MicroRNA-195/497对香烟烟雾提取物诱导的人支气管上皮细胞中TGF-β表达的影响

Effects of microRNA-195/497 on the expression of TGF-β in human bronchial epithelial cells exposed to CSE

摘要:

目的 探讨microRNA-195/497（miR-195/497）在香烟烟雾提取物（CSE）诱导的人支气管上皮细胞中影响转化生长因子-β（TGF-β）表达的作用机制。方法 用3% CSE诱导BEAS-2B细胞模拟慢性阻塞性肺疾病（COPD）的发病机制，实时荧光定量聚合酶链反应（qRT-PCR）检测miR-195、miR-497、SMAD7及TGF-β基因表达。双荧光素酶报告实验验证miR-195、miR-497与SMAD7的靶向关系，将miR-195 mimic和miR-497 mimic转染至3% CSE处理的细胞中，qRT-PCR检测SMAD7的表达。将miR-195 mimic、miR-497 mimic和si-SMAD7转染至3% CSE处理的细胞中，qRT-PCR检测miR-195、miR-497、SMAD7、TGF-β的基因表达，Western blotting检测SMAD7、TGF-β的蛋白表达。结果 3% CSE组miR-195、miR-497 mRNA相对表达量低于正常细胞组（P <0.05），SMAD7、TGF-β mRNA相对表达量高于正常细胞组（P <0.05）。miR-195 mimic组Wt-SMAD7 mRNA相对表达量低于mimic NC组（P <0.05）。miR-195 mimic组与mimic NC组Wt-SMAD7 mRNA相对表达量比较，差异无统计学意义（P >0.05）。miR-497 mimic组Wt-SMAD7 mRNA相对表达量低于mimic NC组（P <0.05）。miR-497 mimic组与mimic NC组Mut-SMAD7 mRNA相对表达量比较，差异无统计学意义（P >0.05）。miR-195 mimic组SMAD7 mRNA相对表达量低于mimic NC组（P <0.05）。miR-497 mimic组SMAD7 mRNA相对表达量低于mimic NC组（P <0.05）。3% CSE组细胞miR-195和miR-497相对表达量较Control组下降（P <0.05），SMAD7及TGF-β蛋白相对表达量升高（P <0.05）；miR-195 mimic + 3% CSE组细胞miR-195相对表达量较3% CSE组升高（P <0.05），SMAD7、TGF-β下降（P <0.05），而miR-497相对表达量无明显差异（P >0.05）；miR-497 mimic + 3% CSE组细胞miR-497相对表达量较3% CSE组升高（P <0.05），SMAD7、TGF-β下降（P <0.05），而miR-195相对表达量无明显差异（P >0.05）；si-SMAD7 + 3% CSE组SMAD7、TGF-β蛋白相对表达量较3% CSE组下降（P <0.05），而miR-195、miR-497无明显差异（P >0.05）。3% CSE组SMAD7、TGF-β蛋白相对表达量较Control组升高（P <0.05），miR-195 mimic + 3% CSE组、miR-497 mimic + 3% CSE组、si-SMAD7 + 3% CSE组较3% CSE组下降（P <0.05）。结论 miR-195/497可通过调控SMAD7的表达影响CSE诱导的人支气管上皮细胞中TGF-β表达。

Abstract:

Objective To explore the mechanism underlying the effects of miR-195/497 on the expression of TGF-β in human bronchial epithelial cells exposed to cigarette smoke extract (CSE).Methods BEAS-2B cells were exposed to 3% CSE to simulate the pathogenesis of chronic obstructive pulmonary disease (COPD), and the expression levels of miR-195, miR-497, SMAD7 and TGF-β genes were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The targeting relationship of miR-195 and miR-497 with SMAD7 was validated via the dual luciferase reporter assay. After transfection of miR-195 mimic and miR-497 mimic into cells treated with 3% CSE, the expression of SMAD7 was detected by qRT-PCR. Following transfection of miR-195 mimic, miR-497 mimic and si-SMAD7 into cells treated with 3% CSE, the gene expressions of miR-195, miR-497, SMAD7 and TGF-β were detected by qRT-PCR, and the protein expressions of SMAD7 and TGF-β were measured via Western blotting.Results Compared with the normal cells, the relative expression levels of miR-195 and miR-497 were lower (P < 0.01), and the relative expression levels of SMAD7 and TGF-β were higher in cells treated with 3% CSE (P < 0.05). The relative expression of Wt-SMAD7 in the miR-195 mimic group was lower than that in the mimic NC group (P < 0.05). There was no difference in the relative expression of Mut-SMAD7 between the miR-195 mimic group and the mimic NC group (P > 0.05). The relative expression of Wt-SMAD7 in the miR-497 mimic group was lower than that in the mimic NC group (P < 0.05). There was no difference in the relative expression of Mut-SMAD7 between the miR-497 mimic group and the mimic NC group (P > 0.05). The relative mRNA expression of SMAD7 in the miR-195 mimic group was lower than that in the mimic NC group (P < 0.05). The mRNA relative expression of SMAD7 in the miR-497 mimic group was lower than that in the mimic NC group (P < 0.05). The expressions of miR-195 and miR-497 were lower (P < 0.05), and the expressions of SMAD7 and TGF-β were higher in cells treated with 3% CSE than those in cells of the control group (P < 0.05). Compared with the 3% CSE group, the expression of miR-195 was higher (P < 0.05), the expressions of SMAD7 and TGF-β were lower (P < 0.05), and the expression of miR-497 was not significantly altered (P > 0.05) in the miR-195 mimic + 3% CSE group. Compared with the 3% CSE group, the expression of miR-497 was higher (P < 0.05), the expressions of SMAD7 and TGF-β were lower (P < 0.05), and the expression of miR-195 was not significantly altered (P > 0.05) in the miR-497 mimic + 3% CSE group. The expressions of SMAD7 and TGF-β in the si-SMAD7 + 3% CSE group were lower than those in the 3% CSE group (P < 0.05), while there was no difference in the expressions of miR-195 and miR-497 between the two groups (P > 0.05). The relative protein expressions of SMAD7 and TGF-β in the 3% CSE group were higher than those in the control group (P < 0.05), whereas those in the miR-195 mimic + 3% CSE group, miR-497 mimic + 3% CSE group, and si-SMAD7 + 3% CSE group were lower compared to those in the 3% CSE group (P < 0.05).Conclusions The miR-195/497 could affect the expression of TGF-β in human bronchial epithelial cells exposed to CSE by regulating the expression of SMAD7.