miR-486-5p靶向MRPL47激活mTOR调控糖酵解在肺癌增殖中的作用研究

The lung cancer incidence of Gejiu tinners ranks the first in the world, however the pathogenesis is still unknown. Previously, we found out a series of mitochondrial ribosomal proteins such as MRPL47 via high-throughput mRNA microarray assays. .Preliminary experiments indicated using MRPL47-shRNA to knok down YTMLC-90 endogenous MRPL47 could inhibit cell proliferation, reducing the protein level of mTOR and reduce ATP and lactic acid production. Also we showed the expression of MRPL47 was significantly higher in Gejiu miners lung cancer than the adjacent tissues, and patients OS was shortened in patients with high MRPL47 expression. Bioinformatics analysis depicts the convergent targeting of the 3`UTRs of MRPL47 mRNA by miR-486-5p. Moreover, qPCR results suggested miR-486-5p expression was lower in Gejiu miners NSCLC, and the expression is negatively correlated with MRPL47. Based on these results, we hypothesize miR-486-5p promotes cancer cells proliferation by targeting MRPL47 which activates mTOR and regulates key enzymes of glycolytic in Gejiu minners lung cancer. In this study, we further use Luciferase Assay and etc. to obtain reliable evidences on the hypothesis so as to identify the key target genes, and to provide a novel scientific proof for targeted therapy on lung cancer.

云南个旧矿工肺癌发病率死亡率均居世界首位，其发病机制远未阐明。通过表达谱芯片筛选出MRPL47等多个线粒体核糖体蛋白在癌组织中高表达。预实验结果显示，敲减内源性MRPL47，可抑制肿瘤细胞增殖，降低mTOR蛋白的表达，导致糖酵解产物ATP和乳酸产量减少。IHC结果显示，MRPL47在个旧矿工肺癌中的表达明显高于癌旁组织，且高表达患者OS缩短。软件预测提示miR-486-5p与MRPL47 mRNA的3'UTR端存在结合位点，qPCR结果显示miR-486-5p在个旧矿工肺癌中表达下调，且与MRPL47表达呈负相关。由此提出个旧矿工肺癌可能的作用机制：miR-486-5p靶向MRPL47激活mTOR调控糖酵解，促进个旧矿工肺癌细胞增殖。本项目拟进一步通过CO-IP、荧光素酶报告基因等技术，旨在获得miR-486-5p靶向MRPL47激活mTOR调控糖酵解，促进个旧矿工肺癌增殖的依据。

肺癌是发病率、死亡率最高的恶性肿瘤。云南地处西南边陲，个旧作为特有的职业性肺癌高发现场，早期流行病学研究显示，锡矿矿工肺癌发病率居全国首位。前期研究结果表明，锡矿开采过程中形成的大量含砷二氧化硅粉尘是导致个旧矿工肺癌高发的重要因素。新近研究表明，独特的职业性致病因素引发的驱动基因改变是重要致癌因素。.利用14例个旧矿工肺癌和癌旁正常组织标本筛选出差异表达基因MRPL47，IHC结果显示，MRPL47在个旧矿工肺癌中的阳性表达率为58.00%，明显高于癌旁组织的阳性表达率14.00%（P＜0.001）。随访结果显示，MRPL47高表达患者OS明显短于低表达患者。功能研究显示，在肺癌细胞中敲减内源性MRPL47，可抑制细胞增殖，降低克隆形成率；裸鼠成瘤结果显示，敲减肺癌细胞内源性MRPL47，可显著抑制肿瘤在裸鼠体内的生长。进一步机制研究显示，在肺癌细胞中敲减内源性MRPL47，mTOR、LAMTOR3的表达降低，糖酵解产物ATP和乳酸产量减少。由此提示，MRPL47激活mTOR调控糖酵解在肺癌中发挥重要作用。.利用数据库miRanda(http://www.microrna.org/microrna/home.do)和Targetscan(http://www.targetscan.org)预测发现：MRPL47 mRNA的3`UTR区存在miR-486-5p的结合位点；荧光素酶报告基因结果显示，过表达miR-486-5p显著抑制MRPL47的3‘UTRs的荧光素酶活性，抑制内源性的miR-486-5p增加荧光素酶活性；Realtime PCR检测发现miR-486-5p在个旧肺癌组织中表达下调，且其表达与MRPL47呈负相关。进一步利用慢病毒过表达肺癌细胞中miR-486-5p，功能研究显示，细胞增殖速率减慢、克隆形成能力降低；裸鼠成瘤结果显示，肿瘤生长明显减慢；机制研究显示，MRPL47的表达降低，mTOR、LAMTOR3的表达降低，糖酵解产物乳酸及ATP产量减少。由此提示：miR-486-5p靶向MRPL47激活mTOR信号通路，调控糖酵解，在个旧矿工肺癌恶性增殖的作用机制。.综合以上实验结果，通过本课题从能量代谢角度阐明miR-486-5p靶向MRPL47激活mTOR信号通路，调控糖酵解，在个旧矿工肺癌恶性增殖的作用机制，为认识肿瘤的发病机制提供科学依据。