miR-181a在胃癌自噬及其顺铂耐药中的作用机制研究

A number of chemotherapy drugs can induce autophagy. This inducible autophagy is a pro-survival mechanism and contributes to the development of acquired drug resistance. Hence, inhibition of autophagy may provide a new combined therapeutic strategy. Only a few drugs are available for inhibiting autophagy activity. Recently, the association between miRNA and cancers has attracted much attention. However, it is unknown if miRNA can inhibit this kind of protective autophagy and reverse drug resistance. In our pilot study, based on miRNA microarray analysis, we identified that miR-181a plays an important role in gastric cancer, and promotes cancer cell growth, invasion, and anti-apoptosis. In addition, autophagy related genes such as ATG5 are the targets of miR-181a. The proposed study will take miR-181a as an example to verify the hypothesis of "miRNA regulates autophagy via targeting autophagy related genes, and subsequently involves in drug resistance". We will analyze the regulation of miR-181a on autophagy pathway, the impact of autophagy on miR-181a via inhibition and overexpression of miR-181a, as well as in the presence of autophagy inducer and inhibitor. Also, we will produce miR-181a lentiviral vectors, and assess the therapeutic effects of miR-181a in combination with cisplatin in vitro and in vivo. The study will provide evidence for a novel strategy to overcome drug resistance.

许多化疗药都能诱导自噬，这种可诱导的自噬是一种促细胞存活的机制，有利于肿瘤获得耐药性。因此，抑制自噬可能成为一种新的肿瘤联合治疗手段。目前可供选择的自噬抑制剂很少。近年miRNA与肿瘤的关系倍受关注，miRNA能否抑制这种保护性自噬并逆转肿瘤耐药尚不明确。我们前期通过基因芯片筛选，发现miR-181a与胃癌关系密切，能促癌细胞增殖、侵袭和抗凋亡；且miR-181a可靶向调控ATG5等多个自噬相关基因。本课题将以miR-181a为代表，在胃癌细胞顺铂耐药株中验证"miRNA通过靶向作用自噬相关基因来调控自噬，并参与肿瘤耐药"的假说。拟通过抑制和过表达miR-181a，结合自噬诱导和抑制剂，分析miR-181a对自噬通路的调控，以及自噬对miR-181a的影响；构建miR-181a慢病毒，通过体内、外实验评价miR-181a与顺铂联用的治疗效果。研究结果将为探索新的耐药解决方案提供实验依据。

肿瘤化疗药物诱导的保护性自噬是肿瘤细胞获得耐药性的一种重要机制，因此，抑制自噬有望成为新的肿瘤联合治疗手段。我们研究发现，miR-181a参与自噬调控，过表达miR-181a能抑制细胞自噬；通过对miR-181a靶基因的筛选和验证，发现ATG5和MTMR3是miR-181a调控自噬通路的主要效应基因。研究还发现，顺铂在诱导自噬的同时，可影响miR-181a的表达水平，且与细胞顺铂耐药性相关；顺铂敏感的胃癌细胞株miR-181a明显升高，而耐药株则变化不明显，提示miR-181a有望成为顺铂耐药性的预测因子。进一步体外细胞实验发现，过表达miR-181a可以有效降低顺铂耐药株SGC7901/DDP细胞的顺铂IC50浓度，抑制细胞增殖，促进细胞凋亡，从而增强顺铂疗效；体内荷瘤裸鼠实验也证实，miR-181a可抑制自噬，增加胃癌细胞对顺铂的敏感性，达到缩小肿瘤体积，提高疗效的目的。本研究发现为miRNAs在肿瘤化疗药物耐药方面提供了实验依据，miR-181a有望成为一种新的化疗药物耐药性预测因子和治疗手段。