MiR-199a-3p对大鼠肝细胞增殖的抑制作用

摘要

目的 探讨miR-199a-3p对体外培养SD大鼠BRL-3A增殖的影响.方法 人工合成miR-199a-3p模拟物、miR-199a-3p阻遏物及其各自阴性对照载体,分别转染BRL-3A肝细胞,转染后12 h、24h、48 h和72h,应用定量PCR分析细胞中Cyclin D1和PCNA mRNA表达水平,免疫组化方法观测Cyclin D1和PCNA阳性细胞比率,流式细胞仪检测细胞各周期比例和CCK 8法检测细胞增殖.实验分4组:mimics组,mi-nc组,in-hibitors组和in-nc组.结果 转染后24h、48 h、72 h,inhibitors组肝细胞Cyclin D1和PCNA mRNA表达水平较in-nc组显著增多(P＜0.05),而mimics组肝细胞Cyclin D1和PCNA mRNA表达水平较mi-nc组显著降低(P＜0.05).inhibitors组肝细胞Cyclin D1和PCNA阳性细胞比率均高于mi-nc组(P＜0.05),而mimics组肝细胞Cy-clin D1和PCNA阳性细胞比率均低于mi-nc组(P＜0.05).inhibitorss组肝细胞G0/G1期细胞比例明显低于in-nc组(P＜0.05),而mimics组肝细胞G0/G1期细胞比例明显高于mi-nc组(P＜0.05).同时,inhibitors组肝细胞S期细胞比例明显低于mi-nc组(P＜0.05)和in-nc组(P＜0.05).inhibitors组肝细胞增殖能力明显高于in-nc组(P＜0.05),mimics组肝细胞增殖能力明显低于mi-nc组(P＜0.05).结论 miR-199a-3p能够延长大鼠BRL-3A肝细胞周期和抑制肝细胞增殖,其作用可能与抑制Cyclin D1和PCNA的表达有关.%Objective To investigate the inhibiting effect of miR-199a-3p on the proliferation of rat liver derived cell line (rat BRL-3a liver cells) in vitro.Methods miR-199a-3p mimics,miR-199a-3p inhibitors and their negative controls were artificially synthesized.Rat BRL-3a liver cells,divided into four groups (Group mimics,Group mi-nc,Group inhibitors,Group in-nc),were respectively transfected by four artificial miRNAs.At 12 hours,24 hours,48 hours,and 72 hours after transfction,Cyclin D1 and PCNA mRNA expression changes were examined by real-time fluorogenic quantitative PCR.At 48 hours after transfction positive cells of Cyclin D1,PCNA were counted in immunohistochemical analysis.Cell cycle was measured by flow cytometry.Cell proliferation was assessed by CCK 8.Results At 12 hours,mRNA expression changes of Cyclin D1 and PCNA had no statistical difference in four groups (P＞0.05).At 24 hours,48 hours,72 hours,Both mRNA and protein expression of Cyclin D1 and PCNA of Group inhibitors were significantly increased (P ＜ 0.05) than that of Group in-nc,on the other hand,Both mRNA and protein expression levels of Cyclin D1 and PCNA of Group mimics were significantly lowered (P ＜0.05) than that of Group mi-nc.In immunohistochemical test,positive cells rate of Cyclin D1,PCNA in Group inhibitors were significantly higher than Group in-nc (P ＜ 0.05),the reverse result happened in Group mimics.G0/G1 phase cells ratio of Group inhibitors was significantly lower than Groups in-nc (P ＜ 0.05),G0/G1 phase cells ratio of Group mimics was significantly higher than that of Group mi-nc (P ＜ 0.05).At the same time,the S phase cells ratio of Group inhibitors was significantly higher than Group in-nc (P ＜ 0.05),the S phase cells ratio of group Group mimics was obviously lower than Group mi-nc (P ＜ 0.05).CCK 8 test showed that cells of Group inhibitors proliferated significantly faster than that of Group in-nc (P ＜ 0.05),cells of Group mimics proliferated obviously slower than than that of Groups mi-nc (P ＜ 0.05).Conclusion MiR-199-a-3 p can prolong the cell cycle and inhibit the cell proliferation in rats BRL-3-a liver cells,and that the effect may be associated with the inhibiting effect of MiR-199a-3p on Cyclin D1 and PCNA expression.