RNA And Its Modification In The Pathogenesis Of Human Hepatocellular Carcinoma

Non-coding RNAs play important roles in cancer biology,providing potential targets for cancer intervention.As a new class of endogenous non-coding RNAs,circular RNAs(circRNAs)have been recently identified in the development,cell function and certain types of pathological responses,generally acting as microRNAs(miRNA)sponge to regulate gene expression.The dysregulation and functions of miRNAs have been extensively investigated in almost every biological process;however,the expression profiles and functions of the newly identified circRNAs in the specific biological activity remain to be further investigated.In the past decade,many non-coding RNAs including miRNAs and long noncoding RNAs(lnc-RNAs)have been revealed to be new kind of biomarkers and targets for cancer diagnosis and therapeutics.Now,identifying the circRNAs deregulation and their roles in cancer attracts much attention as an emerging filed.Hepatocellular carcinoma(HCC)is one of the leading causes of cancer-related death world-wide,especially in China.Identification of potential biomarkers for prognosis predication and screening of targets to design new therapeutic approach will benefit the HCC patients.However,the expression profile and function of circRNAs in human hepatocellular carcinoma(HCC)remain unclear.Here we analyzed the expression profile of human circRNAs in HCC tissues and identified circMTO1 as one circRNA significantly dowregulated in HCC tissues.HCC patients with lower circMTO1 expression had shorter survival.By using bitotin-labeled circMTO1 probe to perform RIP(RNA in vivo precipitation)in HCC cells,together with MiRanda Program prediction showing around 99 miRNAs as possible targets of circMTO1,we identified miR-9 as the circMTOl-associated microRNA.Furthermore,we performed FISH in HCC tumor and matched control tissues,and found that circMTO1 colocalized with miR-9 in the cytoplasm in tumor tissues,and this colocalization was much less in matched control tissues.Functionally,we set up the experimental system to silence or overexpress MTOl in HCC cells and confirmed the specificity of MTO1 silencing and overexpression.We found that knockdown of circMTO1 in HCC could dowregulate p21,the target of oncogenic miR-9,resulting in the promotion of HCC cell proliferation and invasion.Overexpression of miR-9 mimics also photocopied the effects of silencing circMTO1 in HepG2 cells.Thus,circMTO1 could suppress HCC cell line proliferation and invasion,while miR-9 promoted HCC cell proliferation and invasion as an oncogenic miR.Importantly,intratumoral administration of cholesterol-conjugated circMTO1 siRNA promoted HCC tumor growth in vivo,accordingly,the significantly decreased expression of p21 and increased expression of MMP2 and PCNA in SMMC-LTNM tumor tissues was found.In sum,circMTO1 suppresses HCC progression by acting as the sponge of oncogenic miR-9 to promote p21 expression,suggesting circMTO1 as a potential molecule in cancer intervention.The decrease of circMTO1 may serve as a prognosis predictor for poor prognosis of HCC patients.It has been reported that RNA has multiple types of modifications,however,N6-methyladenosine(m6A)modification is most commonly discovered.RNA m6A modification is critical to the splicing,transportation,translation and degradation of RNA,thus contributing to numerous biological processes and the pathogenesis of the disease.Considering that hepatocellular carcinoma(HCC)is one of most death-leading cancers in China and there is no report about RNA m6A modification of HCC to date,we established the experimental system of analyzing RNA m6A modification,and have utilized MeRIP-Seq approach to obtain the expression pattern of human HCC RNA m6A modification.In addition,by Cas9 approach,we have established and screened the stable human hepatocellular carcinoma cell lines deficient of the enzymes for RNA m6A modification including methyltransferases(writer)METTL3/METTL14,demethyltransferases(eraser)FTO/ALKBH5,and related factors(Reader,effector)YTDHF1/YTDHF2/YTHDC1/WTAP.The data provided solid basis for the future study of the important mRNA molecules with the changed m6A modification in the progression of HCC and better understanding of RNA m6A modification in cancer biology.