慢病毒介导的miR-34a过表达增强阿霉素对人肝癌细胞的抑制作用

LV-hsa-mir-34a enhance the inhibitory effects of Doxorubicin on hepatocellular carcinoma ceils

目的构建miR-34a重组慢病毒载体，观察其转染后对肝癌细胞活力、细胞周期和凋亡的影响．并观察转染后对阿霉素体外抑制肝癌细胞生长的影响。方法构建含miR-34a基因的重组慢病毒载体，转染人肝癌细胞并阿霉素干预后，测定细胞活性、检测细胞周期、凋亡及相关蛋白表达水平的变化。结果成功构建了miR．34a的重组慢病毒载体LV-hsa—mir-34a（元件顺序：Ubi—MCS—SV40-EGFP—IRES-puromycin）。经质粒酶切和测序鉴定正确。与对照组比较，重组慢病毒载体转染肝癌细胞后miR-34a表达上调，差异有统计学意义（HepG2：t=15．36，P〈0．01；Hep3B：t=36．75，P〈0．01：Bel-7402：t=24．17，P〈0．01）。与对照组比较，miR-34a表达上调并阿霉素干预后肝癌细胞活力下降（HepG2：t=7．12，P〈0．01；Hep3B：t=8．89，P〈0．01；Bel-7402：t=13．62，P〈0．01），G1期细胞显著增多（HepG2：F=137．65，P〈0．01；Hep3B：F=143．39，P〈0．01；Bei-7402：F=1306．47，P〈0．01），细胞凋亡增加（HepG2：F=386．14，P〈0．01；Hep3B：F=881．94，P〈0．01；Bel-7402：F=885．89，P〈0．01），差异有统计学意义，细胞周期蛋白和凋亡蛋白发生相应改变。结论构建miR．34a重组慢病毒载体并转染肝癌细胞后可高效表达miR-34a，miR-34a高表达可以降低肝癌细胞的恶性生物学行为。miR-34a重组慢病毒载体感染肝癌细胞后，阿霉素对肝癌细胞的抑制作用显著增强。

Objective To construct recombinant lentiviral vector of mieroRNA-34a and observe the cell viability, cell cycle and apoptosis of hepatocellular carcinoma cells transfected with the vector system and treated with Doxorubicin. Methods Recombinant lentiviral vector containing microRNA-34a gene was constructed and transfected into 3 hepatoceUular carcinoma cell lines, and cells were treated with Doxorubicin. The expression of microRNA-34a gene was detected by real-time PCR. The effect of microRNA- 34a overexpression on hepatocellular carcinoma cells proliferation were quantified via MTT assay, cell cycle and apoptosis was evaluated by flow cytometry. Western blotting was used to evaluate the expression of cell cycle and apoptosis related protein. Results The successful construction of microRNA-34a recombinant lentiviral vector was confirmed by plasmid enzyme digestion and DNA sequencing. Compared with the control group, relative expression of microRNA-34a gene in hepatocellular carcinoma cells significantly increased （（HepG2： t=15.36, P〈0.01; Hep3B： t=36.75, P〈0.01; Bel-7402： t=24.17, P〈0.01））. Cells viability decreased （HepG2. t =7.12, P 〈0. 01 ; Hep3B： t =8.89, P 〈0. 01 ; Bel-7402： t = 13.62, P 〈 0.01 ） , G1 phase cells increased significantly（ HepG2 ： F = 137.65, P 〈 0. 01 ; hep3B ： F -： 143.39, P 〈 0. 01 ; Bel-7402 ： F = 1 306.47, P 〈 0. 01 ） and cell apoptosis increased （ HepG2 ： F = 386. 14, P 〈 0. 01 ; Hep3B： F = 881.94, P 〈 0. 01 ; Bel-7402： F = 885.89, P 〈 0. 01 ）. Conclusions MicroRNA-34a recombinant lentiviral vector （LV-hsa-mir-34a） transfected hepatocellular carcinoma cells overexpress microRNA-34a, reduce the malignant biological behavior. MicroRNA-34a recombinant lentiviral vector （ LV- hsa-mir-34a） enhance the in vitro inhibitory effects of Doxorubicin on hepatocellular carcinoma cell lines.