上调microRNA-27b对血小板衍生因子诱导下的人视网膜色素上皮细胞增殖的抑制作用

目的　观察过表达的microRNA-27b(miR-27b)对血小板衍生因子（platelet-derived growth factor,PDGF）诱导下的人视网膜色素上皮细胞（ARPE-19）增殖的抑制作用，探讨miR-27b在ARPE细胞生物学行为中的作用及其调控机制。方法　将miR-27b过表达质粒或空载体转入ARPE-19细胞36 h后，用终浓度为20 μg·L^-1的PDGF预处理ARPE-19细胞5 h。根据转染物不同，将实验分为空白对照组（control组）、miR-27b阴性对照组（miR-27b NC组）、miR-27b模拟物转染组（PDGF+mimics组）和空载体转染组（PDGF+NC组）。实时荧光定量PCR(qRT-PCR)检测转染后各组ARPE-19细胞中miR-27b的表达水平；MTT法测定各组细胞活性；通过流式细胞术评估各组细胞周期的分布变化；Western blot检测各组细胞周期的正向调控因子cyclinD1、CDK4和负向调控因子p21^CIP1和p27^KIP1的表达水平。结果　qRT-PCR检测结果显示:经PDGF处理5 h，与miR-27b NC组相比，PDGF+NC组miR-27b的表达显著降低（P<0.01）；与PDGF+NC组相比，PDGF+mimics组miR-27b的表达增加（P<0.01）。MTT检测结果显示:与miR-27b NC组相比，PDGF+NC组ARPE-19细胞的光密度（D）值增加(P<0.01)；而与PDGF+NC组相比，PDGF+mimics组可明显抑制ARPE-19细胞的D值(P<0.01)。流式细胞术检测结果显示:与空白对照组和miR-27b NC组相比，PDGF+NC组G0/G1期细胞的百分比显著降低，S期细胞的百分比增加（P<0.05）；而与PDGF+NC组相比，PDGF+mimics组miR-27b的过表达显著增加了G0/G1期细胞的百分比，并抑制了细胞增殖（均为P<0.05）。Western blot检测结果显示:与PDGF-NC组相比，PDGF+mimics组miR-27b的过表达可显著降低cyclinD1蛋白和CDK4蛋白的表达，同时增强了p21^CIP1蛋白和p27^KIP1蛋白的表达（均为P<0.05）。结论　上调miR-27b表达可抑制PDGF诱导的ARPE细胞增殖。

Up-regulation of microRNA-27b inhibits PDGF-induced cell proliferation in ARPE-19 cells

Objective　To observe the inhibitory effect of overexpressed miR-27b on the proliferation of human retinal pigment epithelial cells (ARPE-19) induced by platelet-derived growth factor (PDGF), and to explore the role and regulatory mechanism of miR-27b in the biological behavior of human RPE cells.Methods　After transferring miR-27b over-expression plasmid or empty body into ARPE-19 cells for 36 hours, ARPE-19 cells were pretreated with PDGF with final concentration of 20 μg·L^-1 for 5 hours. According to the different transfection materials, the experiment was divided into control group, miR-27b negative control group (miR-27b NC group), miR-27b analog transfection group (PDGF+mimics group) and empty vector transfection group (PDGF+NC group). The expression level of miR-27b in ARPE-19 cells was detected by real-time quantitative PCR (qRT-PCR). The cell activity was measured by MTT and the distribution of cell cycle was evaluated by flow cytometry. Western blot was used to detect the expression levels of CyclinD1, CDK4, p21^CIP1 and p27^KIP1.Results　qRT-PCR showed that, 5 hours after PDGF pretreat, the expression of miR-27b in PDGF+NC group was significantly lower than that in miR-27b NC group (P<0.01), and PDGF+mimics group was significantly higher than PDGF+NC group (P<0.01). The results of MTT showed that the optical density (D) of ARPE-19 cells in PDGF + NC group was higher than that in miR-27b NC group (P<0.01), while PDGF+mimics group significantly inhibited the D value when compared with PDGF+NC group (P<0.01). The results of flow cytometry showed that compared with the control group and miR-27b NC group, the percentage of G0/G1 phase cells in PDGF+NC group decreased significantly, while the percentage of S phase cells increased (P<0.05). Compared with PDGF+NC group, over expression of miR-27b in PDGF+mimics group significantly increased the proportion of cells in G0/G1 phase and inhibited cell proliferation (all P<0.05). Western blot showed that the overexpression of miR-27b in PDGF+mimics group significantly decreased the expression of cyclin D1 and CKD4, and enhanced the expression of p21^CIP1 and p27^KIP1 compared with PDGF+NC group (all P<0.05).Conclusion　Up-regulation of miR-27b expression can inhibit PDGF induced proliferation of ARPE cells.