Yin Yang 1 在肿瘤细胞自噬调控中的作用及其机制

Autophagy plays a critical role in tumorigenesis and progression. However, how autophagy in tumor cells is regulated was not clarified. Our preliminary results found high expression of YY1 (Yin Yang 1) in breast cancer cells. siRNA targeted knockdown of YY1 resulted in autophagy independent down-regulation of p62/SQSTM1, autophagy blockage and growth inhibition. Exogenous p62 could reverse the effect of YY1 knockdown on autophagy and cell growth. In addition, p62 mRNA level was not altered by YY1 knockdown, indicating that microRNA are most likely invovled in the regulation of p62 by YY1. Bioinformatical analysis and further RT-PCR validation indicated that miR372 was the potential microRNA involved in the post-transcriptional regulation of p62 expression by YY1. First, there is a miR372 binding site in the 3-UTR of p62 mRNA. Second, miR372 expression was increased after YY1 knockdown. Third, miR-372 inhibitor resecued YY1-kncokdown induced p62 down-regulation and autophagy blcokage. Based on these preliminary results, we proposed that YY1 affects p62 protein level to regulate autophagy in human cancer cells through controlling the expression of miR372. By revealing such an epigentic contribution of autophagy regulation in human cancer cells, this study will provide us deeper insights into human carcinogenesis and thus is helpful to design better anti-cancer strategies targeting autophagy.

细胞自噬在肿瘤发生发展中发挥着重要作用，但目前自噬的调控机制尚未完全阐明。我们前期研究发现转录调控因子YY1(Yin Yang 1)在肿瘤细胞中高表达；抑制YY1表达导致自噬调控关键因子p62蛋白水平下调，从而阻滞细胞自噬并抑制增殖；但抑制YY1表达并不影响p62 mRNA水平，而外源p62却可逆转YY1表达下调导致的自噬阻滞和增殖抑制，表明转录后翻译前机制如miRNA可能参与YY1对p62的表达调控。进一步实验发现YY1很可能通过调控miR372表达来改变 p62蛋白水平，因为p62 mRNA的3-UTR中存在miR372结合位点；抑制YY1表达后miR372表达上升；抑制miR372则可逆转YY1表达下调导致的p62表达下降及自噬阻滞。本项目拟在以上工作基础上，阐明YY1调控miR372以及p62表达的分子机制，从表观遗传学角度明确自噬调控机制及其与肿瘤的关系，为肿瘤防治提供理论依据。

细胞自噬是细胞通过降解胞内物质维持细胞内稳态的过程，是细胞应对外界环境的一种防御和应激机制。近年来的研究发现，细胞自噬调控紊乱与肿瘤发生发展密切相关。转录因子YY1（Yin Yang 1）在恶性肿瘤发生发展中发挥重要作用，但其在肿瘤细胞自噬的相关调控机制尚未见报道。通过本项目研究，我们发现YY1在乳腺癌中高表达，与乳腺癌发生成正相关（p=0.046）；‘Loss of function’和‘Gain of function’实验证实YY1通过直接调控miR372的转录表达靶向调控SQSTM1的水平，从而参与细胞自噬和增殖，而且YY1和SQSTM1的表达在乳腺癌中呈正相关（p=0.0122）。同时我们也发现在外界环境胁迫下，乳腺癌细胞也通过上调YY1的表达来上调SQSTM1的水平，进而增强细胞自噬抵抗逆境。我们进一步发现YY1主要是通过改变miR372基因启动子区的DNA甲基化水平来调控miR372转录，抑制YY1的表达可以下调miR372基因启动子区DNA甲基化来上调miR372转录，从而靶向抑制SQSTM1表达，阻滞细胞自噬，抑制细胞增殖。本项目发现了一条新的表观遗传学调控机制（YY1-miR372-SQSTM1）参与调控肿瘤细胞自噬，为肿瘤细胞自噬调控异常提供了新的补充，也阐明了一种SQSTM1自噬非依赖的调控模式，也进一步丰富了YY1转录因子的促癌分子机制，为乳腺癌防治提供新的可能分子靶点和理论依据。