MicroRNA-544在肝癌组织中的表达及其对肝癌细胞恶性生物学行为的影响

目的 明确微RNA-544(microRNA-544,miR-544)在肝癌组织中的表达及其对肝癌细胞增殖、凋亡、克隆形成及成球能力的影响,探讨其在肝癌发生、发展中的作用.方法 利用qPCR法检测二乙基亚硝胺(DEN)造模的大鼠肝组织和人肝癌组织与癌旁组织以及成球培养后肝癌干细胞球中miR-544的表达;肝癌细胞株Hep3B转染miR-544mimic或miR-544 inhibitor后,利用CCK-8试剂盒检测细胞增殖情况,平板克隆形成实验观察细胞克隆形成能力,碘化丙啶(PI)染色后流式细胞仪检测细胞凋亡情况.肝癌细胞转染miR-544mimic后成球培养观察细胞形成肿瘤干细胞球能力的变化.结果 MiR544在DEN造模大鼠肝组织及人肝癌组织中的表达分别较未造模大鼠肝组织(P＜0.05)和癌旁组织(P＜0.01)下调.过表达miR-544可抑制肝癌细胞的增殖(P＜0.05,P＜0.01)和平板克隆形成能力(P＜0.05),并促进细胞凋亡(48 h和72 h,P＜0.01);而在肝癌细胞中抑制miR-544的作用可促进肝癌细胞的增殖(P＜0.01)和克隆形成能力(P＜0.05).在富集肝癌干细胞的成球培养中,miR-544的表达下调(P＜0.05);而过表达miR-544可抑制肝癌细胞形成干细胞球(P＜0.05).结论 MiR-544在肝癌组织中表达下调,且可抑制肝癌细胞的多种恶性生物学行为,提示其可能在肝癌的发生、发展中发挥着抑癌作用.

Expression of microRNA-544 in hepatocelluar carcinoma (HCC) and its effect on malignant behaviors of HCC cells

To study the expression of microRNA-544 (miR-544) in hepatocelluar carcinoma (HCC) tissues and its effect on proliferation, apoptosis, colony formation and sphere-forming potential of HCC cells, so as to explore the role of miR-544 in carcinogenesis and development of HCC. Methods Quantitative real-time PCR was used to detect the expressions of miR-544 in liver tissues from rats treated with diethylnitrosamine (DEN), in HCC and adjacent non-tumor tissues from patients, and in HCC spheres after sphere-forming culture. After HCC cell line Hep3B being transfected with miR-544 mimic or miR-544 inhibitor, cell counting kit-8 (CCK-8) was used to detect the proliferation of the HCC cells, colony-formation assay was used to observe the colony-formation ability of the HCC cells, and flow cytometry was used to analyze the cell apoptosis of HCC cells with propidium iodide staining. The sphere-formation assay was performed to determine the sphere-forming potential of HCC cells transfected with miR-544 mimic. Results MiR-544 expression was significantly down-regulated in the HCC tissues of DEN-treated rats (versus the control rats without modelling, P<0.05) and HCC tissues of patients (versus adjacent non-tumor tissues, P<0.01). Overexpression of miR-544 significantly inhibited proliferation (P<0.05, P<0.01) and colony-formation potential (P<0.05) of HCC cells, and also significantly promoted the apoptosis of HCC cells at 72 h (P<0.01). While inhibition of miR-544 in HCC cells significantly promoted the cell proliferation (P<0.01) and colony-formation potential (P<0.05). MiR-544 expression was significantly down-regulated in sphere-forming HCC cells during enrichment culture of liver cancer stem cells (P<0.05), and overexpression of miR-544 inhibited the sphere-formation of HCC cells (P<0.05). Conclusion MiR-544 expression is reduced in HCC tissues and can inhibit the malignant biological behaviors of HCC cells, indicating its inhibitory roles in the development and progression of HCC.