摘要
目的探讨微小RNA-204（miR-204）在非小细胞肺癌（NSCLC）组织中的表达及其对A549细胞增殖、凋亡和迁移能力的影响和对B淋巴细胞瘤-2（bcl-2）的靶向作用机制。方法以45例非小细胞肺癌组织、A549细胞株为研究对象，通过实时定量聚合酶链反应检测miR-204在NSCLC中的表达；转染miR-204模拟物和抑制剂，用细胞计数试剂盒（CCK-8）、Transwell、划痕实验、半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-3活性检测细胞增殖、迁移、凋亡能力变化；构建含有bcl-23’-非编码区野生型和突变型的双荧光素酶报告载体，并通过功能恢复实验，验证miR-204与bcl-2的靶向天系，结果miR-204在非小细胞肺癌组织中的表达（1．28±0．31）较对照正常组织（3．75±0．27）明显降低（P=0．000），bcl-2在癌组织中表达明显增高（3．46±0．40比0．78±0．48，P=0．000），与TNM分期呈显著负相关，与年龄、性别关系差异无统计学意义；miR-204能明显抑制A549细胞的增殖、迁移，并促进细胞凋亡能力（P=0．000）。共转染miR-204和bcl-2野生型3’非编码区双荧比素酶表达载体的A549细胞荧光活性为0．12±0．02，较对照组（0．29±0．03）显著下降（P=0．000），miR-204对细胞的增殖、凋亡、迁移能力的影响可以被过表达bcl-2所恢复。结论miR-204存非小细胞肺癌中表达下调，可能通过靶向调控bel-2的表达影响非小细胞肺癌的增殖、迁移、凋亡能力。
Objective To investigate the expression of mieroRNA - 204 （ miR - 204 ） in non - small cell lung cancer （NSCLC） tissues and the molecular mechanism of targeting B cell lymphoma - 2 （bcl - 2） in the regulation of cell proliferation, apoptosis and invasion in A549 cell line. Methods The expressiun of miR - 204 was detected by real - time polymerase chain reaction in 45 NSCLC tissues. Cell counling kit - 8 （ CCK - 8） assay, Transwell assay and eysteinyl aspartate - specific protease （Caspase） - 3 assay were performed to access cell line proliferation, invasion and apoptosis ability of A549 cell line. The target gene of miR - 204 was analyzed by bioinformaties combined with Dual - lueiferase assay and function recovery assays. Results miR - 204 expression level was down - regulated in NSCLC tissues compared with adjacent normal tissues （ 1.28 ± 0. 31 and 3.75± 0. 27, P = 0. 000） while the expression of bel - 2 was up - regulated in NSCLC tissues （3.46 ±0. 40 and 0. 78 ±0. 48, P =0. 000）. Furthermore, the expression levels were correlated with the pathologic grades. MiR- 204 can significantly decrease the proliferation and iuvasion ability, as well as increase apoptosis （P =0. 000）. Furthermore, a significant reduction of lucit）rase activity was detected in A549 cell after eo - transfected with miR - 204 and Dual - lueiferase expression vector containing bel - 2 wide - type 3 ＇ - untranslated region when compared with muted - type region group （0. 12 ±0. 02 and 0. 29 ±0. 03, P =0. 000）. The biological behavior effect on A549 cell can be recovered through over express the level of bcl - 2. Conclusion miR - 204 is down - regulated in NSCLC, and regulate the proliteration, invasion and apoptosis of A549 cells via targeting bcl -2.
出处
《中华实验外科杂志》
CSCD
北大核心
2017年第7期1160-1162,共3页
Chinese Journal of Experimental Surgery
基金
2015年深圳市科技研发资金基础研究项目（JCYJ20150403091443310）
