Knock out CD44 in reprogrammed liver cancer cell C3A increases CSCs stemness and promotes differentiation.

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.