miRNA-101通过下调COX-2的表达影响肺癌细胞的生物学功能

miRNA-101 influence the biological function of lung cancer cells through downregulate COX-2 expression

目的:探讨miRNA-101(miR-101)在肺癌组织和细胞中的表达及其对人肺癌细胞增殖、克隆形成和成瘤能力的影响。方法:采用Western blotting和实时荧光定量RT-PCR技术分别检测10例患者肺癌组织及相应癌旁组织、人肺癌细胞A549和NCI-H26及正常人胚肺成纤维细胞MRC-5中环氧合酶2(cyclooxygenase-2,COX-2)及miR-101的表达。A549和NCIH26细胞分别转染miR-NC和miR-101构建过表达miR-101的细胞系及其对照,Western blotting、CCK-8和克隆形成实验分别检测过表达miR-101对A549、NCI-H26细胞中COX-2的表达、细胞增殖和克隆形成能力的影响。用A549、A549/NC、A549/101细胞在BALB/c裸鼠皮下构建移植瘤模型,观察过表达miR-101对A549细胞小鼠移植瘤生长的影响。结果:在肺癌组织和A549、NCI-H26细胞中,COX-2的表达明显高于癌旁组织(t=20.03,P=0.001)和MRC-5细胞(t=14.59,P=0.012),而miR-101的表达则相反(组织:t=18.33,P=0.002;细胞:t=28.95,P=0.000)。成功构建过表达miR-101的A549/101、NCI-H26/101细胞系,与A549、NCI-H26细胞相比,A549/101(t=26.03,P=0.000)、NCI-H26/101(t=23.29,P<0.01)细胞内COX-2表达量显著降低,A549/101和NCI-H26/101细胞的活力和克隆形成能力也显著降低(均P<0.05)。A549/101细胞小鼠皮下肿瘤的生长速度较A549细胞和A549/NC细胞显著减慢(F=14.81,P=0.003)。结论:肺癌组织和A549、NCI-H26细胞中miR-101低表达、COX-2高表达,过表达miR-101可以抑制A549、NCI-H26细胞的增殖,其机制可能与下调了COX-2表达有关。

Objective: To investigate the expression of miRNA-101( miR-101) in lung cancer specimens and lung cancer cell lines as well as its impact on the proliferative,colony-forming and tumorigenic capacities of human lung cancer cells. Methods: Cyclooxygenase-2( COX-2) and miR-101 mRNA and protein levels were assessed,respectively,by RTPCR and Western blotting in clinical specimens( lung cancer tissue and the surrounding normal tissue) collected from 10 patients,human lung cancer cell lines A549 and NCI-H226,and human diploid MRC-5 fibroblasts. A549 and NCI-H226 cells were transfected with miR-NC and miR-101 respectively. The transfectants were assessed for COX-2 protein content by Western blotting,proliferative activity by CCK-8,colony-forming capacity by colony formation experiments,and tumorigenic capacity in vivo by xenograft experiments in nude BAL B / C mice. Results: COX-2 protein content was significantly higher in cancer tissue specimens than in both para-cancer tissue specimens( t = 20. 03,P = 0. 001) and human diploid MRC-5 fibroblasts( t = 14. 59,P = 0. 012). In contrast,the expression of miR-101 was down-regulated in lung cancer tissue specimens and cell lines as compared with para-cancer tissue specimens( t = 18. 33,P = 0. 002) and human diploid MRC-5 fibroblasts( t = 28. 95,P = 0. 000). Overexpression of miR101 resulted in a significant decreases in COX-2 expression( t = 26. 03,P = 0. 000) and proliferative( F = 5. 783,P = 0. 017) and colony-forming( Dunnett t test I-J =- 0. 28,P = 0. 035) capacities of A549 and NCI-H226 lung cancer cells in vitro. A549 cells overexpressing miR101 had a remarkably reduced tumorigenic capacity in nude BALB / c mice in vivo( F = 14. 8,P = 0. 003). Conclusion:MicroR-101 is downregulated whereas COX-2 is up-regulated in lung cancer. Over-expression of miR-101 results in significant inhibition of proliferation,colony formation and tumorigenesis of lung cancer cells through down-regulation of COX-2and thus may offer a potential therapeutic option for lung cancer.