common and has the highest fatality rate among gynecological malignancies.Most ovarian cancer cases are diagnosed at an advanced stage due to a lack of overt symptoms.Complete remission can occur for most patients following their initial treatment but there is still a high rate of reoccurrence,with about 75% of patients will relapse within 3 years and are prone to chemotherapy resistance,then the 5-year relative survival rate is only about40%.The occurrence and development of ovarian cancer is an extremely complex process involving multiple factors.It is important clinical significance to study deeply of its pathogenic mechanism and finding effective therapeutic targets.N6-methyladenosine(m6A)modification is the most abundant RNA methylation modification in eukaryotes.It is a dynamic and reversible regulatory process.Its regulatory factors are shared by methyltransferase,demethylase and specific protein which can recognize the modification and realize the regulation of RNA downstream genes.m6 A modification affects various biological effects of cells by regulating the splicing,nuclear export,degradation,and translation of messenger RNA(m RNA).Abnormal expression of m6 A regulatory factors is closely related to the occurrence and development of a variety of tumors.At present,there are still few studies on m6 A modification in ovarian cancer,and METTL14,which is one of the core members of m6 A methyltransferase,has not been reported.First,this study explored the expression of METTL14 in ovarian cancer and its influence on clinicopathological factors and prognosis.Secondly,we analyzed the effect of METTL14 on the level of m6 A modification of ovarian cancer cells,and studied its effect on the proliferation,invasion and migration of ovarian cancer cells,and mechanism.In addition,we used bioinformatics methods to analyze the expression of multiple m6 A regulatory factors in ovarian cancer from a multiomics perspective,and constructed a prognostic model based on m6 A regulatory factors expression.This study provided a preliminary study of the role of m6 A modification and its methyltransferase METTL14 in ovarian cancer,and provided a new theoretical basis for the molecular mechanism of the occurrence and development of ovarian cancer.Part I The expression and clinical significance of m6 A methyltransferase METTL14 in ovarian cancerObjective: To study the expression of METL14 in ovarian cancer and normal ovarian tissues,and analyze the relationship between the expression of METL14 in ovarian cancer and clinicopathological factors and prognosis.Methods: Real-time fluorescent quantitative PCR(q RT-PCR),Western blot,and immunohistochemical methods were used to analyze the expression difference of METTL14 in normal ovarian tissues and ovarian cancer tissues.Then analyzed the relationship between METTL14 expression in ovarian cancer and clinicopathological factors and prognosis.Results: There was no significant difference in the expression of METTL14 m RNA in normal ovarian tissue and ovarian cancer tissue(P>0.05),but the expression of METTL14 protein in ovarian cancer tissue was higher than that in normal ovarian tissue,and the difference was significant(P<0.05).Patients with high METTL14 expression in ovarian cancer compared with those with low METTL14 expression,had a later FIGO stage,higher rates of lymph node metastasis,abdominal cavity metastasis,abdominal effusion,and worse overall survival rate(P<0.05).Conclusion: The expression of METTL14 protein in ovarian cancer tissues had a up-regulation trend.The high expression of METTL14 is related to the poor prognosis of ovarian cancer patients.Part II The effect of METTL14 expression on the proliferation,invasion and migration of ovarian cancer cellsObjective: To study the effect of METTL14 on the level of m6 A modification of ovarian cancer cells;to explore the effect of METTL14 on the proliferation,invasion and migration of ovarian cancer cells and preliminary study of its mechanism.Methods: Lentiviral transfection technology was used to transfect human ovarian cancer cell lines A2780 and SKOV3 to construct an ovarian cancer cell line with stable up-regulation of METTL14;small interfering RNA(si RNA)was used to down-regulate the expression of METTL14 in ovarian cancer cells.The LC-MS/MS method was used to detect the level of m6 A modification in ovarian cancer cells after up/down-regulation of METTL14.CCK8,scratch woundhealing test,and Transwell test were used to detect the effect of up /downregulation of METTL14 expression on the proliferation,invasion and migration of ovarian cancer cells.Then used Western-blot to detect the expression of key proteins of epithelial-mesenchymal transition(EMT)and PI3K-AKT pathway in ovarian cancer cells.Results: Up/down-regulating the expression of METTL14 could affect the m6 A modification level of ovarian cancer cells.The proliferation,invasion and migration of ovarian cancer cells were promoted then up-regulating the expression of METTL14,whereas were inhibited then down-regulating METTL14.Up/down-regulating the expression of METTL14 could affect the expression of key proteins of EMT in ovarian cancer cells and the expression of key phosphorylated proteins in the PI3K-AKT signaling pathway.Conclusion: METTL14 can regulate the level of m6 A modification in ovarian cancer cells and affect the ability of proliferation,invasion and migration of ovarian cancer cells.The mechanism may be achieved by regulating EMT and PI3K-AKT pathways.Part Ⅲ: Clinical significance of a prognostic model based on m6A regulatory factors in ovarian cancerObjective: To analyze the expression of multiple m6 A regulatory factors in ovarian cancer and their influence on the prognosis of ovarian cancer from a multi-omics perspective;construct a risk model based on m6 A regulatory factors and evaluate the prognostic value of the model.Methods: Downloaded ovarian cancer transcriptome,copy number variation(CNV),single nucleotide variation(SNV)data and corresponding clinical data from the TCGA database,and used Perl and R language to organize,analyze and visualize the data.Firstly,Analyzed the copy number variation and mutation of21 m6 A regulatory factors in ovarian cancer,and explored the protein interaction network and m RNA expression correlation network of the m6 A regulatory factors.Secondly,Consensus clustering analysis was used to cluster the ovarian cancer patients into different subtypes base on the expression of m6 A regulatory factors,then analyzed the differences in clinical features and prognosis of different subtypes.Lastly,Lasso and stepwise regression were used to screen variables,then built a Cox regression model,and evaluated the model’s prognostic value through univariate and multivariate analysis.And further analyzed the differences in gene enrichment pathways,tumor microenvironment,and tumor mutation burden between high-risk and low-risk groups.Results: In ovarian cancer,the copy number variation frequency of m6 A regulatory factor was relatively high,which copy number gain more common than copy number loss,and the total mutation rate was relatively low.METTL14,METTL3,and WTAP were the core proteins in the protein interaction network,and the expression of m6 A regulatory factors in ovarian cancer were generally positively correlated.Through cluster analysis,patients with ovarian cancer could be divided into two subtypes.There was heterogeneity between the two subtypes,and the prognosis was different.Seven m6 A regulators were screened out through Lasso and stepwise regression to construct a COX risk regression model.The higher the risk score,the worse the prognosis;the risk score was an independent risk factor affected the prognosis of ovarian cancer,and the evaluation of the prognosis was relatively accuracy.High-risk group genes were mainly enriched in TGF-β signaling pathway,adherens junction signaling pathway,and Wnt signaling pathway.And the high-risk group had a higher stromal score in tumor microenvironment,and lower tumor mutation burden.Conclusion: The m6 A regulatory factor has a relatively high copy number variation frequency and a relatively low mutation rate in ovarian cancer,and the expression of each regulatory factor is generally positively correlated.The risk score constructed based on m6 A regulatory factors may have certain clinical application value for ovarian cancer.