Guo J H, Su C, Jiang S Y, Wang F, Feng X, Wang J T
Tianjin Medical University Eye Hospital, College of Optometry and Ophthalmology, Tianjin Medical University Eye Institute, Tianjin 300384, China.
Center of Stomatology, Shenzhen Hospital, Peking University, Shenzhen 518036, China.
Zhonghua Yan Ke Za Zhi. 2019 May 11;55(5):355-360. doi: 10.3760/cma.j.issn.0412-4081.2019.05.009.
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. 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 test. With the increase of H(2)O(2) concentration, miR-1 decreased (390.80, 0.01) while FN increased (13.16, 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 (21.67, 0.01) and 0.409±0.020 (29.91, 0.01), respectively, compared with untreated control cells (1.000); whereas, the mRNA levels of FN increased to 1.630±0.233 (4.47, 0.011) and 1.903±0.246 (6.15, 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) (17.39, 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 (11.01, 0.01) and 0.584±0.022 (5.57, 0.01), respectively. MiR-1 decreases while FN increased in HTMC under oxidative stress. MiR-1 inhibits FN expression through targeting FN 3'-UTR. .
探讨氧化应激下人小梁网细胞（HTMC）中微小RNA-1（miR-1）的表达及其对纤连蛋白（FN）的调控作用。实验研究。将HTMC分别用0、60、100、200、400 μmol/L过氧化氢（H₂O₂）处理6小时后，置于培养基中培养24小时。采用实时定量PCR检测这些细胞中miR-1和FN mRNA的表达。根据生物信息学分析，预测miR-1的靶基因是FN；构建了pcDNA3/pri-miR-1载体、pcDNA3/增强绿色荧光蛋白（EGFP）-FN-3'UTR载体和pcDNA3/EGFP-FN-3'UTRmut载体。将pcDNA3/pri-miR-1分别与pcDNA3/EGFP-FN-3'UTR或pcDNA3/EGFP-FN-3'UTRmut共转染入HTMC。以pDsRed2-N1作为内参。转染48小时后，用荧光分光光度计检测EGFP和红色荧光蛋白（REP）的吸光度，以探讨miR-1对FN表达的影响。将miR-1过表达质粒转染入HTMC后，用200 μmol/L H₂O₂刺激24小时，然后通过实时PCR、蛋白质印迹法和免疫荧光检测FN mRNA和蛋白水平。数据采用单因素方差分析或检验进行分析。随着H₂O₂浓度的增加，miR-1降低（390.80，0.01）而FN升高（13.16，0.01）。与未处理的对照细胞（1.oo0）相比，200 μmol/L和400 μmol/L H₂O₂刺激的HTMC中miR-1水平分别降至0.608±0.014（21.67，0.01）和0.409±0.020（29.91，0.01）；而FN的mRNA水平分别升至1.630±0.233（4.47，0.011）和1.903±0.246（6.15，0.003），与未处理的对照细胞（1.000）相比。通过生物信息学分析，miR-1可能在FN mRNA 3'-UTR中有候选结合位点。同时，与pcDNA3/pri-miR-1和pcDNA3/EGFP-FN-3'UTR共转染的细胞（0.329±0.015）相比，与pcDNA3/pri-miR-1和pcDNA3/EGFP-FN-3'UTRmut共转染的细胞（0.562±0.018）具有更高的EGFP表达（17.39，0.01）。与对照（1.000）相比，过表达miR-1后FN的mRNA表达和蛋白水平分别降至0.294±0.081（11.01，0.01）和0.584±0.022（5.57，0.01）。在氧化应激下，HTMC中miR-1降低而FN升高。miR-1通过靶向FN 3'-UTR抑制FN表达。
