miR-326-3p对视网膜祖细胞分化调控作用的研究

Regulation of miR-326-3p on the differentiation of retinal progenitor cells

目的探讨微小RNA-326-3p(miR-326-3p)在视网膜祖细胞(RPCs)分化过程中的作用,旨在为干细胞移植治疗视网膜退行性疾病研究提供新的思路。方法将RPCs随机分为对照组(Control组)、miR-326-3p过表达组(Mimics组)和miR-326-3p敲降组(Inhibitors组),在分化培养基中培养7 d后,分别转染miR-326-3p-Control、Mimics和Inhibitors,转染48 h后收取细胞RNA和蛋白,通过qPCR实验和Western blotting实验检测RPCs分化指标胶质细胞标记物(GFAP)、双极神经元标记物(PKC-a)、视杆细胞标记物(Rhodopsin)、视锥和视杆细胞标记物(Recoverin)和中间神经元标记物β3-微管蛋白(β3-tubulin)在3组中的表达量。结果(1)miR-326-3p在视网膜祖细胞分化过程中的表达量明显增加;(2)与Control组相比,Mimics组中miR-326-3p过表达后RPCs分化指标GFAP、PKC-a、Rhodopsin、Recoverin和β3-tubulin表达量明显的增加;而Inhibitors组中敲除miR-326-3p后这些分化指标明显降低。结论miR-326-3p在视网膜祖细胞分化过程中内源性表达量明显增加,并且其对视网膜祖细胞的分化有明显促进作用。

Objective To investigate the role of miR-326-3p in the differentiation of retinal progenitor cells(RPCs),and to provide a new research direction of stem cell transplantation for the treatment of retinal degenerative diseases.Methods Retinal stem cells(RPCs)were randomly divided into the Control group,miR-326-3p overexpression group(Mimics group)and miR-326-3p inhibitors group(Inhibitor group).After treated under differentiation medium for 7 days,RPCs were transfected with miR-326-3p-control,miR-326-3p-mimics and miR-326-3p-inhibitors.Cells were collected after 48 hours of tranfection for qPCR and Western blotting experiments to test the expression levels of RPC differentiation markers,including GFAP(a glial cell marker),PKC-α(a marker for retinal bipolar neurons),Rhodopsin(a marker for rod photoreceptors),Recoverin(a marker for rod and corn photoreceptors),andβ3-tubulin(a pan-neuronal marker).Results(1)The expression level of miR-326-3p was significantly increased during the differentiation of RPCs;(2)Compared with the Control group,the expression levels of RPC differentiation markers including GFAP,PKC-a,Rhodopsin,Recoverin andβ3-tubulin were significantly increased after the overexpression of miR-326-3p,while significantly decreased after miR-326-3p was knocked out in the Inhibitor group.Conclusions The endogenous expression of miR-326-3p significantly increased during the differentiation of retinal progenitor cells,which significantly promoted the differentiation of RPCs.