miR-141在6P22扩增膀胱移行癌中与E2F3/Rb信号通路交互调控机制的研究

Overexpression of E2F3 and inactivation of Rb pathway are obligate events in the development of highly malignant, invasive bladder cancer with 6P22 amplification. Preliminary study showed that expression of miR-141 and miR-200c was lower in this kind of bladder cancer tissue than in bladder cancer tissue without 6P22 amplification. Morover, reduction of CDK1, CDK2 and CDK6 was detected accompanied with increasement of P27kip1 after overexpression of miR-141; overexpresion of miR-141 also resulted in G0/1 phase arrest and inhibition of cell proliferation in TCC-SUP cell lines with 6P22 amplification. Inactivation of Rb was reported to induce epithelial-mesenchymal transition (EMT) by up-regulating ZEB1, which down-regulates the expression of miR-141 and miR-200c. According to this, miR-141 is supposed to participate in regulating proliferation, differentiation and invasion of this type of bladder cancer through interaction with E2F3 and Rb. We aim to investigate the reciprocal regulatory mechanism between miR-141 and E2F3/Rb signal pathway. We look forward to screen key targets and provide new idea to explore personalized therapy for bladder cancer with 6P22 amplification.

6P22扩增膀胱癌恶性程度高、分化差，E2F3过表达和Rb通路失活是其发病过程中必要事件。前期研究证实此类膀胱癌组织中miR-141和miR-200c表达较非6P22扩增膀胱癌组织降低，在6P22扩增的膀胱癌细胞系TCC-SUP中分别过表达此二者后E2F3表达降低。此外，TCC-SUP细胞中过表达miR-141后CDK1、CDK2、CDK6表达均降低，P27kip1表达增高，细胞出现G0/1期阻滞，细胞增殖受到抑制。文献报道Rb失活上调ZEB1表达诱导上皮间质转化（EMT）进程，ZEB1下调miR-141和miR-200c的表达，故推测miR-141可能与E2F3/Rb信号通路交互调控此类膀胱癌的增殖、分化和侵袭。本课题拟探讨miR-141和E2F3/Rb信号通路之间的交互调控机制，筛选关键靶点，为6P22扩增膀胱癌的个性化治疗提供新的思路。

背景：miR-200家族包括miR-141，miR-200a，miR-200b，miR-200c，miR-429，参与调节膀胱癌多项生物学行为，细胞周期缺乏相关研究。主要研究内容：（1）miR-200家族对膀胱癌细胞（T24、TCCSUP、5637）细胞周期的调控。（2）miR-200家族对膀胱癌细胞细胞周期相关蛋白（E2F3，CDK2等）的调控。（3）miR-200家族对膀胱癌细胞增殖调控。（4）miR-200家族对膀胱细胞迁移和侵袭的调控。（5）RB对miR-200家族表达的调控。重要结果：在膀胱癌细胞中转染miR-200b 和miR-200c mimics及对照组。CCK-8实验、流式细胞术检测显示过表达miR-200b 和miR-200c促进细胞增殖、细胞周期G1期向S期转变；Western blot检测E2F3和CDK4蛋白高表达，同时也发现miR-200c靶向结合RECK促进T24、EJ细胞侵袭和迁移。siRb后测miR200家族变化。关键数据：qRT-PCR证实膀胱癌组织中miR-200b和miR-200c明显高表达相比其癌旁组织。qRT-PCR检测转染后miR-200b 和miR-200c的表达水平。CCK-8实验证实过表达miR-200b 和miR-200c的膀胱癌细胞增殖明显加快，周期实验证实G1期细胞比例相对于阴性对照（63.87%）有所下降，下降为54.85%，S期比例从阴性对照的28.57%上升至31.28%。过表达miR-200b 和miR-200c上调E2F3、CDK2、CDK4和P27KIP1等周期相关蛋白的表达水平。报告基因证实RECK的3-UTR 区与miR-200c靶向结合。miR-200c抑制RECK的表达从而促进膀胱癌细胞迁移和侵袭。采用siRNA技术抑制RB基因表达，检测发现miR200b和miR200c表达上调，提示RB基因可能通过调控miR200b和miR200c的表达来参与膀胱癌细胞细胞生物学行为。科学意义：本研究在膀胱癌细胞中证实了miR-200b和miR-200c具有促进G1期进入S期，其机制是通过上调细胞周期关键蛋白CDK2、CDK4、E2F3表达水平，miR-200c靶向抑制RECK表达，促进膀胱癌细胞侵袭和迁移，提示miR-200b和miR-200c是治疗膀胱癌的潜在靶点。