FTO介导的m6A修饰的筛选、鉴定及作为肝癌早期诊断标志物的评价

N6-methyladenosine (m6A) is the most important chemical modification in mRNAs, which mainly occurs in 3’-UTR region or near stop codon, and regulates mRNA stability, splicing and translation. Recent studies have shown that m6A mediated by METTL3/METTL14 is involved in several cancers development and progress, including hepatocellular carcinoma (HCC). However, the role of FTO-dependent m6A in HCC remains to be elucidated. Our data showed that m6A content was increased and FTO decreased in mRNA from HCC tumor tissues compared with the matched tumor-adjacent tissues. Thus, we hypothesize that FTO may induce HCC progress via regulating m6A and m6A can be used as an early diagnostic biomarker for HCC. To verify the hypothesis, cell and mice models with or without FTO knockdown or overexpression will be established to explore FTO functions in HCC; m6A-seq will be utilized to obtain m6A profiling in mRNA to explore new biomarkers for HCC , also the new biomarkers will be further evaluated in clinical samples. These results will provide novel predictive biomarkers for early diagnosis and monitoring the therapy efficiency of hepatocellular carcinoma.

m6A是mRNA上的最主要修饰，具有调控mRNA的剪切、翻译、稳定性等功能，其甲基转移酶METTL3､METTL14介导的m6A修饰参与包括肝细胞癌（HCC）在内的多种肿瘤发生发展过程，然而FTO在HCC的作用尚未见报道。我们发现：HCC患者癌组织和外周血中FTO表达水平降低，m6A修饰水平升高。据此假设：FTO介导的m6A修饰参与调控HCC的发生发展，可作为HCC早期诊断标志物。本项目拟采用FTO敲减/过表达细胞模型和相应的移植瘤动物模型探究肿瘤形成、增殖、侵袭和转移特性，液质联用技术检测mRNA的m6A水平，m6A-seq筛选m6A差异修饰基因；临床样本进一步分析评价m6A差异修饰基因作为HCC发生发展标志物的性能。研究将为HCC早期诊断和治疗监测提供新的标志物。

m6A是mRNA上的最主要修饰，具有调控mRNA的剪切、翻译、稳定性等功能，其甲基转移酶METTL3､METTL14介导的m6A修饰参与包括肝细胞癌（HCC）在内的多种肿瘤发生发展过程，对于FTO在HCC的作用机制研究则很少报道。我们前期发现：HCC患者癌组织和外周血中FTO表达水平降低，m6A修饰水平升高。据此假设：FTO介导的m6A修饰参与调控HCC的发生发展，可作为HCC早期诊断标志物。本项目通过构建FTO敲减/过表达细胞模型，CCK8试验、平板克隆试验、划痕试验和transwell试验等试验发现，过表达FTO可以有效抑制肝癌细胞的增殖、迁移和侵袭能力；而敲除FTO则促进肝癌细胞的增殖、迁移和侵袭。生物信息学发现PLIN1作为FTO下游基因，受到m6A修饰调控，参与肝癌的进程。同时鉴于TARBP在microRNA成熟过程中关键作用，发现了TARBP2-miR-145-SERPINE1轴抑制肝细胞肝癌的发生发展。同时，针对HBV感染是我国肝癌的主要风险因素，我们探索了铁死亡相关基因在HBV相关肝癌作用机制，发现SLC1A5是HBV相关HCC的独立危险因素，并与肿瘤免疫微环境相关，是HBV相关HCC的优秀预测因子，并能为开发创新的可能治疗技术提供理论支持。通过对临床样本研究发现，HCC患者血清中CystatinC含量降低，其编码基因CysC抑制肝癌的发生发展。这些研究将为HCC早期诊断和治疗监测提供新的标志物。