The Functional Mechanism And Clinical Value Of M6A RNA Methylation And MiR-1299/NOTCH3/TUG1 Feedback Loop In Ovarian Cancer

BackgroundOvarian cancer(OC)is the most invasive gynecological tumor in the world.Due to the lack of characteristic symptoms and reliable biomarkers in the early stage,most patients are diagnosed in the late stage and will experience recurrence after the first treatment.Most patients at present benefit from multi-line therapy rather than effective first-line treatment,indicating that the introduction of new treatments to prolong the first progression and overall survival is still a key goal of clinical research.However,the selection of targets and the application of treatment methods depend on a deeper understanding of the mechanism of occurrence and development of OC.Epigenetics is a stable inheritance of gene expression or functional changes by regulating the interaction between genome and environment without changing the basic sequence of DNA.Genetic epigenetic changes are essential for tumorigenesis and often show abnormalities in the early stages of the disease.OC tumorigenesis is largely mediated by epigenetic changes,but the current studies are mainly focused on DNA methylation and histone modification.The role and mechanism of post-transcriptional RNA modification and non-coding RNA regulation in OC are still unknown.N6-methyladenosine(m6A)is the most abundant epigenetic modification in eukaryotic mRNA.It plays a role in many processes such as mRNA processing,transportation,translation and stability,and its dynamic and reversible changes determine cell growth and differentiation.Non-coding RNA(lncRNA),represented by microRNA(miRNA)and long-chain non-coding RNA,is the key regulator of gene expression at the post-transcriptional level.MiRNA is a destabilizer and inhibitor of mRNA.LncRNA can act as a competitive endogenous RNA(ceRNA)or miRNA "sponge" to regulate other key genes in tumors.Therefore,this study is based on the changes of m6A RNA methylation in OC and its clinical application,as well as the regulation of key oncogene by non-coding RNAs.The purpose of this study is to explore new targets for the pathogenesis and treatment of OC from the post-transcriptional epigenetic level.MethodsThis study is divided into two parts.In the first part,methylation detection kit was used to quantitatively detect m6A modification levels in 35 OC tissues and 5 OC cell lines,qPCR and western blot were utilized to detect the expression of m6A regulatory enzymes,and Oncomine and GEPIA websites to analyze the survival and prognosis information of TCGA OC database.Small interfering RNA and overexpression plasmid were used to knock down or increase the expression of m6A demethylase ALKBH5 in OC cells,therefor to detect its effects on tumor cell proliferation,apoptosis,cell cycle,migration and invasion.RNA methylation immunoprecipitation sequencing and transcriptome sequencing were performed on OC cells overexpressing ALKBH5,and the data were used for integrated analysis.The m6A modification level in peripheral blood cells in 31 newly diagnosed OC and 37 healthy controls was detected.The diagnostic efficacy was evaluated by ROC curve,and its correlation with serum tumor marker CA125 was analyzed.In the second part of the study,bioinformatics analysis and dual luciferase reporter assay were used to identify the potential miRNAs and lncRNAs regulating NOTCH3 in OC.qPCR was used to detect the expression of miRNA,lncRNA and mRNA,and western blot to detect protein levels.By transfecting miR-1299 mimics or inhibitors,or infecting TUG1 shRNA lentivirus,the effects of miR-1299 or lncRNATUG1 on tumor cell proliferation and cell cycle were studied in both vivo and vitro experiments.Correlation analysis and cytotoxicity experiments were used to study the effect of NOTCH3 on TUG1 expression.ResultsThe first part of the study found that the level of m6A RNA methylation in OC tissues was significantly increased compared with normal ovarian tissues(0.033%vs.0.02572%,P<0.001).Down-regulation of demethylase ALKBH5,which was related to clinical stage and survival prognosis,mediated the up-regulation of m6A modification.ALKBH5 regulated the dynamic changes of m6A level in OC cell lines.Overexpression of ALKBH5 could inhibit the proliferation of OC cells,promote their apoptosis and cause cell cycle arrest,while knocking down ALKBH5 showed the opposite effect.The results of MeRIP-seq and RNA-seq suggested that the tumor-repressive effect of ALKBH5 was closely related to cell cycle and proliferation-related genes and pathways.The level of m6A modification in peripheral blood cells in OC patients was also significantly increased in comparison with healthy controls(0.085 vs.0.052%,P=0.004),which was positively correlated with the level of serum tumor marker CA125(r=0.330,P=0.046).It displayed a good diagnostic efficiency in distinguishing OC patients from healthy individuals(AUC=0.795,95%CI 0.713-0.876,P<0.001).The second part of the study shows that miR-1299 was a negative regulator of oncogene NOTCH3 in OC,which was significantly down-regulated in OC and closely related to tumor differentiation.The upregulation of miR-1299 significantly inhibited the proliferation,colony formation and DNA synthesis of OC cells,and induced G0G1 cell cycle arrest.Overexpression of miR-1299 in xenotransplantation mouse model could effectively inhibit the growth of tumor in vivo.LncRNATUGl acted as a sponge of miR-1299,competitively adsorbing miR-1299 and up-regulating the expression of NOTCH3,thus promoting the malignant proliferation of OC.In addition,TUG1 was a potential downstream target of NOTCH3,forming a feedback loop of miR-1299/NOTCH3/TUG1 in the progression of OC.ConclusionsDownregulation of demethylase ALKBH5 mediates the increased m6A methylation level in OC and ALKBH5 exerts an anti-tumor effect mainly by inhibiting cell proliferation,inducing apoptosis and causing cycle arrest.Peripheral blood m6A is a potential biomarker of OC.These findings suggest that M6A modification and its regulatory enzymes can be used as potential targets for the diagnosis and treatment of OC.LncRNATUG1 promotes malignant proliferation of OC through competitive adsorption of miR-1299 and up-regulation the level of NOTCH3.This finding enhances the understanding of the pathogenesis of OC caused by key oncogenes regulated by non-coding RNA and is conducive to the development of new diagnostic and therapeutic methods based on miRNA and lncRNA.