缺氧特异性抑制YTHDF2促进肝癌生长的作用及其机制

The mechanism by which hypoxia promotes the progress of hepatocellular carcinoma (HCC) has yet to be further clarified. Our preliminary data showed that the expression of YTHDF2, a reader of m6A, the binding of which leads to the alteration of the translation efficiency and stability of m6A-containing RNAs, was significantly down-regulated specifically by hypoxia in HCC. Overexpressed YTHDF2 in HCC inhibited cell proliferation, colony formation and tumor formation, and suppressed the ERK/MAPK signaling pathway. Moreover, the function of YTHDF2 depended on the synergy between its N-terminal and C-terminal domains. All the above data indicates that hypoxia in HCC could specifically inhibit the expression of YTHDF2, which further affects ERK/MAPK pathway by regulating the RNA stability of its target genes and finally results in tumor progression. Based on this hypothesis, we plan to: 1) confirm the inhibitory effect of hypoxia on YTHDF2 expression and its dependency on HIF1α in several more HCC cell lines, 2) verify that YTHDF2 inhibits cell proliferation in HCC by inactivating ERK/MAPK pathway in vitro and in vivo, 3) identify target genes of YTHDF2 by RNA-seq and m6A-seq and clarify their functions, 4) detect the expression of YTHDF2, CAIX, a marker of hypoxia, and p-ERK in HCC specimens and analyze their clinical significance. Successful accomplishment of this project would be helpful to develop new strategies for prognosis estimation and/or targeted therapy of HCC.

缺氧能促进肝癌进展，但机制待完善。我们预实验发现，缺氧能特异性抑制肝癌细胞m6A结合蛋白YTHDF2的表达；过表达YTHDF2抑制肝癌细胞的增殖、克隆形成及成瘤能力，同时抑制ERK/MAPK通路，且该功能依赖于其N端功能域和C端功能域之间的协同作用。这些结果提示：缺氧能特异性抑制肝癌细胞YTHDF2的表达，后者通过调控其靶基因RNA的稳定性，进而影响ERK/MAPK通路，从而促进肝癌细胞生长。本项目拟：在多种肝癌细胞系中明确缺氧对YTHDF2表达的抑制及其对HIF1α的依赖；在细胞和动物水平进一步验证YTHDF2通过ERK/MAPK通路失活来抑制肝癌细胞生长；通过RNA-seq及m6A-seq鉴定YTHDF2的下游靶基因，并阐明靶基因的作用；肝癌临床标本中检测YTHDF2、CAIX（缺氧标志物）和p-ERK，并分析其临床意义。本项目的完成，将为肝癌患者的预后判断和/或靶向治疗等提供新策略。

缺氧能促进肝癌进展，但机制待完善。基于预实验发现，本项目提出科学假说：缺氧能特异性抑制肝癌细胞YTHDF2的表达，后者通过调控其靶基因RNA的稳定性，进而影响ERK/MAPK通路，从而促进肝癌细胞生长。在此假说驱动下，本项目主要研究内容包括：1.进一步明确缺氧对于YTHDF2表达的抑制作用及其对HIF1α的依赖，2.进一步验证YTHDF2抑制肝癌细胞的生长是通过使ERK/MAPK通路失活，并阐明机制，3.探讨YTHDF2在肝癌中的临床意义。本项目在多种肝癌细胞系中，分别通过缺氧处理和敲低HIF1α，进一步明确缺氧显著抑制YTHDF2的表达及其对HIF1α的依赖；构建多种YTHDF2过表达株，在细胞和小鼠在体水平分别明确YTHDF2对肝癌细胞增殖、克隆形成以及成瘤能力的抑制作用；过表达YTHDF2可显著抑制细胞缺氧引起的ERK磷酸化水平变化，证明了YTHDF2抑制肝癌细胞的生长是通过使ERK/MAPK通路失活；鉴定出YTHDF2的靶基因EGFR，证明YTHDF2直接与EGFR mRNA的m6A位点结合，破坏后者的稳定性，进而抑制ERK/MAPK通路；证实YTHDF2与CAIX或p-ERK的表达均相关，YTHDF2与CAIX或p-ERK联合分析能更好地判断肝癌病人的预后，具有重要临床意义。本项目的完成，将为肝癌患者的预后判断和/或靶向治疗等提供新策略。