摘要
目的:研究微小RNA-200a(miR-200a)对肺癌细胞增殖的影响,并探讨其分子机制。方法:采用Real-time PCR检测15例非小细胞肺癌组织和对应癌旁组织、人肺癌细胞株(A549、NCI-H520、SK-MES-1)及人正常肺支气管上皮细胞株16HBE中miR-200a的表达水平。用CCK-8法检测miR-200a对A549肺癌细胞增殖活性的影响。通过生物信息学方法预测miR-200a可能的靶基因,双荧光素酶报告基因实验结合Real-time PCR和Western blot验证miR-200a对靶基因YAP1的调控作用。CCK-8法检测下调靶基因YAP1对A549肺癌细胞株增殖活性的影响。结果:miR-200a在非小细胞肺癌组织和肺癌细胞系中表达明显降低(P<0.01)。上调miR-200a表达后明显抑制A549肺癌细胞的增殖活力(P<0.01)。双荧光素酶报告基因显示miR-200a可以直接作用于靶基因YAP1的3'-UTR区域抑制荧光素酶活性(P<0.01),Real-time PCR和Western blot检测显示上调miR-200a的表达能够明显下调A549肺癌细胞YAP1 mRNA和蛋白的表达水平(P<0.01)。CCK-8法显示下调YAP1的表达能够明显抑制A549肺癌细胞的增殖活性(P<0.01)。结论:miR-200a通过靶向作用于YAP1基因来抑制肺癌细胞的增殖,从而在肺癌中发挥抑癌基因的功能。
Objective： To investigate the effects of miR-200a on the proliferation of lung cancer cells and to identify its direct target genes. Methods： Real-time PCR was performed to analyze the miR-200a expression in 15 paired clinical specimens of non-small cell lung cancer and adjacent noncancerous tissues, human lung cancer cell lines （A549, NCI-H520, and SK-MES-1）, and one human normal lung bronchial epithelial cell line （16HBE）. The effects of miR-200a on the proliferation of A549 lung cancer cells were detected through CCK-8 method. The candidate target genes of miR-2OOa were identified by bioinformatics screening and then verified by dual luciferase reporter gene assay, real-time PCR, and Western blot. The effects of YAP1 downregulation on the proliferation of A549 lung cancer cell line were also observed through CCK-8 method. Results： The miR-2OOa expression in non-small cell lung cancer tissues and lung cancer cell lines was significantly decreased （P〈0.01）. The upregulation of miR-2OOa expression could significantly inhibit the pro- liferation of A549 lung cancer cells （P〈0.01）. Dual luciferase reporter gene indicated that miR-2OOa could directly affect the 3＇-untrans- lated region of the YAP1 gene to inhibit luciferase activity （P〈0.01）. Real-time PCR and Western blot revealed that the upregulation of miR-2OOa expression could significantly reduce the mRNA and protein expression levels of YAP1 in A549 lung cancer cells （P〈0.01）. CCK-8 method indicated that the downregulation of YAP1 could significantly prevent the proliferation of A549 lung cancer cells （P〈 0.01）. Conclusion： MiR-200a inhibits the proliferation of lung cancer cells by targeting YAP1. Thus, miR-200a elicits tumor suppression effects.
出处
《中国肿瘤临床》
CAS
CSCD
北大核心
2017年第7期311-315,共5页
Chinese Journal of Clinical Oncology
