探討肝細胞過度表現TERT基因在B肝相關肝癌形成過程所扮演的角色

Abstract

B肝炎病毒所引發之肝細胞癌包含多個致病相關的基因體變異。本實驗室發現近70%的男性B肝相關的肝細胞癌中有B型肝炎病毒嵌入-124和-146鹼基位點體突變發生於telomerase reverse transcriptase (TERT)基因的啟動子，進而導致TERT的表現量上升。上述兩種遺傳突變皆發生在早期階段的肝細胞癌，指出其所造成的TERT表現量上升可能為肝癌致癌過程中重要的驅使者。然而先前研究指出TERT的異常表現為一免疫原，會引發免疫攻擊而使TERT異常表現的細胞被清除，我們因此提出一假說，即在早期階段TERT異常表現的肝癌細胞會因其他協同致癌因子作用進行免疫逃脫，因此發揮其致癌功能。為了驗證這個假說，本篇論文利用免疫完全的小鼠，以尾靜脈高壓注射和sleeping beauty (SB) transposon system將帶有螢光素酶報導基因的TERT基因轉染至C57BL/6J公鼠的肝細胞中，後續以活體動物影像系統追蹤嵌入至肝細胞中的TERT基因表現情形。同時我們也將其他協同致癌因子，包括HBx和β-catenin，以transposon的形式一起送入肝細胞中表現，探討其對TERT表現的肝細胞進行免疫逃脫之可能作用。結果發現TERT基因的冷光素酶表達在第15天後開始顯著下降且與有效免疫活性清除的能力有關，包含CD8 and CD4 T cells，顯示TERT可作為免疫原引發免疫的反應，使得表現TERT的肝細胞在15天之後消失。加入β-catenin之後，在第15天的肝臟中仍會看到TERT表現的肝細胞，這些表現TERT的肝細胞同時也會表現β-catenin，顯示β-catenin可能協助TERT(+)的肝細胞在第15天逃脫免疫的攻擊。此外，當再多加入HBx之後，表現TERT的肝細胞似乎有群聚的現象。本研究因此建立一B肝相關肝癌的動物模式，未來將可用於探討此類肝癌細胞中TERT與HBx和β-catenin等致癌因子協同作用，參與B肝相關肝癌形成之分子機制。

Several genetic mutations were accumulated and contributed to hepatitis B virus (HBV) related hepatocellular carcinoma (HCC). Our recent study identified the HBV integrations and somatic mutations at the promoter region of telomerase reverse transcriptase (TERT) gene in around 70% of male HBV related HCC, which leads to an elevation of TERT expression. As both genetic mutations occur at the early carcinogenic stages, suggesting elevation of TERT could be the driver event for liver carcinogenesis. As documented, aberrant TERT expression could be an immunogen to induce active immune surveillance. It thus raises a hypothesis that the TERT elevated hepatocytes might need to escape the immune surveillance for subsequent carcinogenesis. To test this hypothesis, this study have tried to build an immune competent mouse model, using hydrodynamic injection and a sleeping beauty (SB) transposon system to introduce human TERT (hTERT)-luciferase reporter gene into the hepatocytes of C57BL/6J male mice. The expression of integrated hTERT-Luc is followed in vivo by the IVIS imaging system. The transposon based expression plasmids for HBx and β-catenin are delivered with hTERT-Luc at the same time, to examine the effect on the immune escape of hTERT overexpression hepatocytes. The results showed that the expression of integrated hTERT-Luc decreases after 15 days post injection, dependent on the active CD8 and CD4 T cells, supporting hTERT as an immunogen. Expression of β-catenin helps immune escape of the hTERT integrated hepatocytes. Moreover, the expression of HBx might help cluster formation of hTERT(+) hepatocytes. Our study thus established an immune competent animal model for future delineating the carcinogenic mechanism induced by TERT, in collaborating with HBx and β-catenin, in HBV-related male HCC.