上调miR-200a表达对人肝癌细胞MHCC97中边缘群细胞干细胞特性的影响

Effects of miR-200a up-regulation on the characteristics of stem cells of side population in hepatocellular carcinoma cell line MHCC97

目的:探讨转染miR-200a mimics对人肝癌细胞系MHCC97中边缘群细胞(SP)干细胞特性的影响。方法:利用流式细胞技术从MHCC97细胞中分离出SP细胞。转染组为应用脂质体介导转染miR-200a mimics的SP细胞,对照组为未接受转染的SP细胞。采用实时荧光定量RT-PCR检测两组SP细胞miR-200a的表达情况。四甲基偶氮唑盐(MTT)和软琼脂克隆形成实验检测细胞增殖情况。Transwell小室检测细胞迁移能力。裸鼠成瘤实验检测细胞体外成瘤能力。结果:转染miR-200a mimics可明显上调SP细胞miR-200a的表达。MTT结果显示转染组SP细胞的增殖能力明显低于对照组(P<0.05),转染组软琼脂中克隆形成率(24.70±5.54)%明显低于对照组(51.63±7.11)%(P<0.05)。Transwell实验显示转染组细胞穿膜数为(18.52±4.27)个,明显少于对照组(63.34±7.41)个(P<0.05)。裸鼠成瘤实验中转染组成瘤数(1/8)也低于对照组(8/8)(P<0.05)。结论:上调人肝癌细胞系MHCC97中SP细胞miR-200a的表达,能够抑制SP细胞的增殖和迁移,降低体外成瘤能力,即抑制SP细胞的干细胞特性。

Objective：To investigatet the effects of miR-200a up-regulation on the characteristics of stem cells of side population in hepatocellular carcinoma cell line MHCC97. Methods：SP（side population）cells were isolated by fluorescence activated cell sorting（ FACS）from MHCC97 cell line of human hepatocellular carcinoma. The miR-200a mimics was transfected into SP cells by liposome. The expression levels of miR-200a in SP cell pre- and post-tranfection was measured by reverse transcription polymerase chain reaction（ RT-PCR ）. MTT method and Soft agar cloning method was used to evalute the capability of cell proliferation. The invasion and migration capability of cells were evaluated by using transwell chamber model. Tumor cell implantation experiment was done to test the tu-morigenicity. Results：The expression of miR-200a in SP cell was obviously up-regulated transfection of post miR-200a mimics. MTT assay showed that the cell proliferation ability of SP in transfected group was obviously lower than control group（without transfection）（P〈0. 05）. The rate of clone formation was（24. 70±5. 54）% in trans-fected group and it was（51. 63 ± 7. 11）% in control group（P〈0. 05）. Transwell chamber assay demonstrated the cell penetrating number was 18. 52 ＆#177; 4. 27 in transfected group and it was 63.34 ±7.41 in control group（P〈0. 05）. The number of tumors that formed in transfected group（1/8）was significantly lower than in the control group （8/8）（P〈0. 05）. Conclusion：Up-regulation of the miR-200a expression in SP cells of MHCC97 cell line would inhibit their ability of proliferation and migration,decrease their tumor formation ability in vitro,which is indicate the inhibition of the stem cell characteristics of SP cells.