| Background:Hepatocellular carcinoma(HCC)is ranked as the sixth most common neoplasm and the third leading cause of cancer deaths.Liver cancer stem cells(LCSCs)contribute to HCC development,metastasis,and drug resistance.Musashi2(MSI2)plays an essential role in the promotion and maintenance of normal stem cells and cancer stem cells(CSCs).Notch1 signaling pathway is one of the core signaling pathways in regulating CSCs.Both MSI2 and Notch1 signaling are involved in the maintenance of CSCs.However,it is unknown whether MSI2 and Notch1 signaling are involved in the maintenance of CD44v6+LCSCs.Therefore,we investigated the clinical significance and function of MSI2 and its relationship with Notch1 signaling in the maintenance of stemness properties in CD44v6+LCSCs.Methods:The first part,the expression of MSI2 and CD44v6 were detected by fresh specimens and a HCC tissue microarray.The tissue microarray containing 82 HCC samples was used to analyze the correlation between CD44v6 and MSI2.The second part,CD44v6+/-cells were isolated using microbeads sorting from SNU-398 and MHCC-97h HCC cell lines,and the quality of sorting was monitored by flow cytometry.Sphere formation assay,transwell assay,clone formation assay,Sorafenib toxic assay in vitro,and limited dilution of tumor formation in NOD/SCID mice in vivo were used to identify the characteristics of LCSCs.Furthermore,we explored the roles of MSI2 and Notch1signaling in CD44v6+LCSCs by sphere formation assay,transwell assay,clone formation assay in vitro,and xenograft tumor models in vivo.The third part,Notch RT~2PCR Array,Co-immunoprecipitation,and RNA-immunoprecipitation were used to further investigate the molecular mechanism of MSI2 in activating Notch1 signaling.Results:The first part,(1)Western blot showed that the expression of MSI2 and CD44v6in HCC tissues were higher than that of in para-HCC tissues.(2)We found MSI2expression was positively correlated with high CD44v6 expression in HCC tissues,and further correlated with tumor differentiation.Furthermore,both of the expression of MSI2and CD44v6 in HCC tissues positively correlated with the poor prognosis.The second part,(1)CD44v6+cells isolated from SNU-398 and MHCC-97h HCC cell lines exhibited increased self-renewal,proliferation,migration and invasion,resistance to Sorafenib,and tumorigenic capacity,expressed more stemness related genes(Nanog,Oct4,Sox2).(2)MSI2 was elevated in sorted CD44v6+cells than CD44v6-cells.Downregulated MSI2 in CD44v6+LCSCs decreased the ability of self-renewal,invasion and migration,proliferation,tumorigenicity accompanied with decreased expression of stemness-associated genes,while upregulated MSI2 in CD44v6-cells increased the ability of self-renewal,invasion and migration,proliferation,and tumorigenicity accompanied with increased expression of stemness-associated genes.(3)Notch1 signaling was elevated in sorted CD44v6+cells than CD44v6-cells.Inhibition of Notch1 signaling using lentivirus orγsecretase inhibitors RO4929097 in CD44v6+cells significantly attenuated the stemness properties,decreased the expression of the stemness related genes.The third part,(1)Western blot showed that in CD44v6+cells,the key moleculars of Notch1signaling pathway were reduced after the MSI2 gene was down-regulated,while in the CD44v6-cells the key moleculars up-regulated after increasing MSI2 gene.(2)The result of Notch RT~2PCR Array showed that a total of 18 genes showed significantly differential expression between the MSI2 sh RNA1 group and the control group.Of these,12 were upregulated and 6 were downregulated in the MSI2 sh RNA1 expression group compared to the control group.We choose the most significantly differential expressed 10 genes to validate by RT-PCR and found Lunatic fringe(LFNG)was the most significantly differential expressed gene.(3)Down-regulation of LFNG using lentivirus in CD44v6+cells significantly attenuated the stemness properties,decreased the expression of the stemness related genes.(4)Sequential regulation by using lentivirus showed that LFNG was downstream of MSI2 and upstream of Notch1 signaling pathway.(5)Mechanically,MSI2 directly bound to LFNG mRNA and protein,resulting in Notch1 activation.Conclusions:Our study demonstrated that MSI2 was positively related to CD44v6 and poor prognosis.CD44v6 could be a reliable marker of LCSCs.Additionally,MSI2 was highly expressed in CD44v6+LCSCs and maintained the stemness properties of CD44v6+LCSCs.Mechanically,MSI2 bound to LFNG mRNA and protein directly to regulate the expression of Notch1 receptor in Golgi,then activated Notch1 signaling pathway,contributing the maintenance of stemness of CD44v6+LCSCs.Our findings provide a new insight to the recurrence and metastasis of HCC and potential molecular targets for targeted therapy of liver cancer. |
