The Role Of NOR1in Proliferation Of Human Pulmonary Smooth Muscle Cells

Pulmonary artery hypertension (PAH) is one of the main complications of chronicobstructive pulmonary disease (COPD). It is closely associated with the prognosis ofCOPD. The pathogenesis of PAH includes sustained vasoconstrictions,structuralremodeling of the pulmonary artery, thrombosis and so on. Pulmonary artery smoothmuscle cells (PASMCs) play an important role in the process of pulmonary vascularremolding. Neuron-derived orphan receptor1(NOR1) belongs to the nuclear receptorsuper family, composed of NR4A subfamily with Nur77and Nurrl. NOR1is kind of proteincoded by immediate early genes, and it participates widely in pathophysiological processes,including cell proliferations, cell differentiation,cell apoptosis,inflammations, cancer andneurological diseases. In this project, we explored the role and mechanism of NOR1in theproliferation of human PASMCs cultured in vitro.Part (1) The expression of NOR1in human PASMCsObjective: To probe NOR1expression on human PASMCs and its functions.Methods:1. We divided the human PASMCs cultured in vitro into two groups:Control group: cultured by smooth muscle cell media (SMCM) and mixed in0.5%fetalbovine serum (FBS) and FBS group: cultured by SMCM+10%FBS.2. Adopting MTTand PCNA immunohistochemistry staining methods to detect the proliferation of humanPASMCs in each group.3. NOR1mRNA expression and protein expression in each groupwere respectively detected by Real time PCR and Western-Blot.Results:1. The proliferation of PASMCs in FBS group increased obviously thancontrol group after24hours cultivation in vitro, the OD value of FBS group detected by MTT was0.446±0.0243compared to Control group was0.349±0.037(P<0.05); and thepositive ratio of PCNA staining in FBS group detected by immunohistochemistry was0.509±0.044compared to Control group was0.261±0.035(P<0.05).2. The NOR1expression in human PASMCs in FBS group was obviously higher than Control group.After2hours cultivation in vitro, the expression of NOR1mRNA (the folds of control)detected by Real time PCR was3.781±0.442compared to Control group was1(P<0.05);and the expression of NOR1protein(NOR1/β-actin)detected by Western blot was:0.573±0.137compared to Control group was0.112±0.013(P<0.05).Conclusions: The expression of NOR1in human PASMCs obviously increased afterFBS stimulations. At the same time, it accompanied with the proliferation of humanPASMCs, so we speculate that NOR1may participate in regulating proliferation of humanPASMCs. Part (2) The Molecular mechanism of NOR1in human PASMCsproliferationObjective: To investigate the mechanism of NOR1in regulating human PASMCsproliferation.Methods:1. Cultivating human PASMCs in vitro, then transfered NOR1’s antisenseoligonucleotide to human PASMCs throught transfection reagent PEI25K.2. HumanPASMCs were divided into four groups:①Control group: SMCM+0.5%FBS;②FBSgroup: SMCM+10%FBS;③FBS+empty transfection group (FBS+N group): SMCM+10%FBS+transfection reagent without DNA;④FBS+antisense NOR1oligonucleotidetransfection group (FBS+AS group): SMCM+10%FBS+NOR1’s antisenseoligonucleotide.3. Using MTT and PCNA immunohistochemistry staining methods todetect the proliferation of human PASMCs in each group.4. NOR1and CyclinD1mRNA expression and protein expression in each group were respectively detected by Real timePCR and Western-Blot.Results:1. NOR1’s antisense oligonucleotide can effectively suppress the NOR1expression in human PASMCs after cultivating in vitro for2hours. The expression ofNOR1mRNA (the folds of control) in FBS+AS group detected by Real time PCR was:1.893±0.163compared to FBS group was4.021±0.0386(P<0.05); and the expression ofNOR1protein (NOR1/β-actin) in FBS+AS group detected by Western blot was0.430±0.039compared to FBS group was0.870±0.071(P<0.05).2. NOR1’s antisenseoligonucleotide can obviously suppress the proliferation of PASMCs after cultivating invitro for24hours. The OD value of FBS+AS group detected by MTT was0.385±0.046compared to FBS group was0.441±0.039(P<0.05); the positive ratio of PCNA staining ofFBS+AS group detected by immunohistochemistry was0.331±0.044compared to FBSgroup was0.511±0.054(P<0.05).3. Transfection with NOR1’s antisense oligonucleotidefor6hours can decrease the expression of CyclinD1in human PASMCs. The expressionvalue of CyclinD1mRNA (the folds of control) in FBS+AS group was1.513±0.161compared to FBS group was2.310±0.301(P<0.05); the expression value of CyclinD1protein (NOR1/β-actin) in FBS+AS group was0.514±0.068compared to FBS group was0.759±0.083(P<0.05).Conclusions:1. NOR1plays an important role in PASMCs proliferation.2. NOR1participates in PASMCs proliferation by regulating target gene CyclinD1.