血小板源性生长因子诱导下人抗原R对气道平滑肌细胞内转化生长因子β1表达的影响

Human antigen R regulates TGF-β1 expression in airway smooth muscle cells under the stimulation of platelet-derived growth factor

目的探讨血小板源性生长因子（PDGF）诱导下人抗原R（HuR）对气道平滑肌细胞（ASM）内转化生长因子β1（TGF－β1）表达的影响。方法体外培养ASM以PDGF（20 μg/L）分别在0、6、12和24 h刺激ASM以诱导哮喘状态。实时荧光定量PCR检测各时间点细胞内HuR、TGF－β1 mRNA水平，Western印迹法检测各时间点细胞内HuR、TGF－β1的蛋白水平。利用RNA干扰技术特异性阻断HuR表达，Western印迹法检测干扰组和对照组HuR及TGF－β1在PDGF刺激0、6和12 h的表达水平。酶联免疫吸附法检测干扰组和对照组在PDGF刺激0、6和12 h后，细胞培养上清中TGF－β1的蛋白水平。将细胞依次分为对照组、对照＋PDGF 6 h组、干扰组及干扰＋PDGF 6 h组，然后各组依次加入放线菌素D（10 mg/L）刺激0、4、8和12 h，检测各组TGF－β1 mRNA的半衰期。结果PDGF刺激下HuR表达呈现明显的时间依赖性，0、6、12和24 h细胞内HuR mRNA及蛋白相对表达量分别为1.00±0.00、1.35±0.14、1.73±0.17、2.07±0.10及0.51±0.10、0.67±0.05、0.83±0.07、0.95±0.02（均P〈0.05）。TGF－β1的mRNA及蛋白水平也呈时间依赖性，0、6、12和24 h细胞内TGF－β1 mRNA及蛋白相对表达量分别为1.00±0.00、1.27±0.06、1.60±0.10、1.87±0.10及0.72±0.09、0.87±0.07、1.13±0.12、1.33±0.05（6与12 h、12与24 h两两比较，均P〈0.05）。PDGF刺激0 h，干扰组与对照组HuR表达差异无统计学意义（P〉0.05），PDGF刺激12 h，干扰组HuR表达较对照组降低了21.9%（P〈0.05）。对照组0、6、12 h TGF－β1蛋白相对表达量分别为0.70±0.05、0.89±0.06、1.06±0.05，而干扰组0、6、12 h TGF－β1蛋白相对表达量分别为0.67±0.09、0.77±0.03、0.89±0.05（与对照组相应时间点比较，均P〈0.05）。细胞培养上清TGF－β1含量对照组0、6、12 h依次为（773.33±16.32）、（877.97±16.03）、（3 060.34±82.53）ng/L，而干扰组0、6、12 h依次为（277.33±9.93）、（407.77±7.14）、（828.05±11.67）ng/L（与对照组相应时间点比较，均P〈0.05）。放线菌素D刺激细胞，干扰组TGF－β1的RNA半衰期与干扰＋PDGF 6 h组比较差异无统计学意义（P〉0.05），与对照组及对照＋PDGF 6 h组比较差异均有统计学意义（均P〈0.05）。结论PDGF可上调ASM内TGF－β1表达，HuR通过增加mRNA稳定性参与PDGF诱导的哮喘状态下TGF－β1的表达过程。

ObjectiveTo study the role of human antigen R （HuR） regulated transforming growth factor β1 （TGF-β1） expression in airway smooth muscle cells under the stimulation of platelet-derived growth factor （PDGF）.MethodsAirway smooth muscle （ASM） cells were cultured at 37 ℃ and 5% CO2 in dulbecco′s modified eagle medium （DMEM） cell medium. Cells at passages between 4 and 11 were divided into different groups according to the different compounds added. For control group, no compounds were administrated. For PDGF group, cells were stimulated with PDGF （20 μg/L） and cultured for an additional time. Cells were harvested and real-time PCR was used to measure mRNA level and Western blotting to detect protein level of HuR and TGF-β1 in ASM cells for different groups. Cells were divided into HuR siRNA group and control group. RNA-interference was used to determine whether lowering HuR expression could decrease PDGF-induced TGF-β1 expression in HuR siRNA group and control group after the stimulation of PDGF for indicated times. Western blotting analysis was used to test the expression of TGF-β1 after interrupting HuR expression. The concentration of TGF-β1 in the cultured serum of HuR siRNA group and control group for 0, 6, 12 h under the stimulation of PDGF was measured by enzyme-link immunosorbent assay （ELISA）. Cells were divided into control group, control＋ PDGF 6 h group, HuR siRNA group and HuR siRNA＋ PDGF 6 h group, then the half-life of TGF-β1 mRNA in different groups was determined by treating ASM cells with the transcriptional inhibitor actinomycin D （10 mg/L） for 0, 4, 8 and 12 h.ResultsPDGF treatment for 0, 6, 12 and 24 h significantly promoted HuR mRNA and protein expression and the relative levels were 1.00±0.00, 1.35±0.14, 1.73±0.17, 2.07±0.10; 0.51±0.10, 0.67±0.05, 0.83±0.07, 0.95±0.02 （all P〈0.05）. Similar alterations could also be demonstrated at TGF-β1 mRNA and protein. The relative expression was 1.00±0.00, 1.27±0.06, 1.60±0.10, 1.87±0.10; 0.72±0.09, 0.87±0.07, 1.13±0.12, 1.33±0.05 （6 h versus 12 h and 12 h versus 24 h, P〈0.05）. HuR expression decreased 21.9% in HuR siRNA group compared with control group under the stimulation of PDGF for 12 h. HuR silencing also decreased PDGF-induced TGF-β1 over-expression in ASM cells. In the control group, the relative protein levels of PDGF treatment for 0, 6 and 12 h were 0.70±0.05, 0.89±0.06, 1.06±0.05 and the protein levels in HuR siRNA group were 0.67±0.09, 0.77±0.03, 0.89±0.05 （all P〈0.05）. The concentration of TGF-β1 in the cultured serum was measured by ELISA and the outcomes were （773.33±16.32, 877.97±16.03, 3 060.34±82.53） ng/L in control group for 0, 6 and 12 h. Under the same condition, the outcomes of HuR siRNA group were （277.33±9.93, 407.77±7.14, 828.05±11.67） ng/L （both P〈0.05）. Actinomycin D disturbed the process of transcription and the half-life of TGF-β1 mRNA in HuR siRNA group showed no significant change compared with the HuR siRNA＋ PDGF 6 h group （P〉0.05）. However, compared with control group and control ＋ PDGF 6 h group, the half-life of TGF-β1 mRNA in HuR siRNA group showed significant change （P〈0.05）.ConclusionsPDGF can elevate TGF-β1 expression in ASM cells. HuR regulates TGF-β1 expression by promoting its mRNA stability.