MiR-429调节肝细胞癌炎性微环境、分化及增殖

目的 观察miR-429对人肝细胞癌(HCC)炎性浸润、分化以及增殖的影响.方法 利用免疫磁珠法分选人肝癌细胞系HCCLM3细胞,得到EpCAM (CD326)阳性以及阴性的细胞.对两种细胞分别进行miR-429的干扰(AntagomiR-429)与过表达(miR-429mimic),通过流式细胞技术、real-time PCR法检测其干预效果.利用CCK-8法检测miR-429过表达及干扰肝癌细胞系的增殖活性并绘制生长曲线.分别利用干扰miR-429的EpCAM阳性HCCLM3细胞及其对照、过表达miR-429的EpCAM阴性HCCLM3细胞及其对照进行NOD-SCID小鼠皮下荷瘤实验,通过免疫组织化学法检测成瘤后癌组织内炎性细胞浸润情况及增殖分化相关蛋白的表达情况.结果 干扰miR-429的表达能降低EpCAM阳性肝癌细胞的比例,反之,过表达miR-429能促使EpCAM阴性肝癌细胞转化为EpCAM阳性细胞.过表达miR-429可增强EpCAM阴性肝癌细胞在小鼠体内的成瘤能力,而干扰miR-429表达可降低EpCAM阳性细胞成瘤率;与对照相比,过表达miR-429的瘤体组织中出现大量的巨噬细胞,提示miR-429可招募炎性细胞浸润从而促进肿瘤发展;AFP染色提示miR-429与肿瘤的低分化有关;Ki67染色结果证实miR-429在体内能够促进肿瘤组织生长.与对照相比,miR-429在体外也可以促进肝癌细胞的增殖,差异具有统计学意义(P＜0.05).结论 miR-429能够在体外、体内水平影响EpCAM阳性细胞的比例,显著影响肝肿瘤微环境、促进肿瘤细胞的增殖活性并降低其分化潜能.

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

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 over-expression. In vivo study showed that over-expressing miR-429 in EpCAM-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.