The Role And Mechanism Of M6A Methyltransferase METTL14 In Ovarian Cancer Cell Proliferation

Background Ovarian cancer(OC)has the fifth highest fatality rate among all tumors in women and the first among female germline malignancies.Epithelial ovarian cancer(EOC)is the most common pathological type in clinic,and its molecular mechanism is not completely clear.In terms of diagnosis and treatment,it has been faced with the dilemma of lack of new effective biomarkers,high drug resistance rate,and insufficient of precision in therapy,and there has been no breakthrough progress for many years.Therefore,in-depth exploration of the mechanism of occurrence and development of EOC is of great value for the search for new molecular markers and new targets for clinical treatment.N6-methyladenosine(m6A)modification and its regulators play a key role in the occurrence and development of tumors by affectingRNA metabolism,and thus become another new focus in the field of epigenetics.The m6 A modification level in ovarian cancer is also dynamically regulated by related regulators.The abnormal expression of m6A regulators is involved in the autophagy,proliferation,metastasis,stemness maintenance and drug resistance of ovarian cancer cells through m6A-dependent and(or)-independent activities.Methyltransferase-like 14(METTL14)plays a key role inRNA substrate recognition and promoting METTL3 to play an efficient catalytic activity,which is an indispensable member of m6 A methyltransferase complex(MTC).However,the effect of METTL14 on m6 A modification in ovarian cancer and its malignant biological behavior remains unclear.Copy number variations(CNVs)is defined as the existence of a large number of DNA fragment variations greater than 1kb but less than 3Mb in the human genome,including insertions,deletions,duplications,and multi-site compound variations.There are a large number of CNVs in tumors,but their relationship with tumor occurrence and development and the specific molecular mechanism are not very clear.CNVs of m6 A regulators in tumors have also recently attracted extensive attention.The potential application value of CNVs in diagnosis,treatment and prognosis of cancer patients still needs to be explored and confirmed by more studies.In this study,the CNVs of m6 A regulators in ovarian cancer were used as the entry point,and the m6 A methyltransferase METTL14 was screened as the target molecule to be studied to clarify its role in the proliferation of ovarian cancer.By exploring the regulatory relationship between METTL14 and the downstream proliferation gene trophininassociated protein(TROAP),the molecular mechanism of METTL14 involved in regulating the stability of TROAP mRNA was elucidated,which provided the basis for finding specific therapeutic targets in the process of ovarian cancer proliferation.Purposes 1)To clarify the expression and clinical significance of METTL14 in ovarian cancer.2)To study the effect of METTL14 on m6 A modification level and cell proliferation of ovarian cancer.3)To explore the molecular mechanism of METTL14 targeting the proliferation-related gene TROAP to regulate the proliferation of ovarian cancer cells.4)To verify the role of METTL14 as a tumor suppressor gene in a subcutaneous transplanted tumor model of human ovarian cancer in nude mice.Methods 1)Entering the cBio Portal database,the CNVs and mRNA expression data of ovarian cancer were filtered and downloaded,then the correlation relationship between CNVs and mRNA expression of 23 m6 A regulators was analyzed.Based on the mRNA expression profile data of ovarian cancer tissue and normal ovarian tissue in the Gene Expression Omnibus dataset(GEO),the mRNA expression levels of m6 A regulators were evaluated,and the differentially expressed gene METTL14 was screened out as a target molecule for follow-up research.The expression of METTL14 was assessed at mRNA and protein level,and its correlation with the survival and prognosis of ovarian cancer patients was further evaluated.2)Detection of m6 A modification level in ovarian cancer tissue by colorimetric method.The correlation between m6 A modification level and METTL14 expression in ovarian cancer tissue was analyzed based on GEO database and molecular biology experiments.Cytoscape software was used to perform cluster analysis on genes with low m6 A modification levels in ovarian cancer,and to clarify the specific biological process affected by the reduction of m6 A modification levels in ovarian cancer.A lentiviral overexpression system of METTL14 was constructed and the effects of METTL14 overexpression on cell proliferation,clone formation and cell cycle distribution were detected in different ovarian cancer cell lines through MTS cell proliferation assay,plate cloning assay and flow cytometry.3)Screening the proliferation candidate genes negatively correlated with METTL14 in the ovarian cancer database,and using cluster analysis of Cytoscape software to predict that METTL14 may negatively regulate the downstream target gene TROAP.The expression of TROAP in ovarian cancer and its correlation with the expression of METTL14 were analyzed by using GEO database and human protein map database,and further confirmed by q RT-PCR and western blotting experiments.In addition,the expression of TROAP downstream target molecules such as cyclin D1,survivin and p-AKT in METTL14-overexpressing SKOV-3 cells was also detected by qRT-PCR