let-7c对HCCLM3细胞增殖的影响

目的 用瞬时转染的方法 研究let-7c对人肝癌细胞HCCLM3增殖的影响并探讨其机制.方法 用脂质体2000将miRNA转染人人肝癌细胞HCCLM3,设let-7c组转染let-7c,设对照组转染阴性对照miRNA,设空白组不做任何转染.用细胞计数试剂盒CCK-8检测转染后细胞生长增殖的变化,用流式细胞术检测各组细胞周期分布的差异.用Western blot和实时荧光PCR检测转染后细胞周期蛋白(cyclin) D1及其mRNA表达的变化.多组数据的两两比较采用LSD法.结果 let-7c组细胞在转染后48、72h时的吸光度值分别为0.70±0.05、0.77±0.09,低于空白组的0.97±0.10、1.21±0.12和对照组的0.91±0.07、1.12±0.09,各组比较,F值分别为14.431、21.146,P值均＜0.01,差异均有统计学意义.转染后72h,let-7c使癌细胞处在G1期的细胞比例增加.空白组、对照组、let-7c组G1期的细胞比例分别为43.53％±0.86％、44.82％±0.77％、54.52％％±0.13％,各组比较,F=240.739,P＜0.01,差异有统计学意义.let-7c能抑制cyclin D1及其mRNA的表达,空白组、对照组、let-7c组cyclin D1的表达水平分别为0.48±0.09、0.47±0.06、0.23±0.06,各组比较,F=11.316,P＜0.01,差异有统计学意义; mRNA的相对表达量分别为1.03％±0.29％、1.01％±0.11％、0.63％±0.14％,各组比较,F=6.315,P＜0.05,差异有统计学意义.结论 let-7c能抑制HCCLM3细胞的增殖,并使细胞停留在G1期的细胞比例增加.导致这种现象的机制可能为let-7c抑制cyclin Dl及其mRNA的表达.

Effect of let-7c on the proliferation of human hepatocellular carcinoma cell HCCLM3

Objective To investigate let-7c's effect on the proliferation of human hepatocellular carcinoma cell HCCLM3 by transient transfection and the mechanism inside.Methods 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 Kit8(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 perfomed with SPSS 17.0.Results The absorbances of let-7c group were 0.70 ± 0.05,0.77 ± 0.09 at 48h 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 traasfection 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.Conclusion let-7c can inhibit proliferation of HCCLM3 cells and increase the proportion of cells in Gl phase.The mechanism may be that let-7c represses the expressions of cyclin D 1 at both protein and mRNA levels.