TGF-β1调控肺泡上皮细胞PI3K亚基构成变化的研究
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摘要
目的探讨肺泡上皮细胞(AECs)中PI3K亚基的表达情况和TGF-β1是否可以调控PI3K亚基的构成变化。方法采用实时定量PCR法检测AECs中PI3K亚基mRNA的表达情况,予5ng/ml TGF-β1刺激A549细胞0、3、6、12、24和48h不同时间点,实时定量PCR法检测PI3K亚基mRNA的表达变化,Western blot法检测PIK3CD蛋白表达水平。结果 AECs中Ⅰ型PI3K催化亚基以表达PIK3CA、PIK3CB和PIK3CD为主,Ⅰ型PI3K调节亚基以表达PIK3R1、PIK3R2和PIK3R3为主,Ⅱ型PI3K亚基和Ⅲ型PI3K亚基以表达PIK3R4、PIK3C3、PIK3C2A和PIK3C2B为主。TGF-β1刺激可以调控Ⅰ型PI3K催化亚基中的PIK3CD mRNA和蛋白的表达呈时间依赖性升高。结论 AECs中Ⅰ型PI3K亚基、Ⅱ型PI3K亚基和Ⅲ型PI3K亚基均有不同程度表达,TGF-β1经调控PIK3CD的表达导致Ⅰ型PI3K催化亚基的构成变化。
Objective To investigate the expression of phosphoinositide 3-kinase(PI3K) subunits and the regulatory effect of transforming growth factor-β1(TGF-β1) on constituent changes of PI3K subunits in alveolar epithelial cells(AECs). Methods AECs A549 cells were cultured in vitro and stimulated by TGF-β1. The expression of PI3K subunits and the dynamic expression change of PI3K subunits at mRNA and protein levels were measured with RT-PCR and Western blot, respectively. Results The A549 cells expressed classⅠPI3K catalytic subunits including PIK3CA, PIK3CB and PIK3CD; classⅠPI3K regulatory subunits including PIK3R1, PIK3R2 and PIK3R3; class Ⅱ,Ⅲ PI3K subnits including PIK3R4, PIK3C3, PIK3C2A and PIK3C2 B. TGF-β1-stimulated the expression of PIK3CD at mRNA and protein levels in a time-dependant manner. Conclusion ClassⅠ, Ⅱ and Ⅲ PI3K subunits are variously expressed in AECs. TGF-β1 up-regulates the constituent changes in PIK3CD of class Ⅰ PIK3 catalytic subunits.
Objective To investigate the expression of phosphoinositide 3-kinase(PI3K) subunits and the regulatory effect of transforming growth factor-β1(TGF-β1) on constituent changes of PI3K subunits in alveolar epithelial cells(AECs). Methods AECs A549 cells were cultured in vitro and stimulated by TGF-β1. The expression of PI3K subunits and the dynamic expression change of PI3K subunits at mRNA and protein levels were measured with RT-PCR and Western blot, respectively. Results The A549 cells expressed classⅠPI3K catalytic subunits including PIK3CA, PIK3CB and PIK3CD; classⅠPI3K regulatory subunits including PIK3R1, PIK3R2 and PIK3R3; class Ⅱ,Ⅲ PI3K subnits including PIK3R4, PIK3C3, PIK3C2A and PIK3C2 B. TGF-β1-stimulated the expression of PIK3CD at mRNA and protein levels in a time-dependant manner. Conclusion ClassⅠ, Ⅱ and Ⅲ PI3K subunits are variously expressed in AECs. TGF-β1 up-regulates the constituent changes in PIK3CD of class Ⅰ PIK3 catalytic subunits.
引文
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[3]Chen C,Fang X,Wang Y,et al.Preventive and therapeutic effects of phosphoinositide 3-kinase inhibitors on acute lung injury[J].Chest,2011,140(2):391-400.DOI:10.1378/chest.10-3060.
[4]Fang X,Li K,Tao X,et al.Effects of phosphoinositide 3-kinase on protease-induced acute and chronic lung inflammation,remodeling,and emphysema in rats[J].Chest,2013,143(4):1025-1035.DOI:10.1378/chest.12-1040.
[5]Wang X,Adler KB,Erjefalt J,et al.Airway epithelial dysfunction in the development of acute lung injury and acute respiratory distress syndrome[J].Expert Rev Resp Med,2007,1(1):149-155.DOI:10.1586/17476348.1.1.149.
[6]Kasai H,Allen JT,Mason RM,et al.TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition(EMT)[J].Respir Res,2005,9:56.DOI:10.1186/1465-9921-6-56.
[7]Jean S,Kiger AA.Classes of phosphoinositide 3-kinases at a glance[J].J Cell Sci,2014,127(Pt 5):923-928.DOI:10.1242/jcs.093773.
[8]Pons-Tostivint E,Thibault B,Guillermet-Guibert J.Targeting PI-3K signaling in combination cancer therapy[J].Trends Cancer,2017,3(6):454-469.DOI:10.1016/j.trecan.2017.04.002.
[9]Rothschild SI.Targeted therapies in non-small cell lung cancer-beyond EGFR and ALK[J].Cancers,2015,7(2):930-949.DOI:10.3390/cancers7020816.
[10]Willis BC,Borok Z.TGF-beta-induced EMT:mechanisms and implications for fibrotic lung disease[J].Am J Physiol Lung Cell Mol Physiol,2007,293(3):L525-534.DOI:10.1152/ajplung.00163.2007.
[11]Stokes CA,Condliffe AM.Phosphoinositide 3-kinase delta(PI3Kdelta)in respiratory disease[J].Biochem Soc Trans,2018,46(2):361-369.DOI:10.1042/bst20170467.
[12]Chapman HA.Epithelial responses to lung injury:role of the extracellular matrix[J].Proc Am Thorac Soc,2012,9(3):89-95.DOI:10.1513/pats.201112-053AW.
[13]王瑞丽,戴威,李凤琴,等.微囊蛋白1及细胞内钙离子在TGF-β1诱导哮喘大鼠气道平滑肌增殖中的作用[J].浙江医学,2016,38(20):1634-1636,1642.
[14]Michalovich D,Nejentsev S.Activated PI3 kinase delta syndrome:from genetics to therapy[J].Front Immunol,2018,9:369.DOI:10.3389/fimmu.2018.00369.
[15]Ge Q,Moir LM,Trian T,et al.The phosphoinositide 3'-kinase p110delta modulates contractile protein production and IL-6 release in human airway smooth muscle[J].J Cell Physiol,2012,227:3044-3052.DOI:10.1002/jcp.23046.
[16]Mercado N,To Y,Ito K,et al.Nortriptyline reverses corticosteroid insensitivity by inhibition of phosphoinositide-3-kinase-delta[J].J Pharmacol Exp Ther,2011,337(2):465-470.DOI:10.1124/jpet.110.175950.
[17]Low PC,Manzanero S,Mohannak N,et al.PI3Kdelta inhibition reduces TNF secretion and neuroinflammation in a mouse cerebralstrokemodel[J].NatCommun,2014,5:3450.DOI:10.1038/ncomms4450.
[18]Fruman DA,Chiu H,Hopkins BD,et al.The PI3K Pathway in Human Disease[J].Cell,2017,170(4):605-635.DOI:10.1016/j.cell.2017.07.029.