组蛋白去甲基化酶JHDM1D对miRNAs激活膀胱癌细胞p21基因表达的作用及机制研究

Recently, it is discovered that non-coding RNAs (ncRNAs) have potent abilities to induce gene expression by targeting promoter, and termed as RNA activation (RNAa). It has a promising application in cancer therapy caused by decreased expression of tumor suppressors and researches of gene over-expression. We have reported that many micro RNAs (miRNAs), such as miR-1236-3p, can activate p21 expression through RNAa and inhibit bladder cancer cells. However, miR-103b fails to up-regulate p21 expression though it has a higher alignment score. And the exact reason is unclear. Then we analyzed genes expression profile between miR-1236-3p and miR-103b transfection groups using Microarray and subsequent experiments. It was found that both the miRNAs were enriched at targeted regions and miR-1236-3p, other than miR-103b, activated p21 expression depended on up-regulation of histone demethylase JHDM1D. Moreover, JHDM1D was recruited at p21 promoter which miR-1236-3p targeted. In present study, we will compare the differences between miR-1236-3p and miR-103b in induction of p21 expression in bladder cancer cells at several molecular levels, and verify that phenomenon with more miRNAs in order to explore the RNAa mechanism. This study will provide new methods for selecting gene-activating miRNAs and increase success rate.

近来发现，非编码RNA可通过直接靶向作用于基因启动子而诱导基因高表达，这种现象称为RNA激活（RNAa）。其在某些以抑癌基因低表达为主导的肿瘤和基因过表达研究中具有广阔的应用前景。我们既往报道微小RNA，如miR-1236-3p，通过RNAa诱导抑癌基因p21表达并抑制膀胱癌细胞；而评分较高的miR-103b却未能激活p21，具体原因尚不清楚。我们采用表达谱芯片和相关实验发现，两个miRNA都能在p21基因其靶启动子区富集；miR-1236-3p诱导p21激活依赖组蛋白去甲基化酶JHDM1D高表达，并且后者在其靶启动子区富集增加，而miR-103b无此现象。本研究拟在多分子水平比较两个miRNA介导膀胱癌细胞p21表达过程中的不同，并在更多miRNA中验证，寻找JHDM1D在miRNA激活膀胱癌细胞p21的作用规律，探讨RNAa的机制。为筛选活性miRNA提供更多的评估手段，提高筛选效率。

近来发现，非编码RNA可通过直接靶向作用于基因启动子而诱导基因高表达，这种现象称为RNA激活。其在某些以抑癌基因低表达为主导的肿瘤和基因过表达研究中具有广阔的应用前景。我们既往报道微小RNA，如miR-1236-3p，通过RNAa诱导抑癌基因p21表达并抑制膀胱癌细胞；而评分较高的miR-103b却未能激活p21，具体原因尚不清楚。首先我们构建了p21基因启动子活性质粒，体外验证miR-1236-3p可以激活该启动子。ChIP实验证实JHDM1D在miR-1236-3p靶作用区域富集，用siRNA沉默JHDM1D后，其基因激活现象消失。其次，我们采用蛋白芯片分析发现绝大部分膀胱癌中JHDM1D高表达，而正常膀胱粘膜细胞中低表达。但是miR-1236-3p在p21表达过程中对于JHDM1D的招募和其表达水平无明显关系。而miR-103b之所为无法激活p21基因表达，主要原因在于无法招募JHDM1D。这表明组蛋白去甲基化酶JHDM1D在miRNA激活膀胱癌细胞系p21基因表达过程中发挥重要作用。再次，我们检测了RNA激活过程中几个关键的蛋白，如Ago1、RNAP II等，与既往研究结果吻合。此外，我们还研究了saRNA激活E-cadherin基因并抑制肾癌细胞的迁移和侵袭。miR-373激活膀胱癌细胞E-cadherin表达，并抑制细胞增殖和转移能力。我们证实了ApoG2可以通过诱导GSK/AKT复合物的形成，而下调PI3K/AKT信号通路的磷酸化水平，从而促进E型钙粘素和β连环蛋白由胞浆向胞膜转运，最终发挥抗嗜铬细胞瘤迁移和侵袭的作用。dsP53-285通过靶向启动子作用激活肾上腺嗜铬细胞瘤PC12细胞野生型TP53表达，抑制细胞增殖、诱导细胞周期阻滞、凋亡，以及延缓裸鼠移植瘤的肿瘤生长。