Study On The Roles Of RNA N6-methyladenosine Modification And Its Catalytic Gene METTL14 In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the most common cancer worldwide.In China,HCC has become one of the important cancer-related death with the incidence of cancer increased year by year.Accumulating evidence indicates that the poor prognosis of HCC is primarily due to the metastasis and postsurgical recurrence.Despite advances in the early diagnosis and clinical treatment strategies of HCC,the advanced hepatic cancers with postsurgical recurrence or low response to chemotherapeutic agent still have a low 5-year survival rate.The lack of effective interventions and the high prevalence of the cancer demand a better understanding of the molecular mechanism of cancer progression.Recently,the study of RNA epigenetic modification began to rise in various fileds.The N6-methyladenosine(m6A),a predominant internal modification of RNAs,becomes a new research hotspot.RNA m6A modification is prevalent in mRNA and lncRNA in many tissues and organs of higher eukaryotes,but has long been lacking progress due to methods limited and insufficient understanding of its function.However,recent studies find that m6A modification is not static or unalterable as ever know,but can be dynamically regulated in different stages of developmet or different organs.Similar to DNA methylation or histone methylation,RNA m6A modification is catalyzed by methyltransferase and demethylase.The main components of m6A methyltransferase complex,including METTL3,METTL14,WTAP,and KIAA1429,and the demethylase,such as FTO and ALKBH5,are identified with the unremitting efforts of researchers.With the establishment of the MeRIP-seq,high-throughput studies of m6A modifications have become possible.Recent studies find that RNA m6A modification is associated with many biological processes,such as cell development,stem feature maintenance,mitosis control,circadian regulation,fertilization capacity and tumor formation abilities.However,the status of m6A modification and the underlying regulatory mechanisms in HCC remain incompletely understood.In the present study,we sought to investigate the role of m6A modification in HCC and address the mechanisms by which m6A participates in the biology of liver cancer.We first examined the levels of m6A in the total RNAs of tumour tissues,paired adjacent tissues and normal hepatic tissues by using of colourimetric m6A quantification strategy and RNA m6A immunoblot assay.We found m6A levels were decreased in HCC.To investigate the cause of abnormal m6A modification in HCC,we then assessed the key m6A catalytic genes in HCC tissues and found METTL14 was significantly down-regulated in HCC.We inhibited METTL14 expression in SMMC-7721 and Hep-3B cells and found METTL14 knockdown resulted in decreased m6A levels in HCC cell lines.Moreover,METTL14 overexpression restored the reduction in m6A levels induced by METTL14 depletion.We investigate the relationship between METTL14 and clinical characteristics and found a decreasing tendency of METTL14 expression during the malignant transformation from normal liver to hepatitis,cirrhosis and HCC.Kaplan-Meier analysis revealed that patients with reduced METTL14 expression developed more frequent recurrence and had poorer survival.We found METTL14 was distinctly decreased in breast cancer and METTL14 might involved in metastasis process of breast cancer.Through in vivo and in vitro experiments,we demonstrated that METTL14 depletion enhanced metastatic capacity of HCC and over-expression of METTL14 suppressed tumour invasion and metastasis in HCC.Previous studies suggested that m6A modification could mark primary microRNAs for processing by recognizing DGCR8 in a manner dependent on METTL3/m6A.Given the important roles of miRNAs in malignant transformation,we hypothesized that METTL14 might influence tumour metastasis by targeting metastasis-related miRNAs in an m6A-dependent pri-mi RNAs processing manner.We then found METTL14 mediated m6A modification could enhance the recognition of pri-miR126 by DGCR8 and the subsequent processing to mature miRNA.Conclusion: our findings delineated the functions and mechnisms of m6A modification in HCC for the first time,and demonstrated an METTL14-m6A module that regulates the process of pri-miR126 with respect to functional implications in tumour metastasis.Our results suggest that m6A modification plays critical roles in tumour biology and the interaction between METTL14 and miRNA signalling might offer a possible interventional target of HCC.