微小RNA-155-5p通过激活转化生长因子-β/Smad通路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 activating transforming growth factor-β/Smad pathway

目的观察尿道上皮细胞中微小RNA(miR)-155-5p对转化生长因子-β(TGF-β)/Smad信号通路的影响, 从而参与创伤性尿道狭窄炎症和纤维化的调节。方法将miR-155-5p mimics及miR-155-5p inhibitor转染到尿道上皮细胞SV-HUC-1中, 转染24 h后使用酶联免疫吸附试验(ELISA)检测SV-HUC-1细胞中炎性因子肿瘤坏死因子-α(TNF-α), 白细胞介素(IL)-1β和IL-6的水平。qPCR和蛋白印记分别检测血管内皮生长因子(VEGF)、纤维连接蛋白(FN)、Ⅳ型胶原(Collagen Ⅳ)、TGF-β、Smad3和Smad7的mRNA和蛋白表达水平。组间分析采用单因素方差分析。结果 IL-1β、IL-6和TNF-α的水平在转染miR-155-5p mimics后增加(496.27±18.87比255.77±21.40, F=213.184;931.93±78.50比494.90±30.81, F=80.569;915.70±30.02比486.60±25.05, F=361.323, P<0.01), 但在转染miR-155-5p inhibitor后下降(140.10±14.77比294.07±10.26, F=219.855;257.63±33.75比495.47±29.66, F=84.052;275.47±30.31比484.20±25.34, F=83.746, P<0.01)。miR-155-5p mimics增加了SV-HUC-1细胞中VEGF、FN和Collagen Ⅳ的mRNA(1.87±0.08比0.96±0.07, F=251.787;1.78±0.12比0.96±0.06, F=116.780;2.04±0.12比0.98±0.06, F=197.344)和蛋白质水平(1.55±0.05比0.98±0.12, F=60.460;2.28±0.04比1.00±0.11, F=391.095;1.97±0.06比0.99±0.06, F=407.516), 但miR-155-5p inhibitor降低了SV-HUC-1细胞中VEGF、FN和Collagen Ⅳ的mRNA(0.47±0.06比0.99±0.07, F=104.457;0.54±0.06比0.98±0.08, F=58.767;0.39±0.03比0.96±0.07, F=162.360)和蛋白质水平(0.35±0.03比0.98±0.09, F=137.388;0.32±0.05比0.99±0.09, F=127.047;0.43±0.05比0.98±0.06, F=137.224, P<0.01)。转染miR-155-5p mimics后TGF-β和Smad3的mRNA表达水平(2.42±0.07比1.01±0.05, F=817.164;1.87±0.08比0.95±0.02, F=399.340, P<0.01)和蛋白表达水平(1.56±0.13比0.99±0.05, F=51.031;1.27±0.02比0.98±0.07, F=48.519, P<0.05)升高, 转染miR-155-5p inhibitor后下降mRNA水平(0.41±0.03比1.02±0.06, F=249.053;0.38±0.05比1.00±0.04, F=342.250)和蛋白水平(0.14±0.07比1.02±0.08, F=194.139;0.72±0.03比1.02±0.09, F=30.899, P<0.01);而Smad7的mRNA水平(0.43±0.05比0.97±0.02, F=301.655)和蛋白水平(0.20±0.03比0.95±0.04, F=684.122)被miR-155-5p mimics下调并被miR-155-5p inhibitor上调(mRNA水平为2.16±0.07比0.97±0.07, F=433.500;蛋白水平为1.40±0.09比0.98±0.07, F=41.779, P<0.01)。结论 miR-155-5p通过激活TGF-β/Smad信号通路, 从而参与创伤性尿道狭窄炎症和纤维化的调节。

Objective To observe the influence of of microRNA(miR)-155-5p on the transforming growth factor-β(TGF-β)/Smad signaling pathway in urethral epithelial cells,thereby participating in the regulation of inflammation and fibrosis in traumatic urethral stricture.Methods MiR-155-5p mimics and miR-155-5p inhibitor were transfected into urothelial cells SV-HUC-1,and 24 h after transfection,enzyme linked immunosorbent assay(ELISA)was used to detect the inflammatory factors interleukin(IL)-1β,IL-1 and tumor necrosis factor-α(TNF-α)levels.The mRNA and protein expression levels of vascular endothelial growth factor(VEGF),fibronectin(FN),collagenⅣ,TGF-β,Smad3 and Smad7 were detected by qPCR and Western blotting,respectively.Data were expressed as mean±standard deviation(Mean±SD),and one-way ANOVA was used for between-group analysis.Results The levels of IL-1β,IL-6 and TNF-αincreased after transfection with miR-155-5p mimics(496.27±18.87 vs.255.77±21.40;F=213.184,931.93±78.50 vs.494.90±30.81,F=80.569;915.70±30.02 vs.486.60±25.05,F=361.323,P<0.01),but decreased after transfection with miR-155-5p inhibitor(140.10±14.77 vs.294.07±10.26,F=219.855;257.63±33.75 vs.495.47±29.66,F=84.052;275.47±30.31 vs.484.20±25.34,F=83.746,P<0.01).The miR-155-5p mimics increased the mRNA levels(1.87±0.08 vs.0.96±0.07,F=251.787;1.78±0.12 vs.0.96±0.06,F=116.780;2.04±0.12 vs.0.98±0.06,F=197.344)and protein levels(1.55±0.05 vs.0.98±0.12,F=60.460;2.28±0.04 vs.1.00±0.11,F=391.095;1.97±0.06 vs.0.99±0.06,F=407.516)of VEGF,FN and collagenⅣin SV-HUC-1 cells,but miR-155-5p inhibitor decreased the mRNA levels(0.47±0.06 vs.0.99±0.07,F=104.457;0.54±0.06 vs.0.98±0.08,F=58.767;0.39±0.03 vs.0.96±0.07,F=162.360)and protein levels(0.35±0.03 vs.0.98±0.09,F=137.388;0.32±0.05 vs.0.99±0.09,F=127.047;0.43±0.05 vs.0.98±0.06,F=137.224)of VEGF,FN and collagenⅣin SV-HUC-1 cells,P<0.01.The mRNA expression levels(2.42±0.07 vs.1.01±0.05,F=817.164;1.87±0.08 vs.0.95±0.02,F=399.340,P<0.01)and protein levels(1.56±0.13 vs.0.99±0.05,F=51.031;1.27±0.02 vs.0.98±0.07,F=48.519,P<0.05)of TGF-βand Smad3 were increased after transfection with miR-155-5p mimics,but the mRNA levels(0.41±0.03 vs.1.02±0.06,F=249.053;0.38±0.05 vs.1.00±0.04,F=342.250)and protein levels(0.14±0.07 vs.1.02±0.08,F=194.139;0.72±0.03 vs.1.02±0.09,F=30.899)were decreased after transfection with miR-155-5p inhibitor,P<0.01.The mRNA level(0.43±0.05 vs.0.97±0.02,F=301.655)and protein level(0.20±0.03 vs.0.95±0.04,F=684.122)of Smad7 was down-regulated by miR-155-5p mimics,but up-regulated by miR-155-5p inhibitor(mRNA level 2.16±0.07 vs.0.97±0.07,F=433.500;protein level 1.40±0.09 vs.0.98±0.07,F=41.779,P<0.01).Conclusion MiR-155-5p activates the TGF-β/Smad signaling pathway,thereby participating in the regulation of inflammation and fibrosis in traumatic urethral stricture.