MiR-429 regulates inflammatory infiltration, proliferative ability and differentiation of hepatocellular carcinoma

Abstract

Objective To observe the effects of miR-429 on inflammatory infiltration,differentiation and proliferation of hepatocelluar carcinoma(HCC).Methods HCCLM3 cells were sorted into positive and-negative cells using magnetic activated cell sorting(MACS) according to Ep CAM marker.The Ep CAM-positive and negative cells were transfected with AntagomiR-429 and miR-429 mimic to construct over-expressed and down-regulated cells of miR-429,respectively.To validate the effect of transfection,control cells and constructed cells were tested by flow cytometry(FC) and real-time polymerase chain reaction(PCR).The proliferative curves of transfected cells were plotted using cell counting kit-8(CCK8) assay.Furthermore,tumor-bearing mice model was established by subcutaneous inoculation with transfected cells and related control cells into non-obese diabetic,severe combined immunodeficient mice(NOD-SCID).Mice were sacrificed after 6 weeks,and protein expressions of Ki67,AFP and F4 /80 in tumors were determined via immunohistochemistry(IHC) to examine the status of proliferation,differentiation and inflammatory infiltration,respectively.Results The proportion of Ep CAM-positive cells was decreased by down-regulating miR-429,and Ep CAM-negative cells could partially transfer to Ep CAM-positive cells by miR-429 over-expression.In vivo study showed that over-expressing miR-429 in Ep CAM-negative cells cound enhance the tumor forming ability; whereas Ep CAM-positive cells had an inhibited tumor-formation when down-regulating miR-429.Interestingly,F4 /80,a marker of macrophage,was strongly stained in tumor tissue with miR-429 over-expression.It was also found that in tumor tissue,Ki67 and AFP staining was suppressed with miR-429 down-regulation.Meanwhile,compared to the control,miR-429 significantly accelerated the proliferation of HCCLM3 in vitro compared with the control(P 0.05).Conclusion miR-429 can regulate the proportion of Ep CAM-positive cells of HCC in vitro and in vivo,and greatly increase the inflammatory infiltration and proliferative ability,accompanied by decreased differentiation.