miR-449a对食管鳞癌细胞Eca-109增殖、迁移、侵袭的影响及机制

摘要

目的 探讨miR-449a对食管鳞癌细胞Eca-109增殖、迁移、侵袭的影响及其可能的机制.方法 将食管鳞癌细胞Eca-109分为miR-449a组和NC组,分别转染miR-449a-mimics和NC-mimics;采用实时荧光定量PCR法检测两组miR-449a相对表达量,miR-449a组 miR-449a相对表达量明显高于NC组,证实上调miR-449a表达的Eca-109细胞模型建立成功.采用CCK-8法、平板克隆形成试验检测两组细胞增殖能力(分别以培养12、24、48 h吸光度值和克隆数表示),细胞划痕试验检测细胞迁移能力(以细胞之间的距离表示),Transwell小室试验检测细胞侵袭能力(以侵袭到下室的细胞数量表示),流式细胞仪检测细胞周期比例.采用实时荧光定量PCR法和Western blotting法分别检测两组miR-449a直接靶基因E2F3 mRNA及蛋白相对表达量.结果 miR-449a组细胞培养12、24、48 h时的吸光度值及克隆数、细胞之间的距离、侵袭到下室的细胞数量均低于NC组(P<0.05或<0.01).miR-449a组G1期细胞比例高于NC组,S期细胞比例低于NC组(P均<0.05);两组G2/M期细胞比例比较差异无统计学意义(P>0.05).miR-449a组E2F3 mRNA及蛋白相对表达量均低于NC组(P均<0.01).结论 miR-449a可抑制食管鳞癌细胞Eca-109的增殖、迁移和侵袭;抑制靶基因E2F3表达可能是其作用机制.%Objective To investigate the effects of miR-449a on cell proliferation, migration and invasion of esophageal squamous-cell carcinoma cell line Eca-109 and its mechanism.Methods Eca-109 cells were divided into the miR-449a group and the negative control (NC) group, in which miR-449a mimics and NC-mimics were transfected into Eca-109 cells, respectively.Real-time fluorescent quantitative PCR was performed to detect the relative expression of miR-449a in Eca-109 cells.When the expression of miR-449a in Eca-109 cells of the miR-449a group was higher than that of the NC group, indicating Eca-109 cell models with up-regulated miR-449a expression were successfully established.The cell proliferation abilities of the two groups were analyzed by CCK-8 and colony formation assay, which were shown in OD values and cell survival numbers at 12, 24 and 48 h.The cell migration and invasion was measured by Scratch test and Transwell invasion testing system, respectively.Additionally, flow cytometry was performed to detect cell cycle.Real-time fluorescent quantitative PCR and Western blotting were performed to detect the expression of E2F3 mRNA and protein in the two groups.Results At 12, 24 and 48 h, the OD value, the number of survival cells, distance between cells and the number of cells invading into the lower chamber of the miR-449a group were lower than those in the NC group (P<0.05 or P<0.01).The percentage of cells in G1 phase of the miR-449a group was higher than that in the NC group, and the percentage of cells in S phase was lower than that of the NC group (all P<0.05).However, there was no significant difference in the percentage of cells in G2/M phase between the two groups (P>0.05).The relative expression of E2F3 mRNA and protein in the miR-449a group was lower than that in the NC group (all P<0.01).Conclusion miR-449a can inhibit the proliferation, migration and invasion of esophageal squamous-cell carcinoma cell line Eca-109 by inhibiting the expression of target gene E2F3.