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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 construct lentiviral vector of RAB27A gene and investigate the effect of RAB27A on the proliferation of HepG2 hepatocarcinoma cells. METHODS: The full-length RAB27A gene fused with green fluorescent protein was amplified by PCR using pEGFP-C1-RAB27A plasmid as a template. The full length of RAB27A gene was inserted into shuttle vector pENTR / U6 and then cloned into pENTR / U6 vector. DEST, the recombinant lentiviral expression vector pHAGE-GFP-RAB27A-puro was constructed. After sequencing, the recombinant plasmid was co-transfected into HEK-293T cells with packaging plasmid psPAX2 and envelope plasmid pMD2.G for lentivirus packaging. The culture supernatant was collected and concentrated to obtain lentivirus particles infected HepG2 cells. The green fluorescence intensity of HEK-293T cells and lentivirus-infected HepG2 cells was observed under a fluorescence microscope. The expression of RAB27A protein in HepG2 cells was detected by Western blot. The proliferation activity of HepG2 cells stably overexpressing RAB27A was detected by CCK8 assay and plate clone formation assay. Flow cytometry was used to detect the cell cycle distribution of HepG2 cells stably overexpressing RAB27A. Results: The recombinant lentiviral vector was confirmed by double enzyme digestion and sequencing. The titer of recombinant lentiviral vector was high after concentration. The expression of RAB27A protein was significantly up-regulated after HepG2 cells were infected with recombinant lentivirus. The proliferation activity of HepG2 cells (P <0.01). The percentage of cells in S phase was significantly decreased (P <0.01). Conclusion: The recombinant lentiviral vector of RAB27A gene was successfully constructed and the overexpression of RAB27A gene could significantly inhibit the proliferation of HepG2 cells. RAB27A plays an important role in the development and migration of hepatocellular carcinoma.