The Mechanism Of CD44 3’ UTR Enhancing Natural Killer Cells Mediated Cytotoxicity To Liver Cancer Stem Cells

Background:Liver cancer is one of the most common human malignancies in the world,its mortality rate ranks second among all malignant tumors.High rate of recurrence,metastasis and chemoresistance are important factors limiting long-term survival of liver cancer patients.Cancer Stem Cells(CSC)are a special group of tumor cells with characteristics of self-renewal and pluripotency properties,which are closely associated with tumor recurrence,metastasis and drug resistance.CD44 is an important molecular marker of multiple CSCs such as liver cancer and colon cancer,which is closely related to the maintenance of CSC stemness and self-renewal.Natural Killer(NK)cells preferentially kill CSC expressing a high level of CD44 in pancreatic cancer and glioblastoma.In contrast differentiated tumor cells expressing a lower level of CD44 are not sensitive to NK cell mediated cytotoxicity.NK cells cytotoxicity to liver CSC and its relationship with CD44 remain unknown.In addition,the potential recognition targets and regulatory mechanisms of NK cells cytotoxicity to liver CSC are not clear.Our study attempts to investigate whether NK cells could preferentially kill liver CSC,and the potential recognition targets and regulatory mechanisms in this process to provide support for NK cell treatment towards liver CSC to improve liver cancer patients’ prognosis in the future.Methods:1.Oct4,Sox2,Klf4,c-Myc(OSKM)and shMBD3 were ectopically expressed in HepG2 cells to generate induced CSC expressing different level of CD44(CD44high/intiCSC);2.ELISA assay,Cytotoxicity assay,CRISPRi and Antibody blocking were performed to study the relationship between NK cytotoxic effect on iCSC and CD44;3.Western Blot and RT-PCR were used to find possible recognizing targets for NK cytotoxicity to iCSC;4.Gene overexpression,Luciferase reporting,miRNA target site mutation and RNA immunoprecipitation were employed to testify CD44 3’ Untranslated Region(3’UTR)serves as the competing endogenous RNA(ceRNA)to regulate ULBP2 expression in iCSC;5.Tumor xenotransplantation was used to identified CD44 3’UTR functioned as the ceRNA to enhance NK cytotoxicity by regulating ULBP2 expression.Results:1.HepG2 cells were transformed to CD44intiCSC by ectopically expressing OSKM,shMBD3 further increased CD44 expression to generate CD44highiCSC;2.NK cells could recognize and kill liver CSC;3.CD44 knock-down(CD44-kd)reduced NK cell cytotoxic effect on iCSC,proving NK cell cytotoxic effect was positively correlated with the expression of CD44 in iCSC;4.CD44-kd decreased expression of NK cell activation ligand ULBP2 in iCSC;5.NK cells preferentially killed iCSC by recognizing ULBP2;6.CD44-kd reduced the stability of ULBP2 mRNA and increased miRNA-34a expression;7.miRNA-34a could specifically bind with CD44 3’UTR(3021bp and 4564bp)and ULBP2 3’UTR(1048bp)to regulate their expressions;8.CD44 3’UTR over-expression enhanced ULBP2 mRNA stability and up-regulated its protein expression;9.CD44 3’UTR competitively binding miRNA-34a to regulate ULBP2 expression;10.CD44 3’UTR functioned as the ceRNA to enhance NK sensitivity to iCSC by regulating ULBP2 expression.Conclusion:ULBP2 was an important target for NK cells mediated cytotoxicity to iCSC;CD44 3’UTR functioned as a ceRNA competitively binding to miRNA-34a,preventing it decreasing the stability of ULBP2 mRNA,which in turn up-regulated ULBP2 protein expression and ultimately enhanced NK cells sensitivity upon iCSC.