Abstract: 【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.