miR-122慢病毒载体的构建及其在肝癌细胞中的作用

目的构建慢病毒载体并探讨稳定表达的miR-122在肝癌细胞中的作用。方法将miR-122的前体克隆到慢病毒载体pLUNIG上获得稳定表达miR-122的病毒载体,在293T细胞内进行包装,浓缩获得病毒上清。用病毒上清去感染肝癌细胞HepG2,采用MTT法及克隆增生检测对肝癌细胞增殖的影响,并设空载体组为对照进行分析。各组间均数采用t检验。结果慢病毒质粒Lv-miR122经包装浓缩后收获的病毒上清有效的感染肝癌细胞且miR-122表达显著上调,与慢病毒空载体对照组（Lv-Ctrl）相比,差异具有统计学意义（t=10.745,P〈0.01）;稳定表达的miR-122能明显抑制肝癌细胞的增殖,与Lv-Ctrl和HepG2对照组相比差异有统计学意义（P均〈0.05）;而且miR-122能明显抑制肝癌细胞的克隆增生（t=-5.256,P〈0.01）。结论成功的构建了稳定表达miR-122的慢病毒质粒,并使携带的miR-122能稳定表达且具有抑制肝癌细胞增殖的活性。

Construction of lentiviral vector carrying miR- 122 and function of miR- 122 in hepatocellular carcinoma cells

Objective To construct the lentiviral vector carrying miR - 122 （ Lv - miR122 ） and investigate the function of stably expressed miR - 122 in hepatoeellular carcinoma （ HCC ） cells. Methods Pre - miR - 122 was cloned into lentiviral vector pLUNIG to obtain the Lv - miR122 that had stable expression of miR - 122; Lv - miRl22 was packaged in 293T cells along with other three vectors pRRE, pRSV - REV, and pCMV - VSVG, and virus supernatant was obtained by concentration; the virus supernatant was used to infect HCC cell line HepG2. The expression of miR - 122 was measured by Q - RT - PCR. The proliferation of HepG2 cells was analyzed by MTT assay and col- ony formation assay. Blank lentiviral vector （ Lv - Ctrl） - infected HepG2 cells and uninfected HepG2 cells were used as controls. Results The expression of miR - 122 was significantly higher in Lv -miR122 - infected HepG2 ceils than in Lv - Ctrl - infected HepG2 cells （t = 10. 745, P 〈 0. 01 ）. The Lv -miR122 -infected HepG2 cells （with stable expression of miR- 122） had significantly suppressed prolifera- tion compared with Lv - Ctrl - infected and uninfected HepG2 cells （P 〈 0.05 for both comparisons） ; moreover, miR - 122 significantly in- hibited the colony formation of HepG2 cells （t = 5. 256, P 〈 0.01 ）. Conclusion The Lv - miR122 can be successfully constructed, and the carried miR - 122 can be stably expressed and can inhibit the proliferation of HepG2 cells.