and western blotting.4)In the part of exploring the molecular mechanism of METTL14 targeting the proliferation-related gene TROAP to regulate the proliferation of ovarian cancer cells,the m6 A methylation modification sites of TROAP mRNA were predicted using online database.Next,on the one hand,the effect of METTL14 overexpression on the m6 A modification level of TROAP 3’-untranslated region(3’-UTR)was analyzed by Me RIP-q PCR experiment.On the other hand,the effect of METTL14 overexpression on the stability of TROAP mRNA was examined by blocking newRNA synthesis in ovarian cancer cells by administering actinomycin D.The wild-type and mutant luciferase reporter vectors containing the TROAP 3’-UTR m6 A methylation site were constructed,and the specific role of METTL14 in regulating the stability of TROAP mRNA was confirmed by dual-luciferase reporter gene experiments.Based on literature reports,the m6 A reading protein YTHDF2 related to the stability of TROAP mRNA was screened by q RT-PCR and western blotting experiments,and its regulatory effect on the stability of TROAP mRNA was verified.5)The ovarian cancer SKOV-3 cells stably overexpressed METTL14 were used to establish a subcutaneous transplanted tumor model of human ovarian cancer in nude mice,and the growth of the transplanted tumor was evaluated.The expressions of METTL14 and TROAP in tumors were detected by q RT-PCR,western blotting and immunohistochemistry,and the correlation between METTL14 and TROAP was verified in vivo.Results 1)Except for m6 A reading protein IGF2BP1,CNVs of most m6 A regulators in ovarian cancer were positively correlated with their mRNA expression.METTL14 had decreased copy number and expression level in ovarian cancer tissues.Next,we further demonstrated the low expression level of METTL14 in ovarian cancer SKOV-3 and A2780 cells through a variety of biological experiments.Survival analysis demonstrated that low expression of METTL14 predicted poor survival in ovarian cancer patients,revealing that METTL14 may act as a tumor suppressor gene in ovarian cancer.2)The decreased level of m6 A modification in ovarian cancer tissue is related to the decreased expression of METTL14.Cluster analysis of genes with low m6 A modification levels in ovarian cancer showed that those genes were mainly involved in biological events such as ovarian cancer cell proliferation and mRNA stability.The results of MTS cell proliferation assay,plate cloning assay and flow cytometry showed that METTL14 overexpression had a significant inhibitory effect on ovarian cancer cell proliferation,clone formation and cell cycle progression.3)In view of the “writing” function of METTL14 in the regulation of m6 A modification,and one of the most important functions of m6 A modification is to cause mRNA instability,we used the GEO database to screen out the proliferation candidate gene TROAP that is negatively correlated with METTL14 expression in ovarian cancer.Through q RT-PCR and western blot experiments,we confirmed that overexpression of METTL14 inhibited the expression of TROAP and its downstream molecules such as cyclin D1,survivin and p-AKT.Rescue experiments showed that simultaneous overexpression of TROAP in ovarian cancer cells stably overexpressed METTL14 could counteract the inhibition of cell proliferation caused by METTL14 overexpression,while restoring the expression of molecules in the downstream proliferation-related pathways of TROAP.4)In the part of molecular mechanism research,we first predicted the m6 A methylation sites contained in the 3’-UTR of TROAP mRNA using the m6 AVar online website.Next,it was confirmed by Me RIP-q PCR experiments that overexpression of METTL14 significantly increased the m6 A modification level of TROAP mRNA 3’-UTR.At the same time,METTL14-overexpressing ovarian cancer SKOV-3 cells were treated with actinomycin D,and then the half-life of TROAP mRNA was detected.The results showed that overexpression of METTL14 had a strong attenuation effect on TROAP mRNA and inhibited the stability of its mRNA.After mutating the m6 A methylation site of TROAP mRNA 3’-UTR,a dual-luciferase reporter gene experiment was performed.The results showed that the regulatory effect of METTL14 on TROAP mRNA was weakened after this site was mutated,suggesting that METTL14 can indeed regulate the stability of TROAP mRNA through the m6 A methylation site of TROAP mRNA 3’-UTR.After inhibiting the m6 A reader protein YTHDF2,the attenuation effect of METTL14 on TROAP mRNA was weakened,further indicating that METTL14 reduced the stability of TROAP mRNA in an m6A-YTHDF2-dependent manner.5)In animal experiments,overexpression of METTL14 significantly inhibited the growth of human ovarian cancer xenografts in nude mice.The results of q RT-PCR,western blotting and immunohistochemical experiments showed that overexpression of METTL14 significantly reduced the expression of TROAP,suggesting that METTL14 inhibited the expression of TROAP in vivo.Conclusion Decreased METTL14 expression in ovarian cancer is associated with poorer prognosis in ovarian cancer patients.METTL14 plays an important biological role in the proliferation of ovarian cancer cells.Overexpression of METTL14 inhibits the expression of the proliferation-related gene TROAP and its downstream proliferation-related pathway molecules in an m6A-YTHDF2-dependent manner,thereby inhibiting the proliferation of ovarian cancer cells.