C型肝炎病毒非結構性蛋白質NS5A透過MAPK訊息傳遞路徑調控抗氧化防禦相關基因

Abstract

C型肝炎病毒(hepatitis C virus)感染，常會演變成持續性感染並且可能引發肝癌。C型肝炎病毒非結構性蛋白質NS5A，目前已知會影響宿主細胞的生長和存活能力。本實驗室先前的研究顯示，NS5A蛋白質會與宿主細胞PKR (double-strand RNA-activated protein kinase)形成異源雙體並抑制PKR活性，使細胞通過細胞週期G2/M階段受到阻礙而延遲離開，造成染色體不穩定，可能為引發肝癌的原因之一。為了進一步釐清NS5A-PKR訊息傳遞路徑對於肝癌發生是否扮演重要角色，本論文首先於人類肝癌細胞株Huh7建立可穩定表現突變型NS5A蛋白質(NS5A-mPTT)的細胞株。在此Huh7系統中，突變型NS5A-mPTT蛋白質喪失與PKR形成異源雙體的能力，而NS5A蛋白質所造成的細胞週期異常調控及增殖速度變緩慢的現象，在突變型NS5A-mPTT蛋白質存在下則不會發生。使用軟瓊脂培養(soft agar)測試轉型(transformation)能力，結果發現穩定表現NS5A蛋白質的細胞相對於表現突變型NS5A-mPTT的細胞具有較高的轉型能力，顯示NS5A-PKR路徑對於肝癌發生扮演重要角色。過去研究顯示，NS5A蛋白質誘導活性氧化物(ROS)生成，造成DNA損傷(DNA damage)，可能因此促使細胞走向癌化。而細胞內抗氧化防禦機制主要由Nrf2 (NF-E2-related factor-2)及small Maf (v-maf musculoaponeurotic fibrosarcoma oncogene family)異源雙體所調控。本論文在可穩定表現C型肝炎病毒1b strain之次基因體複製體細胞株(HCV subgenomic replicon)發現DNA損傷標記物8-hydroxyguanosine明顯增加。根據軟體分析發現small Maf家族成員中MafF啟動子上擁有可受NS5A蛋白質調控之轉錄因子的結合位置。利用反轉錄酶-即時聚合酶連鎖反應顯示，C型肝炎病毒1b strain之次基因體複製體，NS5A及突變型NS5A-mPTT細胞中，MafF基因表現量均下降。由MAPK及PI3K抑制劑的實驗結果推測，NS5A可能透過PKR-p38及ERK訊息傳遞路徑抑制MafF的基因表現，且此二路徑相互影響，此一立論進一步在螢光素酶分析(Luciferase reporter assay)得到了證實。相反的，在C型肝炎病毒1b strain之次基因體複製體細胞株發現Nrf2蛋白質的表現量及活性卻增加，可能是病毒調控細胞存活的作用機制之一。當細胞大量表現NS5A蛋白質時，再處理H2O2之後，確實會使細胞累積更大量DNA損傷。由本論文結果推測，NS5A蛋白質透過PKR-p38及EKR訊息傳遞路徑抑制MafF 基因表現而導致抗氧化防禦(antioxidant defense)能力降低，使DNA 受到傷害，可能是促使C型肝炎病毒感染細胞走向癌化的方式之一。

Hepatitis C virus (HCV) often causes a persistent infection and virus-associated hepatocellular carcinoma. The viral nonstructural protein 5A (NS5A) is involved in the control of cell growth and viability. Results from our earlier studies indicated that NS5A can form heterodimer with PKR and down-regulate the PKR activity. Consequently, it causes cell cycle G2/M exit delay and leads to chromosome instability. The phenomenon provides insights into the molecular mechanisms of HCV-associated hepatocarcinogenesis. To clarify the importance of NS5A-PKR signaling pathway in HCV-associated hepatocarcinogenesis, Huh7 cells stably expressed the NS5A mutant, NS5A-mPTT, which has lost the ability of binding to PKR were generated in this study. NS5A de-regulated cell cycle and reduced the rate of cell proliferation, which were not observed in Huh7 cells constitutively expressing the NS5A-mPTT. Soft agar assay revealed a higher transformation ability of Huh7 cells constitutively expressing NS5A than those expressing NS5A-mPTT. This suggests a potential role of NS5A-PKR signaling pathway in the HCV-associated hepatocarcinogenesis. NS5A protein has been reported to induce reactive oxygen species (ROS) production that leads to DNA damage and possibly hepatocarcinogenesis. Heterodimer formation of Nrf2 (NF-E2-related factor-2) and small Maf (v-maf musculoaponeurotic fibrosarcoma oncogene family) is a major paradigm for the cellular antioxidant defense. In this study, the level of DNA damage marker 8-hydroxyguanosine signal was found to be significantly enhanced in Huh7 cells constitutively expressing the HCV subgenomic replicon of genotype 1b. Computer analysis revealed cis-elements of NS5A-regulated transcriptional factors on the promoter of MafF gene, a member of the small Maf family. RT-real-time PCR demonstrated down-regulation of MafF gene expression in Huh7 cells constitutively expressing the HCV subgenomic replicon of genotype 1b, the NS5A protein, or the NS5A-mPTT mutant protein. Luciferase reporter assay and RT-real-time PCR analysis carried out in the presence of MAPK, JNK or PI3K inhibitor further demonstrated that NS5A down-regulated MafF gene expression via the PKR-p38 and ERK signaling pathways. On the other hand, the expression level and activities of Nrf2 were up-regulated in the HCV subgenomic replicon cells, which may be involved in the cell viability. Furthermore, elevated DNA damage was detected in cells over-expressing NS5A upon H2O2 treatment. In conclusion, HCV NS5A protein down-regulates MafF gene expression via PKR-p38 and ERK signaling pathways, and attenuates the ability of antioxidant defense, leading to DNA damage. This could be one of the mechanisms involved in HCV-associated hepatocarcinogenesis.