目的：探索微RNA（microRNA,miRNA/miR）-29家族对淋巴结侵袭性非小细胞肺癌（non-small cell lung cancer,NSCLC）细胞增殖、侵袭的影响及潜在的分子机制。方法：TCGA数据库分析磷酸酶和张力蛋白同源基因（phosphatase and tension homology deleted from chromosome 10, PTEN）mRNA在是否有淋巴结转移的NSCLC患者组织中的表达水平;蛋白质印迹检测非淋巴结转移NSCLC细胞A549以及淋巴结转移NSCLC细胞H1299中PTEN蛋白水平;生物信息学预测调节PTEN的miRNA,实时定量反转录PCR（quantitative reverse transcriptase-mediated PCR,qRT-PCR）检测miR-29家族在A549和H1299中的表达水平;生物信息学预测miR-29家族与PTEN mRNA 3’ 非翻译区（untranslated region,UTR）结合位点,且通过双荧光素酶报告基因系统验证;转染miR a/b/c类似物或者抑制子,蛋白质印迹检测细胞中PTEN蛋白表达水平;用CRISPR Cas9技术构建miR-29家族敲低的稳转细胞;蛋白质印迹检测磷酸化Akt（phosphorylated Akt,p-Akt）、Akt、磷酸化黏着斑激酶（phosphorylated focal adhesion kinase,p-FAK）、FAK表达水平,qRT-PCR检测存活蛋白mRNA表达水平以及蛋白质印迹检测存活蛋白;细胞活力检测试剂盒（cell counting kit-8,CCK-8）检测细胞增殖及Transwell检测细胞侵袭的改变。结果：TCGA数据库分析PTEN mRNA的均值在无淋巴结转移NSCLC患者癌组织中表达高于有淋巴结转移的患者（7.916比7.242,P=0.026 8）;PTEN蛋白在A549细胞表达水平高于H1299细胞（3.1倍）;而在H1299细胞中miR-29家族分别是A549细胞的4、4.4和4.1倍（P<0.01）;荧光素酶报告基因实验验证了miR-29家族可靶向结合PTEN mRNA 3’UTR区的位点。A549细胞转染miR a/b/c类似物 36 h后,PTEN蛋白表达水平下降,H1299细胞转染miR a/b/c 抑制子 36 h后,PTEN蛋白表达分别恢复25%、21%和62%。与对照组相比,敲低miR-29家族后H1299细胞中p-Akt与p-FAK水平降低,存活蛋白 mRNA水平下调35%以及蛋白表达水平也相应下调70%;敲低miR-29家族后H1299细胞增殖活性下降,同时细胞的侵袭能力降低75%。结论：NSCLC细胞中miR-29家族通过下调PTEN,致p-Akt、p-FAK信号通路异常活化,进而上调存活蛋白表达,促进NSCLC细胞增殖和淋巴结侵袭。

Objective To investigate the regulation and the underlying molecular mechanism of microRNA(miRNA/miR)-29 family on the cancer cell proliferation and invasion of human lymph-node invasive non-small cell lung cancer (NSCLC). Methods TCGA data base was used to analyze phosphatase and tension homology deleted from chromosome 10 (PTEN) and the mRNA expression between the non-lymph node invasive and lymph node invasive tissue in NSCLC patients. The PTEN protein expression in non-lymph node invasive NSCLC A549 cells and lymph node invasive NSCLC H1299 cells was detected by Western blotting. Three different online software were used to predict the miRNAs targeting PTEN mRNA. Real-time quantitative reverse transcriptase mediated (qRT-PCR) was used to detect miR-29 family expression in A549 cells and H1299 cells. The binding sites between PTEN mRNA 3’ untranslated region(UTR) and miR-29 were predicted by using database and confirmed by luciferase report assays. miR-a/b/c mimics or inhibitors were transfected to A549 or H1299 cells, and the PTEN protein expression was detected by Western blotting. miR-29 family knock-down cells were established by CRISPR cas9 technology. Cell counting kit-8(CCK-8) assay was used to detect the difference of proliferation between negative control group (NC group) and miR-29 family knock-down group, and transwell invasion chamber test was used to detect the difference in invasion. p-Akt,Akt, phosphorylation focal adhesion kinase（p-FAK）, FAK and survivn expression was detected by Western blotting. Survivin mRNA expression was measured by qRT-PCR. CCK-8 method was used to detect the cell proliferation and cell invasion was detected by transwell method. Results The PTEN mRNA expression in the non-lymph node invasive NSCLC patients was higher than that in lymph node invasive NSCLC patients by analyzing the TCGA database. The PTEN protein expression was higher in A549 cells than that in H1299 cells, while miR-29 family expression was lower in A549 cells than that in H1299 cells. The luciferase report assays confirmed that PTEN mRNA 3’UTR was the target of miR-29 family. The PTEN protein expression was decreased after the A549 cells transfected miR a/b/c mimics for 36 h, and PTEN protein expression was increased after H1299 cells transfected miR a/b/c inhibitors for 36 h. The miR-29 family were knocked down in H1299 cells by CRISPR cas9 technology and the proliferation and invasion were significantly decreased compared with NC group. Compared with NC group, the phosphorylation level of Akt, surviving mRNA and protein expression, and the phosphorylation level of FAK were all decreased after miR-29 family was knocked down. Conclusions The miR-29 family promoted the proliferation and invasion of lymph node invasive NSCLC cells by decreasing PTEN expression and abnormally activating p-Akt and p-FAK signaling pathway.