miR-451对肺癌细胞的增殖抑制及其靶基因的初步鉴定.docVIP

miR-451对肺癌细胞的增殖抑制及其靶基因的初步鉴定 武天慧1,彭睿2，彭惠民，姚莉1，孙艳1，尹频1，文利1，张政1（400016 重庆，重庆医科大学：细胞生物学及遗传学教研室1，生物信息学教研室2，实验教学中心第三军医大学高原军事医学系军事医学地理学教研室） [摘要] 目的 探讨非小细胞肺癌细胞增殖的作用及调控机制。方法Lipofectamine 2000转染已构建成功的miR-451真核表达载体至非小细胞肺癌细胞A549，通过MTT检测细胞增殖状况，运用生物信息学技术miR-451预测靶基因分析靶基因PSMB8构建含有PSMB8 3′ UTR野生型和突变型质粒，通过双荧光素酶检测A549细胞PSMB8表达水平，western blot检测PSMB8和NOS2的表达情况。结果 MTT结果显示过表达miR-451能显著抑制A549细胞增殖。miR-451组细胞阻滞在G2期，而细胞凋亡未见明显差异。GO分析发现PSMB8、CAB39、YWHAZ等17个基因，PSMB8主要参与细胞代谢调控，细胞周期与细胞增殖; KEGG信号途径分析结果显示靶基因主要涉及microRNA在细胞周期、PI3K-AKT、mTOR等信号通路。在这17个miR-451预测靶基因中，进一步用生物信息学研究发现含有miR-451的结合位点，且在多个物种中呈高度保守。荧光素酶活性检测发现，过表达miR-451 可抑制PSMB8荧光素酶活性表达。 Western blot结果发现过表达miR-451的A549细胞PSMB8呈下调，而低表达miR-451的正常支气管上皮16HEB细胞PSMB8呈上调。Western blot和细胞荧光免疫化学检测发现炎症因子NOS2在肺癌中表达也降低。结论 miR-451可能通过靶向炎症相关基因PSMB8/NOS2调控非小细胞肺癌细胞增殖，并参与肺癌的发生与发展。 [关键词] 肺癌；微RNA；细胞增殖 [中图法分类号] R394.2 [文献标志码] A ioinformatics Analysis on MicroRNAs in lung cancer and the Study on the Mechanism of Cell Proliferation in A549 Wu Tianhui1*, Peng Rui2*, Luo Yongjun3,Yao Li1, Sun Yan1, Yin Pin1, Wen Li 1, Peng Hui-Min4, Zhang Zheng1 (1Department of Cell Biology and Genetics, 2Department of Bioinformatics, 4Experimental Teaching Center, Chongqing Medical University, Chongqing, 400016; 3Department of Military Medical Geography, College of High Altitude Military Medicine, Third Military Medical University, Chongqing, 400038, China) [Abstract] Objective To explore the effects of miR-451 on cell proliferation in non-small cell lung cancer. Methods The eukaryotic expression vector of miR-451 (pGenesil-miR-451) was transfected into A549 cells using Lipofectamine 2000, cell proliferation was tested by MTT, and cell apoptosis and cell cycle were examined by flow cytometry assay in A549. Using bioinformatics analysis was used for miR-451 target prediction. The target gene functional analysis was carried on by GO and KEGG pathway analysis. Among the candidate genes, PSMB8 was chosen. The luciferase gene expression vectors containing PSMB8