Role Of RELM-βregulatory Signal Pathway Ca²⁺ /Calcineurin/NFATc3 In The Proliferation Of Human Hypoxic Pulmonary Artery Smooth Muscle And Its Mechanism

Objective : To investigate the mechanism of RELM-β regulation of Ca2+/Calcineurin/NFATc3 signaling pathway leading to proliferation of human pulmonary artery smooth muscle cells.By interfering with RELM-β gene expression,the effects of Calcineurin/NFATc3 signaling pathway and human pulmonary artery smooth muscle cells proliferation were observed.By adding recombinant RELM-β protein and pathway inhibitors to human pulmonary arterial smooth muscle cells,the expression of Calcineurin/NFATc3 in the pathway and the effect on the prooliferation of pulmonary artery smooth muscle cells.Methods:(1)In the first part of the experiment,under the conditions of normoxia and hypoxia and the expression of RELM-β gene,the expression of RELM-β and the effect of RELM-β on Ca2+/Calcineurin/NFATc3 signaling pathway and proliferation of human pulmonary artery smooth muscle cells were observed.Divide into eight groups:1.Normoxic group 2.Hypoxia group3.Lentivirus no-load silencing normoxic group 4.Lentivirus no-load silencing hypoxia group 5.Lentivirus silencing normoxia group 6.Lentivirus silencing hypoxia group 7.Lentivirus overexpression normoxia group 8.Lentivirus overexpression hypoxia group.Human pulmonary artery smooth muscle cells were grown logarithmically and synchronized by cell cycle in culture medium of 10% FBS + 1% double antibody for 24 hours.The detected by QT-PCR and western bolt methods,RELM-β,Calcineurin,NFATc3 expression levels,using the EDU kit todetect the proliiferation of human pulmonary artery smooth muscle cells.(2)The second part of the experiment explores the mechanism of RELM-β regulation of Ca2+ /Calcineurin/NFATc3 signaling pathway leading to proliferation of human pulmonary artery smooth muscle cells.Divide into eight groups:1.Control group 2.After adding normal saline,add recombinant human RELM-β protein group 3.Add Calciumion chelating agent BAPTA-AM followed by reconbinant human RELM-β protein group 4.Add Calcineuvin/NFAT inhibitor cyclosporine A followed by reconbinant human RELM-β protein group.The detected by QT-PCR and western bolt methods,Calcineurin,NFA Tc3 expression levels,using the EDU kit todetect the proliiferation of hu man pulmonary artery smooth muscle cells.Results:(1)The first part of the experiment: the effect of hypoxia on the expression of RELM-β and the proliferation of HPASMCs QT-PCR and Western bolt results showed that Compared with the normoxic group,the mRNA and protein expressions of RELM-β,Calcineurin and NFATc3 were increased in the hypoxic group(P<0.05);Compared with the lentiviral empty-loaded normoxic group,the mRNA and protein expressions of RELM-β,Calcineurin and NFATc3 were decreased in the Lentivirus silencing normoxic group(P<0.05);Compared with the Lentivirus silencing normoxia group,the mRNA and protein expression of RELM-β,Calcineurin and NFATc3 was no significant change in the Lentivirus silencing hypoxic group(P>0.05);Compared with the Lentivirus no-load silencing hypoxia group,the mRNA and protein expressions of RELM-β,Calcineurin and NFATc3 in the Lentivirus silencing hypoxia group were significantly decreased(P<0.05);Compared with the Lentivirus no-load silencing normoxia group,the mRNA and protein expressions of RELM-β,Calcineurin and NFATc3 were increased in the Lentivirus no-load silencing hypoxia group(P<0.05);Compared with Lentivirus no-load silencing normoxic group,the mRNA and protein expressions of RELM-β,Calcineurin and NFATc3 in lentivirus overexpressing normoxic group were increased(P<0.05);Compared with Lentivirus silencing normoxia group,the mRNA and protein expressions of RELM-β,Calcineurin and NFATc3 in lentivirus overexpressing normoxic group were increased(P<0.05);Compared with lentivirus overexpressing normoxia group,the mRNA and protein expressions of RELM-β,Calcineurin and NFATc3 were increased in lentivirus overexpressing hypoxia group(P<0.05);Compared with Lentivirus silencing hypoxia group,the mRNA and protein expressions of RELM-β,Calcineurin and NFATc3 were increased in lentivirus overexpressing hypoxia group(P<0.05);Compared with the Lentivirus no-load silencing hypoxia group,the mRNA and protein expressions of RELM-β,Calcineurin and NFATc3 were increased in lentivirus overexpressing hypoxia group(P<0.05).EDU immunofluorescence results show: Compared with the normoxic group,the cell proliferation of in the hypoxic group was increased(P<0.05);Compared with the lentiviral empty-loaded normoxic group,the cell proliferation of in the Lentivirus silencing normoxia group was decreased(P<0.05);Compared with the Lentivirus silencing hypoxia group,the cell proliferation of the lentiviral empty-loaded hypoxia group was significantly was increased(P<0.05),but the cell proliferation of the Lentivirus silencing normoxia group was not significantly changed(P>0.05);Compared with the lentiviral empty-loaded normoxic group,the cell proliferation of the lentivirus overexpressing normoxia group was increased(P<0.05);Compared with the lentiviral overexpressing normoxia group,the cell proliferation of the lentivirus overexpressing hypoxia group was increased(P<0.01).(2)The second part of the experiment explores the mechanism of RELM-β regulation of Ca2+ /Calcineurin/NFATc3 signaling pathway leading to proliferation of human pulmonary artery smooth muscle cells.QT-PCR and Western bolt results showed that Compared with the control group,the expression of mRNA and protein of Calcineurin and NFATc3 in the After adding normal saline,add recombinant human REM-β protein group was significantly increased(P<0.01).Compared with After adding normal saline,add recombinant human REM-β protein group,the expression of mRNA and protein of Calcineurin and NFATc3 in the Add Calcium ion chelating agent BAPTA-AM followed by reconbinant human RELM-β protein group were significantly decreased(P<0.01);Compared with After adding normal saline,add recombinant human REM-β protein group,the expression of mRNA and protein of Calcineurin and NFATc3 in the Add Calcineurin/NFAT inhibitor cyclosporine A followed by reconbinant human RELM-β protein group were significantly decreased(P<0.01).EDU immunofluorescence results show: Compared with the control group,the cell proliferation of the After adding normal saline,add recombinant human REM-β protein group was increased(P<0.01).Compared with the After adding normal saline,add recombinant human REM-β protein group,the cell proliferation of the Add Calcium ion chelating agent BAPTA-AM followed by reconbinant human RELM-β protein group were significantly decreased(P<0.01);Compared with the After adding normal saline,add recombinant human REM-β protein group,the cell proliferation of the Add Calcineurin/NFAT inhibitor cyclosporine A followed by reconbinant human RELM-β protein group were significantly decreased(P<0.01).Conclusions: 1.Hypoxia can incresae the expression of RELM-β、Calcineurin、NFATc3 and can promote the proliferation of human pulmonary artery smooth muscle cells;2.RELM-β leads to proliferation of human pulmonary artery smooth muscle cells by regulating Ca2+/Calcneurin/NFATc3 signaling pathway.