miR-589-5p inhibits MAP3K8 and suppresses CD90+ cancer stem cells in hepatocellular carcinoma

BACKGROUND Cancer stem cells (CSCs) are important in the tumorigenesis and progression of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) play crucial roles regulating CD133+ and EpCAM+ CSCs in HCC, although it is unclear whether miRNAs regulate CD90+ CSCs in HCC. METHODS The miRNA profiles of CD90+ and CD90- HCC cells were analyzed using a miRNA microarray and quantitative real-time PCR (qRT-PCR). CSC characteristics were examined by qRT-PCR and Western blot of pluripotency-associated genes, clone and sphere formation assay, transwell migration assay, and nude mice tumorigenicity assay. miR-589-5p mimic transfection was used to overexpress miR-589-5p in vitro. The CD90 and miR-589-5p expressions of HCC samples were detected by immunohistochemistry and qRT-PCR, respectively. RESULTS miR-589-5p and miR-33b-5p were down-regulated in CD90+ cells. Overexpression of miR-589-5p suppressed CD90+ CSC characteristics such as Oct4, Sox2 and Nanog expression, a high likelihood of forming cell spheres, high invasiveness and high tumorigenicity. Luciferase reporter assays demonstrated that miR-589-5p directly binds to the 3'-untranslated region of mitogen-activated protein kinase kinase kinase 8 (MAP3K8) mRNA, and exogenous miR-589-5p down-regulated MAP3K8 expression. In addition, siRNA inhibition of MAP3K8 also suppressed CD90+ CSC characteristics, even in the absence of miR-589-5p overexpression. In HCC tissues, miR-589-5p expression was inversely correlated with CD90 expression, and high CD90 expression and low miR-589-5p expression were positively correlated with vascular invasion and recurrence and significantly decreased disease-free and overall survival by clinical analysis. CONCLUSION In HCC, miR-589-5p down-regulates the stemness characteristics of CD90+ CSCs in part by silencing MAP3K8. CD90 and miR-589-5p expression predict HCC outcomes and might be novel molecular targets for HCC treatment.