miR-203下调Bmi-1基因对肺腺癌细胞侵袭转移的影响

目的 探讨miR-203在肺腺癌中的表达，并分析其与肺腺癌细胞侵袭转移的关系及其分子机制。方法 实时定量PCR检测40例肺腺癌患者肿瘤组织中miR-203的相对表达水平及其与临床病理特征之间的关系；实时定量PCR检测H1650、A549、H1975、SPC-A-1肺腺癌细胞株中miR-203表达水平；生物信息学软件预测miR-203潜在的靶基因；脂质体2000介导miR-203模拟物、Bmi-1基因或Bmi-1 siRNA转染H1975细胞株；Western blotting检测Bmi-1蛋白水平；双荧光素酶报告基因验证miR-203是否作用于Bmi-1 mRNA的3’UTR 区预测靶位；Transwell小室侵袭实验检测H1975细胞株的侵袭转移能力。结果 40例肺腺癌组织中miR-203的相对表达量为0.065±0.013；肺腺癌miR-203表达与淋巴结转移有关，而与其他临床病理参数均无关；miR-203在肺腺癌细胞株H1650、A549、H1975、SPC-A-1中的相对表达量分别为0.280±0.102、0.308±0.168、0.167±0.073和0.287±0.096。生物信息学软件预测Bmi-1是miR-203的潜在靶基因；过表达miR-203可明显降低Bmi-1蛋白表达水平；双荧光素酶报告基因检测证明miR-203可作用于Bmi-1基因mRNA的 3’UTR区预测靶位。过表达miR-203+Bmi-1 siRNA可显著抑制肺腺癌细胞株H1975的侵袭迁移能力；在miR-203过表达的H1975细胞株中同时过表达Bmi-1可恢复其侵袭能力。结论 miR-203可通过下调Bmi-1基因表达抑制H1975肺腺癌细胞株的侵袭转移，是一种潜在抑制转移的miRNA分子。

The effect of miR-203 on the migration and invasion of lung adenocarcinoma cells via targeting Bmi-1

Objective To investigate the expression of miR-203 in lung adenocarcinoma and analyze the relationship between miR-203 and migration and invasion of lung adenocarcinoma cells. The involved molecular mechanisms are also initially explored. Methods miR-203 was detected in lung tissues of 40 patients with lung adenocarcinoma by real time PCR. The expression of miR-203 was detected in lung adenocarcinoma cell lines H1650, A549, H1975, SPC-A-1 by real time PCR. The potential target gene of miR-203 was predicted by online bioinformatic softwares. Pre-miR-203 mimics, Bmi-1 gene and Bmi-1 siRNA were transfected into H1975 cell line by lipofectamine 2000. The Bmi-1 protein level was analyzed by Western blotting. The predicted miR-203 binding site in Bmi-1 3’-untranslated region（ UTR） was validated by dual-luciferase reporter gene assay. The migration ability of H1975 cells was determined by Transwell assay. Results The relative expression of miR-203 in lung adenocarcinoma tissues was 0？065 ＆#177; 0？013. The expression level of miR-203 in the lymph node metastasis group was lower than those in the non-metastasis group. The relative expression of miR-203 in H1650, A549, H1975 and SPC-A-1 cell lines were 0？280 ＆#177; 0？102, 0？308 ＆#177; 0？168, 0？167 ＆#177; 0？073 and 0？287 ＆#177; 0？096, respectively. Bmi-1 was a potential target gene of miR-203 predicted by miRanda and TargetScan. The Bmi-1 protein level was remark-ably decreased in the pre-miR-203 mimics group. Dual-luciferase reporter gene assay validated the predicted miR-203 binding site of Bmi-1 3’ UTR. Overexpression of miR-203 significantly inhibited the migration and invasion of H1975 cells, whereas the cell migration and invasion ability could be restored by overexpression of Bmi-1. Conclusion miR-203 can suppress the migration of lung adenocar-cinoma cell line H1975 via down-regulating Bmi-1 expression. miR-203 might be a potential tumor metastasis suppressor miRNA.