title: [Human antigen R regulates TGF-β1expression in airway smooth muscle cells under the stimulation of platelet-derived growth factor].
abstract: Objective:To 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).Methods:Airway smooth muscle (ASM) cells were cultured at 37 ℃ and 5% CO2in 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-β1in 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-β1expression 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-β1after interrupting HuR expression. The concentration of TGF-β1in 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-β1mRNA 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.Results:PDGF 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 (allP<0.05). Similar alterations could also be demonstrated at TGF-β1mRNA 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-β1over-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 (allP<0.05). The concentration of TGF-β1in 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 (bothP<0.05). Actinomycin D disturbed the process of transcription and the half-life of TGF-β1mRNA 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-β1mRNA in HuR siRNA group showed significant change (P<0.05).Conclusions:PDGF can elevate TGF-β1expression in ASM cells. HuR regulates TGF-β1expression by promoting its mRNA stability.
authors: Department of Respiratory Medicine, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, China.
authors: N
authors: N
authors: Wang
authors: D
authors: D
authors: Yan
authors: X M
authors: XM
authors: Gu
authors: Y
authors: Y
authors: Liu
authors: S J
authors: SJ
authors: Jiang
mesh_terms: D002478:Cells, Cultured; D006801:Humans; D032389:Myocytes, Smooth Muscle; D010982:Platelet-Derived Growth Factor; D034622:RNA Interference; D012333:RNA, Messenger; D011946:Receptors, Antigen; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1
chemical_list: D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D011946:Receptors, Antigen; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1