慢病毒载体介导RAB27A基因过表达对人HepG2肝癌细胞增殖的影响

Construction of Lentiviral Expression Vector Expressing Human RAB27A and Investigation Its Effect on the Proliferation of HepG2 Cell Lines

目的:构建RAB27A基因慢病毒表达载体,并研究RAB27A对人HepG2肝癌细胞增殖能力的影响。方法:以pEGFP-C1-RAB27A质粒为模板,PCR扩增出融合绿色荧光蛋白的RAB27A基因全长,酶切后插入穿梭载体pENTR/U6,再应用Gateway技术,基因重组到表达载体pHAGEEF1α-puro-DEST上,构建得到重组慢病毒表达载体pHAGE-GFP-RAB27A-puro。测序鉴定序列正确后,将其与包装质粒psPAX2和包膜质粒pMD2.G共转HEK-293T细胞进行慢病毒包装。收集并浓缩培养上清以获得慢病毒颗粒感染HepG2细胞。荧光显微镜下观察HEK-293T细胞和慢病毒感染HepG2细胞绿色荧光强度;Western blot检测稳定感染HepG2细胞株RAB27A蛋白表达水平;CCK8和平皿克隆形成实验检测稳定过表达RAB27A的HepG2细胞增殖活力的变化;流式细胞术检测稳定过表达RAB27A的HepG2细胞周期分布情况。结果:经双酶切及测序结果证实重组慢病毒表达载体构建正确;浓缩后病毒滴度较高;重组慢病毒感染HepG2细胞后,细胞外源RAB27A的蛋白表达水平显著上调,HepG2细胞的增殖活力和克隆形成能力受到明显抑制(P<0.01),S期细胞分布比例明显降低(P<0.01)。结论:RAB27A基因重组慢病毒表达载体构建成功,外源过表达RAB27A基因可显著抑制HepG2细胞增殖能力。RAB27A在肝细胞癌发生发展和迁移中扮演了重要角色。

Objective： To over express RAB27A by lentiviral system in human hepatocellular carcinoma （HCC） cell line HepG2 and investigate the effect. Methods：GFP-RAB27A protein coding gene sequences were amplified from plasmid pEGFP-C1-RAB27A, the PCR products were digested by double restriction enzymes and cloned into shuttle plasmid pENTR/U6, then pHAGE-GFP-RAB27A vector were constructed by Gateway technology. Recombinant plasmid pHAGE-GFP-RAB27A was mixed with the packaging plasmid （psPAX2） and the envelope plasmid （pMD2. G）and then co-transfected into HEK-293T cells. HepG2 cells were transfected with concentrated recombinant lentivirus particle. Western blot analysis were used to detect RAB27A expression in HepG2 cell lines. The proliferative activity was determined by CCK-8 and colony forming assay and the cell cycle analysis by flow cytometry. Results：The recombinant lentiviral expression vector pHAGE-GFP-RAB27A was constructed correctly. After transfected to HEK-293T, the lentivirus was successfully prepared. Strong green fluorescence was observed in transfected HepG2 cells under fluorescent microscope which showed the recombinant lentivirus expressing. The RAB27A protein was over expressed showed by Western-blot. The proliferation of infected HepG2 showed inhibited, and the percentage of S-phase cells was obviously decreased （P 〈 0.01 ）. Conclusion： The exogenous over expressed RAB27A was found inhibited the proliferation of transfected HepG2 cell lines. RAB27A is a key protein in the hepatocellular carcinogenesis and proliferation.