微小RNA-155-5p通过靶向BTB-CNC异体同源体1促进尿道上皮细胞的炎症和纤维化反应  

MicroRNA-155-5p promotes inflammatory and fibrosis response of urethral epithelial cells by targeting BTB and CNC homology 1

作　　者：贾江华[1] 孟庆松[1] 张明[1] 杜蕾[1] 尹跃伟 薛文勇[1] 齐进春[1] Jia Jianghua;Meng Qingsong;Zhang Ming;Du Lei;Yin Yuewei;Xue Wenyong;Qi Jinchun(Department of Urology,the Second Hospital of Hebei Medical University,Shijiazhuang 050000,China)

机构地区：[1]河北医科大学第二医院泌尿外科,石家庄050000

出　　处：《中华实验外科杂志》2022年第5期888-891,共4页Chinese Journal of Experimental Surgery

基　　金：河北省省级科技计划 (20377724D)。

摘　　要：目的观察尿道上皮细胞中微小RNA(miR)-155-5p通过靶向BTB-CNC异体同源体1(BACH1)促进尿道上皮细胞的炎症和纤维化反应,从而参与尿道狭窄的发生和发展。方法生信分析结果BACH1可能是miR-155-5p的直接靶标,然后将miR-155-5p mimics或mimics NC与pmiR-Glo-BACH1-WT或pmiR-Glo-BACH1-MUT共转染到SV-HUC-1细胞中,转染48 h后,检测荧光素酶活性。将BACH1小干扰RNA(siRNA)转染到SV-HUC-1细胞中,pcDNA3.1-BACH1转染到SV-HUC-1细胞中。转染24 h后,用酶联免疫吸附测定(ELISA)检测肿瘤坏死因子-α(TNF-α),白介素-1β(IL-1β)和白介素-6(IL-6)水平,应用实时定量反转录聚合酶链反应(RT-qPCR)检测血管内皮生长因子(VEGF)、纤维连接蛋白(FN)、IV型胶原(collagen IV)的信使核糖核酸(mRNA)水平,同时应用蛋白质印迹法(Western blot)检测VEGF、FN和CollagenⅣ的蛋白质水平。两组间分析采用单因素方差分析。结果miR-155-5p和pmiR-Glo-BACH1-WT共转染的细胞,与miR-155-5p和pmiR-Glo-BACH1-MUT共转染的细胞比较的荧光活性明显下降(FLUCR与RLUCR比率为0.54±0.05比0.95±0.07,F=73.65,P<0.05)。然而,无论转染pmiR-Glo-BACH1-MUT或pmiR-Glo-BACH1-WT,模拟对照细胞中的荧光强度都无明显变化(比率为1.03±0.04比1.01±0.03 F=0.548,P>0.05)。IL-1β、IL-6和TNF-α水平在BACH1敲低细胞中增加(分别为615.57±29.44比285.37±23.97,F=226.939;818.53±41.16比502.63±24.38,F=130.824;596.63±41.16比366.57±28.84,F=88.349),P<0.01,但在BACH1过表达细胞中下降(分别为122.70±15.94比298.87±12.44,F=227.675;282.50±33.16比511.30±39.76,F=58.585;129.83±9.31比374.80±25.40,F=245.926,P<0.01)。此外,VEGF、FN和CollagenⅣ的mRNA(分别为1.65±0.08比0.96±0.06,F=149.555;2.00±0.12比0.97±0.07,F=160.243;1.65±0.06比0.96±0.02,F=369.388,P<0.01)和蛋白质水平(分别为1.50±0.10比1.01±0.09,F=38.382;1.42±0.10比0.99±0.09,F=31.339;1.30±0.06比0.95±0.05,F=62.642,P<0.01)也因BACH1敲低而增加,但因SV-HUC-1细胞中BACH1过表达而下�Objective To observe that microRNA(miR)-155-5p promotes the inflammatory and fibrotic responses of urothelial cells by targeting BTB and CNC homology 1(BACH1),thereby participating in the occurrence and development of urethral strictures.Methods Bioinformatics analysis found that BACH1 may be a direct target of miR-155-5p,and then miR-155-5p mimics or negative mimics were co-transfected with pmiR-Glo-BACH1-WT or pmiR-Glo-BACH1-MUT 48 h after transfection into SV-HUC-1 cells,luciferase activity was detected.BACH1 small interfering RNA(siRNA)was transfected into SV-HUC-1 cells,negative siRNA was used as control,pcDNA3.1-BACH1 was transfected into SV-HUC-1 cells,pcDNA3.1 was used as control.At 24 h after transfection,the levels of interleukin(IL)-1β,IL-6 and tumor necrosis factor-α(TNF-α)were detected by enzyme linked immunosorbent assay(ELISA),the mRNA levels of vascular endothelial growth factor(VEGF),fibronectin(FN)and collagenⅣwere detected by quantitative polymerase chain reaction(qPCR),and the protein levels of VEGF,FN and collagenⅣwere detected by Western blotting.The data were expressed as mean±standard deviation(SD),and one-way ANOVA was used for analysis between two groups.Results Cells co-transfected with miR-155-5p and pmiR-Glo-BACH1-WT showed significantly decreased fluorescence activity compared to cells co-transfected with miR-155-5p and pmiR-Glo-BACH1-MUT(the ratio of FLUCR/RLUCR:0.54±0.05 vs.0.95±0.07,F=73.650,P<0.05).However,the fluorescence intensity in mock control cells did not change significantly regardless of transfection of pmiR-Glo-BACH1-MUT or pmiR-Glo-BACH1-WT(ratio:1.03±0.04 vs.1.01±0.03,F=0.548,P>0.05).IL-1β,IL-6 and TNF-αlevels were increased in BACH1 knockdown cells(615.57±29.44 vs.285.37±23.97,F=226.939;818.53±41.16 vs.502.63±24.38,F=130.824;596.63±41.16 vs.366.57±28.84,F=88.349,respectively,P<0.01),but decreased in BACH1-overexpressing cells(122.70±15.94 vs.298.87±12.44,F=227.675;282.50±33.16 vs.511.30±39.76,F=58.585;129.83±9.31 vs.374.80±25.40,F=245.926,P<

关 键 词：微小RNA-155-5p 尿道狭窄 炎性因子 

分 类 号：R695[医药卫生—泌尿科学]