miRNA-451表达质粒的构建及对人A549肺癌细胞侵袭转移及凋亡的影响

Construction of miRNA-451 expression plasmid and its effect on invasion,metastasis and apoptosis in human A549 lung cancer cells

目的:探讨miRNA-451对人A549肺癌细胞侵袭转移及凋亡的影响。方法:Hsa-miRNA-451序列通过miRBase数据库查找,根据此序列设计出2条寡核苷酸片段,将目的基因经过退火处理后克隆至真核表达载体pGenesil-1.1质粒中,从而构建成为miRNA-451真核表达载体(pGenesil-1.1-miRNA-451质粒)。将pGenesil-1.1-miRNA-451及pGenesil-1.1-control质粒分别转染人A549肺癌细胞,经过G418的筛选,建立pGenesil-1.1-miRNA-451及pGenesil-1.1-control稳定表达的细胞株。流式细胞术分析检测细胞凋亡的情况;Transwell小室检测miRNA-451对细胞侵袭能力的影响;细胞划痕实验检测细胞的迁移情况。结果:经测序证实miRNA-451真核表达质粒构建成功,与未处理的人A549肺癌细胞和稳定表达pGenesil-1.1-control的细胞相比,稳定表达了pGenesil-1-miRNA-451的人A549肺癌细胞的侵袭和迁移能力均明显受到抑制影响,而细胞凋亡无明显变化(P>0.05)。结论:miRNA-451对人A549肺癌细胞侵袭转移具有显著的抑制作用,但对细胞凋亡作用不明显。

Objective：To explore of the effect of miRNA-451 on invasion, metastasis and apoptosis of human A549 lung cancer cells. Methods：Hsa-miRNA-451 sequence was found in miRBase database and two oligonucleotides were designed according to the sequence. The targeted gene was annealed and cloned to eukaryotic expression vector plasmid pGenesil-1.1 thus miRNA--451 eukaryotic expression vector was built （pGenesil- 1.1 -of miRNA -451 plasmid ）. PGenesil- 1.1 -miRNA -451 and pGenesil- 1.1 -control plasmid were transfected into human A549 lung cancer cells and stably expressed pGenesil-1.1-miRNA-451 and pGenesil-1.1-control cell lines were established after being selected by G418. Flow eytometry was employed to detect cell apoptosis, Transwell chamber to detect miRNA-451 cell invasion and cell scratch assay to detect cell metastasis. Results：Sequencing showed that miRNA-451 eukaryotic expression plasmids were successfully constructed. Cell invasion and metastasis were inhibited in stably expressed pGenesil-1.1- miRNA-451 cell lines compared with those in untreated human A549 lung cancer cells and stably expressed pGenesil-1.1-control cell lines, however,there was no change in cell apoptosis（P〉0.05）. Conclusion：miRNA-451 has significant inhibitory effect on invasion and metastasis of human A549 lung cancer cell, however, not on cell apoptosis.