Gong Fang-xiao, Xia Jing-lin, Yang Bi-wei, Xu Xiao-jing, Wu Wei-zhong
Liver Cancer Institute and Zhongshan Hospital, Fudan University, Shanghai 200032, China.
Zhonghua Gan Zang Bing Za Zhi. 2011 Nov;19(11):853-6. doi: 10.3760/cma.j.issn.1007-3418.2011.11.014.
To investigate let-7c's effect on the proliferation of human hepatocellular carcinoma cell HCCLM3 by transient transfection and the mechanism inside.
Lipofectamine 2000 was used to transfect miRNAs into HCCLM3 cells. The cells were divided into three groups, let-7c group: let-7c was transfected, negative control group: negative control miRNA was transfected, blank control group: nothing was transfected. The proliferation of HCCLM3 cells was evaluated using Cell Counting Kit-8 (CCK-8). The cell cycles of each group were assayed by flow cytometry. Western blot and Real time PCR were used to analyze the protein and mRNA expressions of cyclin D1. Statistical analysis was performed with SPSS 17.0.
The absorbances of let-7c group were 0.70 ± 0.05, 0.77 ± 0.09 at 48 h and 72 h after transfection, lower than that of blank control group (0.97 ± 0.10, 1.21 ± 0.12) and negative control group (0.91 ± 0.07, 1.12 ± 0.09), 48 h: F = 14.431, P < 0.05, 72 h: F = 21.146, P < 0.05. The flow cytometry at 72 h after transfection revealed that let-7c increased the percentage of cells in G1 phase. The percentage of blank control group was 43.53% ± 0.86%, the negative control group was 44.82% ± 0.77%, and the let-7c group was 54.52% ± 0.13%, F = 240.739, P < 0.05. let-7c suppressed expressions of cyclin D1 at both protein and mRNA levels. The protein levels of cyclin D1 were 0.48 ± 0.09, 0.47 ± 0.06 and 0.23 ± 0.06 (F = 11.316, P < 0.05) in blank control group, negative control group and let-7c group, respectively. The mRNA levels were 1.03% ± 0.29%, 1.01% ± 0.11% and 0.63% ± 0.14% (F=6.315, P < 0.05) in the above three groups, respectively.
Let-7c can inhibit proliferation of HCCLM3 cells and increase the proportion of cells in G1 phase. The mechanism may be that let-7c represses the expressions of cyclin D1 at both protein and mRNA levels.
通过瞬时转染研究let-7c对人肝癌细胞HCCLM3增殖的影响及其内在机制。
采用Lipofectamine 2000将微小RNA转染至HCCLM3细胞。细胞分为三组，let-7c组：转染let-7c；阴性对照组：转染阴性对照微小RNA；空白对照组：未进行转染。使用细胞计数试剂盒-8（CCK-8）评估HCCLM3细胞的增殖情况。通过流式细胞术检测各组细胞周期。采用蛋白质免疫印迹法（Western blot）和实时荧光定量聚合酶链反应（Real time PCR）分析细胞周期蛋白D1（cyclin D1）的蛋白质和信使核糖核酸（mRNA）表达。使用SPSS 17.0进行统计学分析。
转染后48 h和72 h，let-7c组的吸光度分别为0.70±0.05、0.77±0.09，低于空白对照组（0.97±0.10、1.21±0.12）和阴性对照组（0.91±0.07、1.12±0.09），48 h：F = 14.431，P < 0.05；72 h：F = 21.146，P < 0.05。转染后72 h的流式细胞术结果显示，let-7c增加了G1期细胞的百分比。空白对照组为43.53%±0.86%，阴性对照组为44.82%±0.77%，let-7c组为54.52%±0.13%，F = 240.739，P < 0.05。let-7c在蛋白质和mRNA水平均抑制cyclin D1的表达。空白对照组、阴性对照组和let-7c组cyclin D1的蛋白质水平分别为0.48±0.09、0.47±0.06和0.23±0.06（F = 11.316，P < 0.05）。上述三组的mRNA水平分别为1.03%±0.29%、1.01%±0.11%和0.63%±0.14%（F = 6.315，P < 0.05）。
Let-7c可抑制HCCLM3细胞增殖并增加G1期细胞比例。其机制可能是let-7c在蛋白质和mRNA水平均抑制cyclin D1的表达。
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