The Effect And Mechanism Of RELM? Regulating Ca²⁺/Calcineurin/NFATc3 Signaling Pathway On Proliferation Of Hypoxic Rat Pulmonary Artery Pulmonary Artery Smooth Muscle Cells

Objective:To study whether RELM?regulates the Ca²⁺/Calcineurin/NFATc3signaling pathway on proliferation of hypoxic rat pulmonary artery pulmonary artery smooth muscle cells.Methods:The experiment is divided into two parts:?1?PART ?:The effect of hypoxia and silencing of RELM?and overexpression of RELM?on RELM?,calcineurin and NFATc3 expression and the cell proliferation rate of PASMCs.This experiment was divided into 8 groups:Normoxia group,Hypoxia group,Lentivirus empty normoxia group?Short as empty normoxia group?,Lentivirus empty hypoxia group?Short for empty hypoxia group?,Lentivirus knockdown RELM?normoxia group?Short for knockdown normoxia group?,Lentivirus knockdown RELM?hypoxia group?Short for knockdown hypoxia group?,Lentivirus overexpress RELM? normoxia group?Short for overexpress normoxia group?,Lentivirus overexpress RELM?group?Short for overexpress hypoxia group?.The expression levels of RELM?,Calcineurin and NFATc3 were detected by Western blot and QT-PCR and the proliferation of rPASMCs was detected by EDU.?2?PART ?:To verify the effects of RELM?on Ca²⁺/Calcineurin/NFATc3 pathway and on cell proliferation of rPASMCs,the cells were divided into 4 groups:control group,Normal saline with RELM?group,BAPTA-AM(Ca²⁺chelating agent)with RELM?group,Cyclosporine A?Calcineurin inhibitor?with RELM?group.The expression levels of RELM?,Calcineurin and NFATc3 were detected by Western blot and QT-PCR and the proliferation rate of rPASMCs was detected by EDU.Results:?1?PART ?:The effect of hypoxia and silencing of RELM?and overexpression of RELM?on RELM?,calcineurin and NFATc3expression and the cell proliferation rate of PASMCs:Compared with normoxia group,the expressions of RELM?,Calcineurin,NFATc3 were significantly increased in hypoxia group?P<0.05?;and the cell proliferation was also significantly increased in hypoxia group.Compared with empty normoxia group,the expressions of RELM?,Calcineurin,NFATc3 were significantly increased in overexpress normoxia group?P<0.05?;and the cell proliferation was also significantly increased in overexpress normoxia group.Compared with empty normoxia group,the expressions of RELM?,Calcineurin,NFATc3were significantly decreased in hnockdown normoxia group?P<0.05?;and the cell proliferation was also significantly decreased in knockdown normoxia group.Compared with empty hypoxia group,the expressions of RELM?,Calcineurin,NFATc3 were significantly decreased in knockdown hypoxia group?P<0.05?;and the cell proliferation was also significantly decreased in knockdown hypoxia group.Compared with overexpress normoxia group,the expressions of RELM?,Calcineurin,NFATc3 were significantly increased in overexpress hypoxia group?P<0.05?;and the cell proliferation was also significantly increased in overexpress hypoxia group.Compared with empty hypoxia group,the expressions of RELM?,Calcineurin,NFATc3 were significantly increased in overexpress hypoxia group?P<0.05?;and the cell proliferation was also significantly increased in overexpress hypoxia group.Compared with overexpress hypoxia group,the expressions of RELM?,Calcineurin,NFATc3 were significantly increased in knockdown hypoxia group?P<0.05?;and the cell proliferation was also significantly increased in knockdown hypoxia group.Compared with knockdown normoxia group,the expressions of RELM?,Calcineurin,NFATc3 were significantly increased in overexpress normoxia group?P<0.05?;and the cell proliferation was also significantly increased in overexpress normoxia group.Compared with empty normoxia group,the expressions of RELM?,Calcineurin,NFATc3 were significantly increased in empty hypoxia group?P<0.05?;and the cell proliferation was also significantly increased in empty hypoxia group.Compared with knockdown normoxia group,the expressions of RELM?,Calcineurin,NFATc3 were not significantly changed in knockdown hypoxia group?P>0.05?but the cell proliferation was significantly increased in knockdown hypoxia group.?2?PART ?:To verify the effects of RELM?on Ca²⁺/Calcineurin/NFATc3 pathway and on cell proliferation of rPASMCs:Compare Normal saline and RELM?group with control group:Western blot and QT-PCR results indicats increased expression of RELM?,Calcineurin,NFATc3?P<0.05?;EDU results indicats that the cell proliferation increased.Compare Ca²⁺chelating agent and RELM?group with Normal saline and RELM?group:Western blot and QT-PCR results indicats decrease expression of Calcineurin,NFATc3?P<0.05?;EDU results indicats that the cell proliferation decrease.Compare Calcineurin inhibitor and RELM?group and RELM?group:Western blot and QT-PCR results indicats decrease expression of RELM?,Calcineurin,NFATc3?P>0.05?;EDU results indicats that the cell proliferation decrease.?P>0.05?.Conclusions:1?Hypoxia leads to increasing express of RELM?,Calcineurin and NFATc3,leading to proliferation of pulmonary artery smooth muscle cells.2?Intervention of RELM?may affects the proliferation of rat pulmonary artery smooth muscle cells through Ca²⁺/Calcineurin/NFATc3.