非小细胞肺癌组织中高表达的miR-1269对肺癌细胞A549生物学行为的影响

Effect of high expression of miR-1269 in non-small cell lung cancer tissues on biological characteristics of lung cancer A549 cells

目的:探讨miR-1269在非小细胞肺癌(non-small-cell lung cancer, NSCLC)组织中的表达和对人NSCLC A549细胞生物学特性的影响及其作用机制。方法:选取2017年1月至2018年1月在河北医科大学第四医院胸外科进行首次手术切除的34例新鲜NSCLC癌组织及相应的癌旁组织,应用qRT-PCR检测NSCLC癌组织及相应的癌旁组织中miR-1269的表达水平。将miR-1269 mimics和mimics NC转染至A549细胞后,应用MTS实验、细胞划痕实验和Transwell实验检测A549细胞增殖、迁移和侵袭能力,流式细胞术检测其细胞周期的变化。生物信息学软件预测miR-1269的靶基因,并通过Western blotting和双荧光素酶报告基因实验验证miR-1269对靶基因的调控作用。采用Western blotting检测已转染的A549细胞的细胞周期依赖性激酶抑制剂p21、细胞周期蛋白D2以及上皮-间质转化(epithelial-mesenchymal transition,EMT)相关蛋白ZEB2和E-cadherin的表达情况。结果:NSCLC癌组织中miR-1269的表达水平显著高于对应的癌旁组织(2.81±2.27 vs 1.61±1.36,P<0.05)。miR-1269 mimics转染组A549细胞的增殖、迁移和侵袭能力显著高于空白对照组和mimics NC转染组(均P<0.01)。转染miR-1269-mimics的A549细胞S期细胞比例明显高于mimics NC转染组[(46.54±1.57)%vs(23.32±3.15)%,P<0.01]。miR-1269可与FOXO1基因3’端非翻译区的结合位点相结合,且过表达miR-1269后,A549细胞中FOXO1的表达水平及荧光素酶报告基因的活性均明显降低(均P<0.01)。miR-1269 mimics转染组A549细胞中p21、E-cadherin蛋白表达水平降低(均P<0.05),而CyclinD2、ZEB2蛋白表达水平升高(均P<0.05)。结论:miR-1269在NSCLC组织中表达上调,并且能够促进A549细胞的增殖、迁移、侵袭能力和影响细胞周期进程;其作用机制可能与靶向调控抑癌基因FOXO1的表达有关。

Objective: To investigate the expression of miR-1269 in non-small-cell lung cancer(NSCLC) tissues, and to explore its effect on the cellular biological characteristics of NSCLC A549 cells and the underlying mechanism. Methods: 34 pairs of NSCLC tissues and the corresponding adjacent para-cancerous tissues obtained from the patients, who underwent surgery in the Department of Breast Surgery, the Fourth Hospital of Hebei Medical University from Jan. 2017 to Jan. 2018, were collected for this study. The expression level of miR-1269 in above tissue specimens was examined by real-time fluorescent quantitative PCR. After transfection with miR-1269 mimics and mimics NC(negative control), the proliferation, migration and invasion of A549 cells were detected by MTS, Wound healing and Transwell assay, respectively; and the changes in cell cycle distribution of A549 cells were examined by flow cytometry.The bioinformatics tool was used to predict the possible target gene of miR-1269, and the regulation effect of miR-1269 on target gene was then validated by Western blotting and Dual-luciferase reporter assay. In the meanwhile, the protein expressions of cyclin dependent kinase inhibitor p21, Cyclin D2, and EMT-related proteins(E-cadherin and ZEB2) in the transfected A549 cells were measured by Western blotting. Results: The expression level of miR-1269 in NSCLC tissues was significantly higher than that in paracancerous tissues(2.81±2.27 vs 1.61±1.36, P<0.05). The capacities of proliferation, migration and invasion of A549 cells in miR-1269 mimics transfection group were significantly higher than those in mimics NC group and blank control group(all P<0.01). And the cell proportion at S-phase in miR-1269-mimics group was obviously higher than that in mimics NC group [(46.54±1.57)% vs(23.32±3.15)%,P<0.01].Bioinformatics analysis showed that miR-1269 could combine with 3’UTR of FOXO1 gene. After transfection with miR-1269 mimics,the expression level and luciferase activity of FOXO1 protein in A549 cells were significantly reduced(all P<0.01). Moreover, the protein expressions of p21 and E-cadherin were significantly decreased after over-expression of miR-1269(all P<0.05), while the expressions of ZEB2 and Cyclin D2 were up-regulated(all P<0.05). Conclusion: The expression level of miR-1269 in NSCLC tissues was significantly increased, and it could enhance the proliferation, cell cycle progression, migration and invasion of A549 cells. The possible mechanism may be related to its targeted regulation of FOXO1.