人pre-miR-145真核表达载体的构建及其对胃癌细胞游走、侵袭活性的影响

Construction of eukaryotic expression vector of human pre-miR-145 and its effects on the migration and invasion of gastric cancer in vitro

目的构建人微小RNAl45（miR-145）前体的真核表达载体，探讨其对胃癌细胞生物学行为的影响。方法根据人pre-miR-145序列，合成两条互补的寡核苷酸链，退火后连接人pPG-miR-EGFP载体，转化扩增后进行测序鉴定；重组质粒在阳离子脂质体Lipofectamine2000的介导下，瞬时转染人胃癌细胞株SGC-7901，实时定量聚合酶链反应（PCR）检测细胞中miR．145表达水平，黏附实验和Transwell小室实验检测癌细胞游走、侵袭能力。结果所构建的pPG—miRl45-EGFP载体中插入基因片段与设计序列完全匹配；重组质粒转染SGC．7901细胞后，miR-145表达分别上调20．45倍（P〈0．01），细胞黏附能力分别下降51．5％（P〈0．01），细胞侵袭能力分别下降30．4％（P〈0．01）。结论成功构建人pre—miR-145真核表达载体，转染胃癌细胞过表达后，能抑制癌细胞的游走和侵袭能力。

Objective To construct the eukaryotic expression vector of miR-145 and explore its effects on the biological behavior of gastric cancer. Methods According to the pre-miR-145 sequence, two complementary oligonucleotide strands were synthesized and inserted into pPG-miR-EGFP vector, which was validated by nucleic acid sequencing. Under the induction of Lipofectamine 2000, the recombinant was transfected into SGC-7901 cells. The expression level of miR-145 was detected by real-time reverse tran- scription-polymerase chain reaction （RT-PCR）. The cell adhesion and transwell assays were applied for the detection of in vitro migration and invasion of cancer cells. Results Sequencing analysis confirmed the cor- rectness of the construction. Compared with negative control,the expression of miR-145 was upregulated by 20.45 times （ P 〈 0. 01 ）. Meanwhile, the cellular adhesion was downregulated by 51.5 % （ P 〈 0. 01 ）, and the invasive activity was downregulated by 30. 4% （P 〈 0. 01 ）. Conclusion The expression vector of pre- miR-145 was successfully constructed,and induced the miR-145 overexpression in gastric cancer cells,resuiting in decrease of migration and invasion of cancer cells.