人miR-29b过表达慢病毒载体的构建及其对A549细胞迁移的影响

To Construct Lentiviral Vector with Over-expressing mi R-29b and To Explore Its Effect on Cell Migration in A549 Cell

[目的]构建能高效表达人mi R-29b的慢病毒载体,研究其对人肺腺癌A549细胞迁移的影响。[方法]PCR扩增mi R-29b前体序列,克隆入慢病毒表达载体,测序鉴定。包装好的慢病毒感染A549细胞,流式细胞仪分选后用荧光定量PCR检测mi R-29b表达水平,划痕实验观察过表达mi R-29b对A549细胞迁移的影响。[结果]测序结果证明成功构建人mi R-29b过表达慢病毒载体。mi R-29b过表达慢病毒感染效率为88.56%,使A549细胞中mi R-29b表达升高2.78倍。与A549细胞相比,稳定过表达mi R-29b的A549-mi R-29b细胞的迁移能力显著性降低。[结论]成功构建mi R-29b过表达慢病毒载体,过表达mi R-29b可抑制A549细胞的迁移,为进一步研究mi RNA对肿瘤转移的调控作用奠定了基础。

[Purpose] To construct the lentiviral vector with overexpressing mi R-29 b,and to explore its effect on migration in human lung adenocarcinama cells A549. [Methods] The precursor of mi R-29 b was amplified by PCR and inserted into a lentiviral vector and sequenced. The recombinant lentiviral vector was further packaged for generating lentiviral to infect A549 cells. Sta-ble mi R-29 b overexpressed A549-mi R-29 b cells were sorted by flow cytometry and detected the expression of mi R-29 b by real-time PCR. Wound healing assay was used to evaluate the effect of overexpressed mi R-29 b on cell migration of A549 cells. [Results] DNA sequencing demonstrated that mi R-29 b lentiviral vector was successfully constructed. The transduction efficiency in human A549 cells reached a level of 88.56%. The expression level of mi R-29 b increased 2.78 folds in infected A549 cell. Compared to A549 cell group,the migration abilities of A549-mi R-29 b cells significantly decreased. [Conclusion] The mi R-29 b lentiviral vector is constructed successfully.Overexpression of mi R-29 b could suppress migration abilities in A549 cell. It constructs the foundation of mi RNA for regulation in the further research on tumor metastasis.