The Vitro Experimental Study Of CD133+CD44+EpCAM+Huh-7 Human Liver Cancer Cell Subsets And Their Stemness Biological Characteristics

Objective: This study to detect single surface marker expression and multiple surface marker co-expression of CD133,CD44,EpCAM and CD90 in SK-Hep-1 human liver cancer cell lines and Huh-7 human liver cancer cell lines by using flow cytometry.Screening out higher percentage positive cells of multiple surface marker co-expression and the corresponding negative cells of multiple surface marker co-expression,to research regeneration ability,proliferation,differentiation and drug resistant ability of the two kinds of cell subsets in vitro experiment.Methods:1.The SK-Hep-1 human liver cancer cells and the Huh-7 human liver cancer cells were cultured in vitro by using DMEM.single surface marker expression and multiple surface marker co-expression of CD133,CD44,EpCAM and CD90 were detected in SK-Hep-1 human liver cancer cell lines and Huh-7 human liver cancer cell lines by using flow cytometry,Huh-7 human liver cancer cells were selected for study in vitro experimental.2.The ability of Sphere-forming were observed CD133+CD44+EpCAM+Huh-7 human liver cancer cell subsets and CD133-CD44-EpCAM-Huh-7 human liver cancer cell subsets culturing in DMEM/F12.3.The proliferation ability of CD133+CD44+EpCAM+ Huh-7 human liver cancer cell subsets was compared with that of CD133-CD44-EpCAM-Huh-7 human liver cancer cell subsets by using plate cloning formation experiment.4.The drug resistance ability were detected of CD133+CD44+EpCAM+ Huh-7 human liver cancer cell subsets and CD133-CD44-EpCAM-Huh-7 human liver cancer cell subsets by using cell counting kit-8(CCK-8).5.The expression of proteins of CD133,CD44 and EpCAM in CD133+CD44+EpCAM+ Huh-7 human liver cancer cell subsets and CD133-CD44-EpCAM-Huh-7 human liver cancer cell subsets were detected by using western blot(WB).6.The expressions of stemness related genes Nanog and Sox2 in CD133+CD44+EpCAM+ Huh7 human liver cancer cell subsets and CD133-CD44-EpCAM-huh7 human liver cancer cell subsets were detected by using Real-time fluorescence quantitative polymerase chain reaction(qr-PCR).Result:1.In the SK-Hep-1 human liver cancer cells and the Huh-7 human liver cancer cells,the positive expression rates of CD133,CD44,EpCAM and CD90 were48.52%,31.99%,43.93%,0.37% and 0.21%,36.19%,10.08%,84.72%,respectively.The co-expression rates of CD133+CD44+EpCAM+ human liver cancer cells subsets were11.88% and 0.97%,respectively.2.CD133+CD44+EpCAM+ human liver cancer Huh-7cell subsets had stronger self-renewal ability than CD133-CD44-EpCAM-Huh-7 human liver cancer cell subsets in vitro.3.CD133+CD44+EpCAM+ Huh-7 human liver cancer cell subsets had stronger proliferation ability than CD133-CD44-EpCAM-Huh-7 human liver cancer cell subsets in vitro.4.CD133+CD44+EpCAM+Huh-7 human liver cancer cell subsets had stronger drug resistance ability than CD133-CD44-EpCAM-Huh-7 human liver cancer cell subsets in vitro.5.The expression level of CD133 protein,CD44 protein and EpCAM protein in CD133+CD44+EpCAM+ Huh-7 human liver cancer cell subsets was significantly different from that in CD133-CD44-EpCAM-Huh-7 human liver cancer cell subsets.6.The expressions levels of stemness related genes Nanog and Sox2 in CD133+CD44+EpCAM+Huh-7 human liver cancer cell subsets were higher than those in CD133-CD44-EpCAM-Huh-7 human liver cancer cell subsets.Conclusion: In this study,it was found that CD133+CD44+EpCAM+ Huh-7 human liver cancer cell subsets had high proliferation,self-renewal,differentiation and drug resistance in vitro.CD133+CD44+EpCAM+ Huh-7 human liver cancer cell subsets may represent a subsets of liver cancer stem cells,and CD133+CD44+EpCAM+ marker may be a more specific and reliable marker for the sorting of liver cancer stem cells.However,the stemness biological characteristics of CD133+CD44+EpCAM+ Huh-7 liver cancer stem cell subsets are needed to further confirm in vivo experiments.