title: Knock out CD44 in reprogrammed liver cancer cell C3A increases CSCs stemness and promotes differentiation.
abstract: CD44 is a widely known cancer stem cells marker in various cancers and validated to function in tumor growth, survival and tumor metastasis. In this study, we first established C3A-derived liver cancer stem cells by OSKM method [OCT4, SOX2, KLF4, and c-MYC], termed C3A-induced cancer stem cells (C3A-iCSCs) which acquired self-renewal and stemness abilities. Then we found CD44 was positive in C3A-iCSCs and mainly located in cell nuclear. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) results showed nuclear CD44 combined promoter regions of c-MYC and SOX2. These results suggested that CD44 participated in C3A-iCSCs transcriptional regulation. To explore CD44 overall influence in liver cancer stem cells, CD44 was knocked out in C3A-iCSCs using CRISPR/Cas9 technology. Our results showed a dramatic increase in the expression of stem cell markers OCT4, SOX2 and NANOG in CD44- C3A-iCSCs compared with that in CD44+ C3A-iCSCs. Tumor derived from CD44- C3A-iCSCs also displayed well-differentiated tumor cells compared to CD44+ C3A-iCSCs, which suggested CD44- C3A-iCSCs derived tumor cells exhibited lower malignant degree. Our data indicated nuclear CD44 in liver cancer stem cells is responsible for the poorly differentiated highly malignant tumor cells by maintenance of low stemness state.
authors.affiliation: Department of Cell Biology, Stem Cell Research Center, Department of Basic Medical Sciences, Peking University Health Science Center, Beijing, People's Republic of China.
authors.forename: Shuo
authors.initials: S
authors.lastname: Han
authors.affiliation: Beijing DongFang YaMei Gene Science and Technology Research Institute, Beijing, People's Republic of China.
authors.forename: Jinhai
authors.initials: J
authors.lastname: Guo
authors.affiliation: Department of Cell Biology, Stem Cell Research Center, Department of Basic Medical Sciences, Peking University Health Science Center, Beijing, People's Republic of China.
authors.forename: Yinan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Organ Failure Research, Co-Innovation Center for Organ Failure Research, Guangdong Provincial Research Center of Artificial Organ and Tissue Engineering, Second Department of Hepatobiliary Surgery, ZhuJiang Hospital, Southern Medical University, Guangzhou, People's Republic of China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Cell Biology, Stem Cell Research Center, Department of Basic Medical Sciences, Peking University Health Science Center, Beijing, People's Republic of China.
authors.forename: Qihua
authors.initials: Q
authors.lastname: He
authors.affiliation: Department of Cell Biology, Stem Cell Research Center, Department of Basic Medical Sciences, Peking University Health Science Center, Beijing, People's Republic of China.
authors.forename: Peng
authors.initials: P
authors.lastname: Li
authors.affiliation: Department of Cell Biology, Stem Cell Research Center, Department of Basic Medical Sciences, Peking University Health Science Center, Beijing, People's Republic of China.
authors.forename: Mingzhi
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Department of Cell Biology, Stem Cell Research Center, Department of Basic Medical Sciences, Peking University Health Science Center, Beijing, People's Republic of China.
authors.forename: Haojie
authors.initials: H
authors.lastname: Sun
authors.affiliation: Department of Cell Biology, Stem Cell Research Center, Department of Basic Medical Sciences, Peking University Health Science Center, Beijing, People's Republic of China.
authors.forename: Ruizhi
authors.initials: R
authors.lastname: Li
authors.affiliation: State Key Laboratory of Organ Failure Research, Co-Innovation Center for Organ Failure Research, Guangdong Provincial Research Center of Artificial Organ and Tissue Engineering, Second Department of Hepatobiliary Surgery, ZhuJiang Hospital, Southern Medical University, Guangzhou, People's Republic of China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.affiliation: Beijing DongFang YaMei Gene Science and Technology Research Institute, Beijing, People's Republic of China.
authors.forename: Wotan
authors.initials: W
authors.lastname: Zeng
authors.affiliation: Beijing DongFang YaMei Gene Science and Technology Research Institute, Beijing, People's Republic of China.
authors.forename: Jinwen
authors.initials: J
authors.lastname: Liu
authors.affiliation: Beijing DongFang YaMei Gene Science and Technology Research Institute, Beijing, People's Republic of China.
authors.forename: Lejian
authors.initials: L
authors.lastname: Lian
authors.affiliation: State Key Laboratory of Organ Failure Research, Co-Innovation Center for Organ Failure Research, Guangdong Provincial Research Center of Artificial Organ and Tissue Engineering, Second Department of Hepatobiliary Surgery, ZhuJiang Hospital, Southern Medical University, Guangzhou, People's Republic of China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Department of Cell Biology, Stem Cell Research Center, Department of Basic Medical Sciences, Peking University Health Science Center, Beijing, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Shen
mesh_terms: D001665:Binding Sites; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D065150:Cellular Reprogramming; D000067470:Cellular Reprogramming Techniques; D015972:Gene Expression Regulation, Neoplastic; D055786:Gene Knockout Techniques; D005838:Genotype; D056945:Hep G2 Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D000071317:Nanog Homeobox Protein; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
chemical_list: D014408:Biomarkers, Tumor; C497483:CD44 protein, human; D018398:Homeodomain Proteins; D018960:Hyaluronan Receptors; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; C489427:MYC protein, human; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D016271:Proto-Oncogene Proteins c-myc; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors
keywords: C3A; CD44; CRISPR/Cas9; induced liver cancer stem cells; transcriptional regulation