MicroRNA-96对人视网膜色素上皮细胞增殖与迁移的影响

目的：探讨MicroRNA-96（miR-96）对人视网膜色素上皮（RPE）细胞增殖和迁移的影响。方法：实验研究。通过RNA原位杂交检测人胚胎眼（20周）石蜡切片中RPE层miR-96的表达情况。将miR-96和阴性对照（NC）通过阳离子脂质体介导转染人眼RPE细胞，采用细胞增殖实验（MTS）、流式细胞术和Transwell实验分别检测细胞增殖、细胞周期以及细胞迁移能力。通过生物信息学及Western blot法确定miR-96作用的靶基因。应用Western blot检测miR-96对细胞增殖迁移相关信号通路蛋白（Akt、ERK）及细胞周期相关蛋白（p-Cdc2、CyclinD2、p-Rb）表达的影响。组间数据比较采用独立样本t检验。结果：MiR-96在人眼RPE细胞中有表达。MTS结果显示，转染NC、miR-96后，人眼RPE细胞的相对增殖速率分别为100%、74%±2%，差异有统计学意义（t=42.174，P=0.002）。流式细胞术检测结果显示，转染miR-96后阻滞在G1期的RPE细胞显著多于转染NC后，且差异有统计学意义（t= -18.444，P=0.003）。Transwell实验结果显示，与转染NC相比，转染miR-96能显著抑制RPE细胞的迁移，差异具有统计学意义（t=6.754，P=0.002）。进而，明确了MITF是miR-96作用的靶基因。Western blot检测结果显示，转染miR-96后细胞中细胞周期相关蛋白p-Rb（t=11.211，P=0.002）、p-Cdc2（t=9.133， P=0.003）、CyclinD2（t=7.542，P=0.005）以及迁移相关信号通路蛋白p-ERK（t=16.699，P<0.001），p-Akt（t=23.552，P<0.001）的表达水平均降低。结论：miR-96通过作用于靶基因MITF，并调控细胞周期和迁移相关蛋白的表达从而抑制人RPE细胞的增殖和迁移。

MicroRNA-96 Affects the Proliferation and Migration of Human Retinal Pigment Epithelial Cells

Objective: To investigate the effects of microRNA-96 (miR-96) on the proliferation and migration of human retinal pigment epithelial (RPE) cells. Methods: In this experimental research, the expression of miR-96 was detected in the RPE layer in paraffin sections of the human embryonic eye (20 weeks) by RNA in situ hybridization. MiR-96 or a negative control (NC) was transfected into RPE cells by a lipofectamineRNAiMAX reagent. MTS, flow cytometry, and transwell assays were used to evaluate the effects of miR-96 on cell proliferation, cell cycle, and cell migration. The target gene of miR-96 was predicted by bioinformatics and verified by Western blot. The expression of cell cycle-related proteins in RPE cells was detected by Western blot analysis. Data were analyzed by an independent t test. Results: MiR-96 was highly expressed in human RPE cells. MTS assay showed that the proliferation of RPE cells was inhibited by miR-96 (t=42.174, P=0.002). Flow cytometry revealed that miR-96 resulted in a G1 phase arrest in RPE cells (t=-18.444, P=0.003). Transwell assay also revealed that miR-96 inhibited the migration of RPE cells (t=6.754, P=0.002). Furthermore, MITF was identified as the target gene of miR-96. In addition, the expression of cell cycle-related proteins p-Cdc2, CyclinD2, p-Rb, as well as p-Akt and p-ERK proteins, had significantly decreased in RPE cells transfected with miR-96 (p-Rb: t=11.211, P=0.002; p-Cdc2: t=9.133, P=0.003; CyclinD2: t=7.542, P=0.005; p-ERK: t=16.699, P<0.001; p-Akt: t=23.552, P<0.001). Conclusions: MiR-96 inhibits human RPE cell proliferation and migration by targeting MITF and regulating the expression of cell cycle and migration-related proteins.