| BackgroundHepatocellular carcinoma(HCC)is resistant to chemo-radio therapy and has a high recurrence rate after surgery,which leads to treatment failure of HCC.Cancer stem cells(CSCs)are a small group of cells with properties of self-renewal ability,unlimited growth,pluripotency and migration,and are considered responsible for the recurrence,metastasis and resistance to chemo-radio therapy of HCC.Therapy targeting CSCs provides a novel strategy to develop HCC treatment.Previous studies have shown that CD90,Ep CAM,CD133,CD24,OV-6 and CD44 can be used as CSCs markers in HCC.However,it remains unclear which marker is optimal for clinical application.Micro RNAs(miRNAs)are a large group of small,noncoding RNAs that are 17–27 nucleotides in length.As negative regulators of post-transcriptional gene expression,miRNAs bind to the 3?-untranslated region(UTR)of the target m RNA by completely or incompletely base pairing and suppress protein expression through m RNA degradation or translational repression.miRNAs play key roles in physiological processes of embryonic stem cells.In human solid tumors,many miRNAs have been found to participate in regulation of CSC population and stemness properties including proliferation,migration,invation and cellular differentiation,stem cell self-renewal and resistance to chemo-radio therapy.However,it remains unclear which miRNA participates in regulation of CD90+ CSCs in HCC.Mitogen-activated protein 3 kinase 8(MAP3K8)is a member of the serine/threonine protein kinase family,and is closely related to tumorigenesis and cancer progression in various human tumors.MAP3K8 has variable effects on tumors,but is predominantly considered a tumor-promoting oncogene in several tumor types,such as breast cancer,colon cancer,renal cell carcinoma,endometrial cancer,gastric cancer,nasopharyngeal carcinoma,thymoma and lymphoma.MAP3K8 has Intricate interaction with NF-κB pathways,which has been found to be constitutively activated in CSCs from a variety of cancers and participate in the maintenance,expansion,proliferation and survival of CSCs.These evidences suggest that MAP3K8 might be involved in regulation of CSCs.However,it remains unclear whether MAP3K8 is involved in HCC,espetially in HCC CSCs.Methods1 The expressions of CSCs surface markers were examined in HCC cell lines by flow cytometry.Cell-sphere formation was used to test the impact of stem cell culture environment on CD90 expression.The stemness features of CD90+ CSCs were examined using q RT-PCR,clone and sphere formation assays,cell invasion and migration assays and tumorigenesis in nude mice.2 CD90+ CSC-associated miRNAs were scanned by miRNA microarrays and were comfirmed by q RT-PCR.CD90+ CSCs were transfected with mi R-589-5p mimics to overexpress mi R-589-5p,and CD90+ CSCs features were examined after transfection.3 Downstream target proteins of mi R-589-5p were predicted by in cilico prediction.The direct inhibition of MAP3K8 by mi R-589-5p were tested using luciferase gene reporter,q RT-PCR and Western blot.4 MAP3K8 si RNAs were used to suppress MAP3K8 and CD90+ CSCs features.5 Immunohistochemistry(IHC)and q RT-PCR were used to examine expression levels of CD90 and mi R-589-5p in primary HCC,respectively.Finally,the relationship of the expression levels of CD90 and mi R-589-5p with the HCC clinical outcomes were explored.Results1 Among all of the tested CSC markers,only CD90 was steadily expressed in a small population,even under cell sphere formation conditions.CD90+ HCC cells exhibited CSCs features,such as stemness-associated gene expression,self-renewal ability,a high invasiveness and high tumorigenicity.2 miRNA microarrays showed that three miRNAs were up-regulated and six miRNAs were down-regulated in the CD90+ CSCs.q RT-PCR further proved that mi R-589-5p and mi R-33b-5p were down-regulated in the CD90+ CSCs.Transfection of mi R-589-5p mimics overexpressed mi R-589-5p and suppressed the CD90+ CSCs features,such as stemness-associated gene expression,self-renewal ability,a high invasiveness and high tumorigenicity.3 Luciferase gene reporter showed that mi R-589-5p could directly bind to MAP3K8 m RNA 3’-UTR.Exogenous mi R-589-5p inhibited MAP3K8 expression both at m RNA and protein levels in CD90+ CSCs.4 Transfection of MAP3K8 si RNA suppressed MAP3K8 and the CD90+ CSCs features,such as stemness-associated gene expression,self-renewal ability and a high invasiveness.5 In primary HCC tissues,CD90 was expressed in a small population.CD90 expression was inversely correlated with mi R-589-5p expression,and high CD90 expression and low mi R-589-5p expression were positively correlated with vascular invasion and recurrence and significantly decreased disease-free and overall survival.Conclusions1 CD90 is an ideal marker to identify the CSC population in HCC.CD90+ HCC cells exhibite CSCs features,such as stemness-associated gene expression,a greater self-renewal ability,a high invasiveness and high tumorigenicity.2 mi R-589-5p is down-regulated in CD90+ CSCs and suppresses CD90+ CSCs features.3 mi R-589-5p functions through binding to the MAP3K8 m RNA 3’-UTR to suppress CD90+ CSCs features.4 CD90 expression is inversely correlated with mi R-589-5p expression in primary HCC tissues.High CD90 expression and low mi R-589-5p expression are associated with a poor clinical prognosis in HCC.CD90 and mi R-589-5p are predictors for HCC prognosis. |
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