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 (H(2)O(2)) 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 H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) 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(1.000). 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=11.01, 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. (Chin J Ophthalmol, 2019, 55: 355-360).
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 H(2)O(2)对HTMC进行刺激6 h；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/L H(2)O(2)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H(2)O(2)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 H(2)O(2)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=11.01，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。（中华眼科杂志，2019，55：355-360）.
Keywords: Fibronectins; MicroRNAs; Oxidative stress; Trabecular meshwork.