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黑色素瘤抗原A3基因过表达对人肝癌细胞株的影响

Effects of over-expression of melanoma antigen gene A3 on human hepatocellular carcinoma cell lines

摘要:

目的 观察黑色素瘤抗原A3(MAGE-A3)基因过表达对人肝癌细胞株增殖、凋亡、迁移及侵袭的影响,探讨MAGE-A3基因在肝细胞癌发生发展过程中的作用.方法 将MAGE-A3重组慢病毒表达载体(LV-MAGE-A3)感染人肝癌细胞株HepG2和MHCC-97H;逆转录-聚合酶链反应(RT-PCR)和Western blot技术检测MAGE-A3 mRNA和蛋白在肝癌细胞中的表达;四甲基偶氮唑蓝(MTT)、平板克隆形成实验、Transwell迁移及侵袭实验检测MAGE-A3基因过表达前后肝癌细胞的增殖、迁移及侵袭能力的变化;原位末端脱氧核糖核酸转移酶标记DNA链断端分析法(TUNEL)检测肝癌细胞的凋亡,流式细胞术(FCM)分析细胞周期的改变.结果 病毒感染肝癌细胞120 h荧光达到最强且稳定,感染效率可达80%.RT-PCR和Western blot检测证实经LV-MAGE-A3感染的细胞高效表达MAGE-A3 mRNA与蛋白.HepG2和MHCC-97H细胞经LV-MAGE-A3感染后,迁移细胞数达到(187.00±13.52)、(121.00 ±4.16)个,侵袭细胞数达到(164.00±9.64)、(59.50±1.00)个,与对照组比较差异有统计学意义(P<0.01);FCM检测发现S期细胞比例增加,G1期细胞减少.肝癌细胞的生长增殖、克隆形成能力和凋亡指数在各组间差异无统计学意义(P>0.05).结论 MAGE-A3基因的过表达能提高肝癌细胞的迁移和侵袭能力,提示MAGE-A3基因与肝细胞癌的进展相关.

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abstracts:

Objective To observe effects of over-expression of melanoma antigen gene (MAGE) A3 on the progression,apoptosis,metastasis and invasion in human hepatocellular carcinoma cell lines,and investigate its effect on hepatocellular carcinoma (HCC) progession.Methods The lentivirus vector with recombinant MAGE-A3 gene (LV-MAGE-A3) were constructed and transfected into HepG2 and MHCC-97H cell lines and cell only or transfected with empty vector were as controls.The expression of green fluorescent protein was observed by inverted fluorescent microscope to detect tranduced efficiency.MAGE-A3 gene and protein expression was assayed by reverse transcription-polymerase chain reaction (RT-PCR) and Western-Blot technology.The effects of MAGE-A3 over-expression on proliferation,migration and invasion of HCC were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays,clone formation assays and transwell technique.Cell apoptosis was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) and cell cycle was analysed by flow cytometry (FCM).Results After lentivirus vector transfection for 120 hours,the expression of green fluorescent protein was the strongest and most stable in cells.The transfection efficiency was up to 80%.After transfected with LV-MAGE-A3,the expression of MAGE-A3 gene and the protein in HepG2 and MHCC-97 cells was higher than that of controls.The cell numbers of migration and invasion were 187.00 ± 13.52,121.00 ±4.16 in HepG2 and 164.00 ±9.64,59.50 ± 1.00 in MHCC-97,which significantly increased compared to control group (P <0.01).The cell numbers of S stage increased by FCM.However,the cell proliferation,colony-forming ability and apoptosis of HCC cell lines had no statistical significance between LV-MAGE-A3 group and control (P > 0.05).Conclusion MAGE-A3 gene promote the abilities of migration and invasion in HCC cell lines.It suggests that MAGE-A3 expression is closely corelated to HCC progression.

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