微小RNAlet-7c慢病毒载体的构建及过表达研究

Construction of microRNA let-7c lentiviral vector and its overexpression

目的构建微小RNAlet-7c重组慢病毒载体，验证转染该载体系统后对肝癌细胞增殖的影响。方法构建表达含494bplet-7c基因的重组慢病毒载体，检测let-7c及对照组病毒滴度分别为5．01×10E8TU／ml、5．15×10E8TU／ml；转染HepG2细胞，以荧光定量聚合酶链反应（PCR）对let-7c表达水平进行检测；细胞计数试剂盒（CCK-8）试验评价let-7c过表达后对HepG2细胞增殖的影响。结果构建的let-7c慢病毒载体经质粒酶切和测序鉴定正确；转染HepG2细胞72h后，let-Tc表达上调312倍；CCK-8法检测显示HepG2细胞增殖受到明显抑制（P〈0．01）。结论成功构建let-7c重组慢病毒载体并感染HepG2细胞后高效表达成熟let-7c，抑制了HepG2肝癌细胞的增殖。

Objective To construct recombinant lentiviral vector of miRNA let-7c and verify its effect on liver cancer cell proliferation. Methods Recombinant lentiviral vector containing 494 bp let-7c gene was constructed. The titers of Let-To and the controls were 5.01 × 10E8 and 5.15×10E8 TU/ml respectively. Let-To lentiviral vector was transfected into HepG2, and the expression of let-7c was detected by using real time polymerase chain reaction （PCR）. Cell counting Kit-8 （ CCK-8 ） method was used to testify HepG2 cell proliferation after the vector transfection. Results The successful construction of let-7c lentiviral vector was confirmed by plasmid enzyme digestion and DNA sequencing. Seventy-two h after the vector transfection, the expression of mature let-Tc was increased by about 312 folds, compared to blank vector transfection. The over-expression of let-7c significantly suppressed HepG2 cell proliferation （ P 〈 0. 01 ）. Conclusion The let-Tc recombinant lentiviral vector was successfully constructed. After the vector was transfected to HepG2 cells, mature let-7c was effectively expressed and suppressed the HepG2 liver cancer cell proliferation.