Study On The Molecular Mechanisms Of Self-renewal Associated Genes And Incrnas In The Maintenance Of Liver Cancer Stem Cells

Liver cancer is one of the five most common cancers all over the world. Accounting for 460 thousand morbidity and 420 thousand mortality every year in China, liver cancer is a major killer of health and serves as a national disease. Cancer stem cells are a small population cells in tumor bulk, with properties of self-renewal and differentiation into a new tumor. Cancer stem cells account for tumor initiation, metastasis, drug resistance and recurrence. Accordingly, it is indispensable to eliminate cancer stem cells in tumor treatment. However, the biological mechanisms of liver cancer stem cells are largely unknown.Our previous works showed that CD 133 and CD 13 served as good surface markers of liver cancer stem cells. We isolated liver cancer stem cells (CD13+CD133+) and liver cancer non-stem cells (CD13CD133), followed by transcriptome microarray. We explored two genes (C8orf4, Zic2) and lncRNAs (Lnc-β-Catm,LncBrm) in liver cancer stem cell self-renewal regulation, and then investigated the molecular mechanism.Transcriptome analyses and clinical sample validation showed that C8orf4 was weakly expressed in liver cancer cells, especially in liver cancer stem cells. C8orf4 deficient cells showed impaired sphere formation capacity and tumor initiation capacity. Notch signaling was activated in C8orf4 deficient cells and inactivated in C8orf4 overexpressed cells. Yeast two hybrid (Y2H) showed the interaction between C8orf4 and Notch2 intracellular domain (N2ICD). Confocal and nucleocytoplasmic separation showed that C8orf4 participated in the nuclear translocation of N2ICD. Notch2, Nrarp, Heyl knockdown cells showed attenuate sphere formation and tumor initiation capacity. Additionally, Notch2 signaling was related to clinical severity and prognosis.Zic2 was highly expressed in liver tumors, especially in liver cancer stem cells. Zic2 deficient cells showed impaired sphere formation and tumor initiation capacity. Through realtime PCR, we found Zic2 deficient cells showed reduced expression of Oct4 and Oct4 target genes. Through Y2H and co-IP, we found Zic2 interacts with NURF complex, which played a critical role in Oct4 promoter activation. Altogether, Zic2 recruits NURF complex and drives Oct4 expression, finally promotes liver cancer stem cell self-renewal maintenance.Long noncoding RNAs (LncRNAs) emerge as important regulators in gene expression and a variety of biological processes. Using transcriptome microarray analysis, we identified Lnc-β-Catm, a lncRNA highly expressed in liver tumors and liver cancer stem cells, was required for liver cancer stem cell self-renewal. Lnc-β-Catm interacted with P-catenin and promoted its stability. We found Lnc-β-Catm promoted the interaction between β-catenin and EZH2, a methyltransferase with the capacity to methylate P-catenin in liver cancer stem cells. Lnc-fi-Catm promoted β-catenin methylation, which further inhibited its phosphorylation and ubiquitination. Using primary samples, we confirmed that high expression levels of Lnc-β-Catm and Wnt/β-catenin targets were related to HCC clinical severity and prognosis. In conclusion, Lnc-β-Catm promotes the interaction between β-catenin and EZH2, increases β-catenin methylation and stability, activate Wnt/β-catenin signaling and finally drives liver cancer stem cell self-renewal.We also found LncBrm was highly expressed in liver cancer stem cells. LncBrm was required for liver cancer stem cell self-renewal. LncBrm depleted cells showed decreased Yapl expression levels and reduced Yap1 promoter activation, and vice versa. LncBrm interacted with Brm, a core component of Baf complex, blocked the assembly of Brm-embedded SWI/SNF complex and increased the Brgl-embedded SWI/SNF complex, which activated Yap1 promoter through Klf4-dependent manner. We then proved that LncBrm and Yap1 played essential roles in liver cancer stem cell self-renewal.In conclusion, we identified two genes (C8orf4, Zic2) and two lncRNAs (Lnc-β-Catm, LncBrm), confirmed their roles in live cancer stem cell self-renewal, and uncovered the molecule mechanism.