MiR-429 regulates inflammatory infiltration, proliferative ability and differentiation of hepatocellular carcinoma / 第二军医大学学报

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 EpCAM marker. The EpCAM-positive and negative cells were transfected with AntagomiR- 429 and miR-429mimic 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 EpCAM-positive cells was decreased by down-regulating miR-429, and EpCAM-negative cells could partially transfer to EpCAM-positive cells by miR-429 ove- expression. In vivo study showed that over-expressing miR-429 in EpC AM-negative cells cound enhance the tumor forming ability; whereas EpCAM-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 EpCAM-positive cells of HCC in vitro and in vivo, and greatly increase the inflammatory infiltration and proliferative ability, accompanied by decreased differentiation.