乙肝病毒X蛋白通过血管生成素核转位影响tRNA半分子富集的作用机制

HBV infection leads to wide spectrum of liver disease ranging from acute to chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC). The viral transactivator HBx plays a pivotal role in the initiation and maintenance of hepatic inflammatory processes through interactions with components of the HCC microenviroment, which is attribute to the promotion of angiogenesis by HBx, and the mechanism is not clear. Angiogenin (ANG) has a dual role in promoting the proliferation of tumor cells and promoting angiogenesis of new blood vessels , but whether the effect is realized through tRNA halves produced by ANG shearing tRNA is very little known. We found that tiRNA was abundant in hepatitis tissue of HBV, however, the abundant of tiRNA in cancer tissue of HCC was significantly decreased, which could be correlated with the subcellular expression localization of HBx and ANG. Further study demonstrated that HBx can promote nuclear translocation of ANG. These findings suggest that HBx may regulate the angiogenesis and cell proliferation of HCC by inhibiting the tiRNA abundant whcih was induced by nuclear translocation of ANG and making the disorder of rRNA biogenesis. To verify this hypothesis, the vitro models based on human hepatoma cell lines transfected with HBV whole-genome and HBx respectively were used in this study, which are benefit for understanding that tiRNA abundant is an innate defence mechanism to prevent oncogenic translation and providing a molecular target for anti angiogenesis in the treatment of HCC.

乙肝病毒(HBV)的慢性感染是肝细胞癌(HCC)发生的主要风险因素。乙肝病毒X蛋白(HBx)在HBV诱发HCC发生的炎癌转化过程中起重要作用与其促进肝癌新生血管生成有关，机制不清。血管生成素(ANG)具有促进肿瘤细胞增殖和促进新生血管生成的双重作用，该作用是否通过其剪切生成的tRNA半分子(tiRNA)实现？目前知之甚少。我们前期发现tiRNA在HCC组织中富集显著减少，而在癌旁肝炎组织中富集明显，这种变化可能与HBx和ANG亚细胞定位存在相关性。进一步发现HBx能促进ANG核转位。这些提示HBx可能通过刺激ANG核转位所诱发的tiRNA富集抑制和rRNA生物发生失调实现对肝癌新生血管生成和癌细胞增殖的调控。本课题将以HBx、HBV全基因组稳转肝癌细胞系为模型结合临床资料验证这一提示，以其理解tiRNA富集是细胞应对不利生存环境的一种天然自我保护机制，并为抗肝癌血管生成提供新的分子靶点。

乙肝病毒X蛋白(HBx)作为一个多功能的调节因子，具有促进肝癌新生血管生成的作用，在HBV诱发HCC发生的炎癌转化过程中扮演着关键角色。我们绘制了乙肝病毒性肝癌中失调的piRNA表达谱，筛选了tRNAGly-(GCC)来源的piR25783通过竞争性结合IMP1从而使β-catenin的mRNA不稳定性增加，进而通过影响Wnt/β-catenin信号通路参与肝细胞肝癌生长与转移的调节。进一步我们构建HBx-ncRNA-肝癌相关基因网络作用图,发现一个长链非编码RNA:Linc01612作用显著。Linc01612在肝细胞癌中扮演抑癌基因角色，能够显著抑制肝癌细胞的增殖与侵袭迁移能力，促进肝癌细胞的凋亡。Linc01612通过竞争性结合miR-494上调ATF3的表达，进而抑制MDM2介导的p53泛素-蛋白酶体途径的降解，提高p53蛋白稳定性并激活p53通路。同时，Linc01612通过加强YBX1与NEDD4L的互作，促进YBX1泛素-蛋白酶体途径的降解，进而抑制YBX1及其下游基因Snail和c-Myc的表达，抑制HCC进展。我们在研究HBx作用靶蛋白时，发现了一个新的靶点：DNASE1L3。这个蛋白通过调节与DNA损伤压力相关的衰老相关分泌表型来阻止肿瘤血管生成。另外，DNASE1L3通过上调JAK/STAT通路抑制剂PTPN2与HK2结合，抑制HK2发生磷酸化，从而抑制肿瘤细胞糖酵解进程，同时，DNASE1L3可与CEBPβ相互作用，促进p53表达，激活下游靶基因TIGAR抑制糖酵解。HBx通过上调ZNF384结合DNASE1L3启动子区负性调控DNASE1L3表达水平。最后，我们发现了HBx作用的一个未注释的miRNA（暂定名为miR-MTCO3P38），该miRNA源自假基因MTCO3P38的5’末端，在HCC组织中下调，并与HCC患者的总体生存率显著相关。MiR-MTCO3P38通过STAT3/PTTG1/MYC通路抑制肝细胞癌的进展。这些研究结果提示HBx通过非编码RNA生物发生失调实现对肝癌新生血管生成和细胞增殖的调控,为抗肝癌血管生成提供新的分子靶点。