转录因子CTCF对人肝癌干细胞及细胞增殖的影响

目的 探讨CCCTC结合因子 （CTCF）蛋白对人肝癌细胞 （HepG2）的肿瘤干细胞的影响以及对HepG2和CNE1 （鼻咽癌细胞系）细胞生存能力的影响。方法 构建pEGFP-N1/CTCF、CTCF shRNA和GFP-shRNA质粒，转染至HepG2和CNE1细胞后，以RT-PCR及Western blot 检测CTCF mRNA和蛋白的表达。采用流式细胞术检测转染CTCF-shRNA质粒后48 h的HepG2细胞中携带表面抗原CD90肿瘤干细胞的比例，以转染GFP-shRNA的HepG2细胞和野生型HepG2细胞为对照。采用MTT方法分别检测转染CTCF过表达重组质粒及CTCF-shRNA质粒后24、48、72 h HepG2和CNE1细胞的生存能力。 结果 pEGFP-N1/CTCF转染后增加HepG2和CNE1细胞中CTCF mRNA和蛋白的表达（与pEGFP-N1相比，P<0.05），CTCF-shRNA转染则降低表达（GFP-shRNA相比，P<0.05）。流式细胞仪检测结果显示，CD90 +细胞的检出率在转染CTCF-shRNA质粒细胞 〔(1.733 0±0.417 7)%〕高于野生型HepG2细胞 〔(0.575 0±0.062 9)%〕及转染对照GFP-shRNA质粒细胞 〔(0.350 0±0.086 6)%〕 （P<0.05）；MTT实验结果显示CTCF的表达改变对HepG2和CNE1 细胞的生存能力的影响不明显 （P>0.05）。结论 CTCF可抑制人肝癌干细胞生成，对HepG2和CNE1细胞生长无明显影响。

Effects of CTCF on Human Liver Cancer Stem Cells and Cell Proliferation

【Abstract】 Objective To explore the effects of CCCTC-binding factor (CTCF) on human liver cancer stem cells (HepG2) and cell proliferation of HepG2 and Nasopharyngeal carcinoma cell line (CNE1). Methods The pEGFP-N1/CTCF、CTCF-shRNA and GFP-shRNA plasmids were constructed and transfected into HepG2 and CNE1 cells, and RT-PCR or Western blot were performed to detect the mRNA or protein levels of CTCF. The subpopulation of CD90+ cancer stem cells in HepG2 cells transfected with CTCF-shRNA plasmid or GFP-shRNA plasmid (as transfection control) were assayed by flow cytometry with the wild type HepG2 cells as control. Proliferation of cells transfected with CTCF-overexpression orCTCF-shRNA plasmid was evaluated by MTT assay. Results The levels of both mRNA and protein of CTCF were increased in pEGFP-N1/CTCF transfected HepG2 and CNE1 cells compared to that in pEGFP-N1 transfected cells (P <0.05), and decreased in CTCF-shRNA transfected cells compared to that in cells transfected with GFP-shRNA (P<0.05). The results of flow cytometry demonstrated that, detection rate of CD90+ cells in cells transfected with CTCF-shRNA plasmid 〔(1.733 0±0.417 7)%〕was obviously higher than that of wild-type HepG2 cells 〔(0.575 0±0.062 9)%〕 and cells transfected with GFP-shRNA plasmid 〔(0.350 0±0.086 6)%〕 (P<0.05). The results of MTT analysis showed that, alteration of CTCF had no effect on cancer cell proliferation (P>0.05). Conclusion CTCF inhibits human liver cancer stem cells but no effect on cell proliferation.