JSRV-NM囊膜蛋白激活致癌信号通路并调节细胞自噬的研究

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA). The envelope protein(ENV) of JSRV could transform cells in vitro and also could induce tumors in vivo experiments. As previous reported, it has been identified that JSRV-NM-ENV could induce NIH3T3 cells malignant transformation in vitro, but the molecular mechanism of transformation was not clear. The previous results were used as the breakthrough point, the activated mechanism of PI3K/Akt/mTOR and MAPK pathway in JSRV-NM-ENV transformed cells in vitro was used as the research target, the autophagosome structure of cells was detected through the transmission electron microscopy technology, the levels of mRNA and protein of Beclin1 and LC3 in JSRV-NM-ENV transformed cells was also detected, in order to explore how JSRV-NM-ENV induced malignant transformation through the modulation of autophagy by PI3K/Akt/mTOR and MAPK pathway. Then the natural infected OPA lung tissues in vivo and the tumorigenesis experiment in mouse were used to verify the complex tumorigenic molecular mechanism of ENV. The research was important for the theoretical and practical significance to verify the pathogenesis of OPA and seek out the target for preventing and controlling it.

绵羊肺腺瘤病毒（JSRV）是引起绵羊肺腺瘤病（OPA）的病原，其病毒的囊膜蛋白（ENV）具有体内外转化细胞而致瘤的罕见特征。我们前期的研究已证实JSRV-NM的ENV可引起体外NIH3T3细胞发生恶性转化，但机制不明。本项目以原有研究结果为切入点，通过研究PI3K/Akt/mTOR和MAPK信号通路在经JSRV ENV转化的体外NIH3T3细胞和绵羊绒毛膜滋养层细胞中的激活机制为靶点，结合透射电镜技术检测转化细胞中的自噬体结构和检测自噬因子Beclin1和LC3的mRNA和蛋白表达水平以及添加信号通路抑制剂调控通路激活为调节点，深入探究JSRV是如何通过调控信号通路以及自噬水平的变化来诱导其恶性转化的，然后通过体内实验在自然感染OPA病羊肺组织中和小鼠体内致瘤得到验证，从而揭示ENV致瘤的复杂机制。其研究成果对探明绵羊肺腺瘤病的发病机理以及找到防控该病的靶点具有重要的理论意义和实践意义。

绵羊肺腺瘤病毒（Jaagsiekte sheep retrovirus，JSRV）囊膜蛋白（Envelope，ENV）可在体外诱导细胞发生恶性转化和在体内介导肿瘤的形成。JSRV ENV 如何打破宿主细胞平衡引起转化，需要从细胞稳态角度探讨致瘤机制。自噬（Autophagy）是维持细胞稳态的重要手段。目前，探讨 JSRV ENV 与自噬关联的研究仍是空白。因此，本研究针对自然感染 OPA 肺组织进行 RNA-seq 分析发现OPA 肺组织中差异转录本与16个受细胞自噬调控的生物学功能模块相关，并与 PI3K/Akt-mTOR、MAPK 等调控自噬的信号通路高度关联，同时Western blot法检测p-Akt、p-mTOR、p-MEK1/2、p-ERK1/2、p-p38、p-JNK在OPA肺组织中的磷酸化水平极显著高于健康肺组织，Beclin-1和LC3 II/I在OPA肺组织中的蛋白表达均极显著低于健康肺组织中的表达。同时qPCR法和Western blot法检测Beclin-1和LC3 II/I 在JSRV ENV诱导转化的NIH3T3细胞中的表达均极显著低于对照组。当阻断MAPK信号通路和Akt/mTOR信号通路后，JSRV ENV诱导转化的NIH3T3细胞和绵羊绒毛膜滋养层细胞（STCs）中，Beclin-1和LC3 II/I的表达水平与对照组相比显著上调。从而说明JSRV ENV激活了靶细胞的MAPK和Akt/mTOR 信号通路，并通过MAPK和Akt/mTOR信号通路的调节，抑制Beclin-1和LC3 II/I的表达。将JSRV env 全基因序列同源重组入 pCMV-dR8.91 后包装、纯化，构建过表达ENV慢病毒质粒。感染 STCs 及 NIH 3T3 细胞 72h 后，细胞增殖、迁移、侵袭能力均显著增强（p < 0.05），10d 即可在软琼脂中形成明显集落，将转化细胞注入裸鼠皮下 28d 后形成明显肿瘤。