摘要：
Objective To investigate the expression of microRNA-1 (miR-1) and its regulatory function on fibronectin (FN) in human trabecular meshwork cells (HTMC) under oxidative stress.Methods Experimental study.After HTMC were treated with 0,60,100,200,400 μmol/L hydrogen peroxide (H2O2) for 6 h,respectively,the cells were placed in culture medium for 24 h.The expression of miR-1 and FN mRNA in these cells were detected by real-time quantitative PCR.According to bioinformatics analysis,the target gene of miR-1 is predicted to be FN;pcDNA3/pri-miR-1 vectors,pcDNA3/enhanced green fluorescent protein (EGFP)-FN-3'UTR vectors and pcDNA3/EGFP-FN-3'UTRmut vectors were constructed.pcDNA3/pri-miR-1 were co-transfected with pcDNA3 / EGFP-FN-3'UTR or pcDNA3 / EGFP-FN-3'UTRmut respectively into HTMC.pDsRed2-N1 was taken as internal reference.After 48 h transfection,the absorbance of EGFP and red fluorescent protein (REP) was detected with fluorescence spectrophotometer to explore the effect of miR-1 on FN expression.HTMC was stimulated with 200 μmol/L H2O2 for 24 h after overexpression plasmid of miR-1 was transfected into it,and then FN mRNA and protein levels were detected via real time PCR,Western blotting and immunofluorescence.Data were analyzed via one-way analysis of variance or t test.Results With the increase of H2O2 concentration,miR-1 decreased (F=390.80,P＜0.01)while FN increased (F=13.16,P＜0.01).The level of miR-1 in HTMC stimulated by 200 μmol/L and 400 μmol/L H2O2 decreased to 0.608 ± 0.014 (t=21.67,P＜0.01) and 0.409 ±0.020 (t=29.91,P＜0.01),respectively,compared with untreated control cells (1.000);whereas,the mRNA levels of FN increased to 1.630±0.233 (t=4.47,P=0.011) and 1.903±0.246 (t=6.15,P=0.003),respectively,compared with untreated control cells(l.000).Through bioinformatics analysis,miR-1 might have candidate binding site in FN mRNA 3'-UTR.Meanwhile,these cells co-transfected with pcDNA3/pri-miR-1 and pcDNA3/EGFP-FN-3'UTRmut (0.562±0.018) had higher EGFP expression than cells co-transfected with pcDNA3/pri-miR-1 and pcDNA3/EGFP-FN-3'UTR (0.329±0.015) (t=17.39,P＜0.01).Compared with the control (1.000),after overexpressing miR-1 the mRNA expression and the protein level of FN decreased to 0.294±0.081 (t=1 1.01,P＜0.01) and 0.584±0.022 (t=5.57,P＜0.01),respectively.Conclusions MiR-1 decreases while FN increased in HTMC under oxidative stress.MiR-1 inhibits FN expression through targeting FN 3'-UTR.%目的 探讨氧化应激下微小RNA-1 (miR-1)在人小梁网细胞(HTMC)中的表达及其对纤维连接蛋白(FN)的表达调控作用.方法 实验研究.分别用0、60、100、200、400 μmol/L H2O2对HTMC进行刺激6h;刺激后的细胞在体外培养24 h,随后采用实时荧光定量PCR检测各组细胞miR-1和FN的表达情况.根据生物信息学分析预测出miR-1的靶基因为FN;分别构建pcDNA3/pri-miR-1、pcDNA3/增强型绿色荧光蛋白(EGFP)-FN-3'-非翻译区(3'-UTR)和pcDNA3/EGFP-FN突变体3'-UTR(FN-3'UTRmut)载体并转染HTMC,使用红色荧光蛋白(RFP)表达质粒pDsRed2-N1作为内参,转染48 h后检测EGFP和RFP的吸光度,以探究miR-1对FN表达的影响.过表达miR-1载体转染HTMC后用200 μmol/LH2O2刺激24 h,采用实时荧光定量PCR法、蛋白免疫印迹法和荧光免疫组织化学染色分别检测FN mRNA和蛋白的表达变化.采用单因素方差分析以及两样本t检验对数据进行统计学分析.结果 随着H2O2浓度升高,miR-1的水平逐渐下降(F=390.80,P＜0.01),而FN的水平逐渐升高(F=13.16,P＜0.01).与对照(1.000)相比,200 μmol/L和400 μmol/L H2O2刺激后的HTMC中miR-1水平分别下降至0.608±0.014(t=21.67,P＜0.01)和0.409 ±0.020 (t=29.91,P＜0.01),FN的mRNA水平分别上升至1.630±0.233(t=4.47,P=0.011)和1.903 ±0.246 (t=6.15,P=0.003).通过生物信息学分析,预测到miR-1在FN的mRNA 3'-UTR上存在可能的结合位点,双荧光检测发现:pcDNA3/pri-miR-1和pcDNA3/EGFP-FN-3'UTRmut共转染的细胞EGFP表达(0.562±0.018)高于pcDNA3/pri-miR-1和pcDNA3/EGFP-FN-3'UTR共转染细胞(0.329±0.015)(t=17.39,P＜0.01).与对照(1.000)相比,过表达miR-1时FN的mRNA表达量下降至0.294±0.081(t=1 1.01,P＜0.01);蛋白水平减少至0.584±0.022(t=5.57,P＜0.01).结论 氧化应激下HTMC中miR-1水平降低,而FN的表达升高;miR-1通过靶定FN的3'-UTR来抑制FN的表达.
