HB-EGF Promotes Hypoxia-Induced Rat Pulmonary Artery Smooth Muscle Cell Proliferation By Upregulating CCND1 Expression

Objective:To investigate whether heparin binding epidermal growth factor like growth factor(HB-EGF)mediates the proliferation of pulmonary artery smooth muscle cells(PASMCs)under hypoxia and whether it regulates the expression of cyclin D1(CCND1).Methods:The pulmonary arteries of healthy male Sprague Dawley rats of SPF grade were isolated in vitro,and the smooth muscle layer was taken for primary culture.First of all,the rat PASMCs was cultured at 1%,2%,5%,10% and 21% oxygen for 24 hours,48 hours and72 hours,respectively.RT-PCR and western blot detect the expression of HB-EGF and CCND1.The rat HB-EGF si RNA and CCND1 si RNA sequences were designed and transfected.The experiment was divided into five groups: normoxia group,hypoxia group,hypoxia + NC si RNA group,hypoxia + HB-EGF si RNA group,hypoxia +CCND1 si RNA group.In order to detect the proliferative activity of rat PASMCs cultured under different conditions,MTT method was used.In order to detect the expression of HB-EGF and CCND1 in each group,RT-PCR and Western blot were used.Results:Firstly,the primary PASMCs of rats were successfully extracted and cultured well,which were confirmed to be the required cells by α-SMA morphological identification.After 24 hours,48 hours and 72 hours of culture,it was found that the cell proliferation activity was the strongest under the concentration of 1% O2,so the subsequent experiment was set as 1% O2.MTT assay showed that the proliferation activity of rat pulmonary artery smooth muscle cells was the highest in hypoxia group and hypoxia +NC si RNA group.Compared with hypoxia group,the proliferation activity of rat pulmonary artery smooth muscle cells in hypoxia + HB-EGF si RNA group and hypoxia+ CCND1 si RNA group was significantly inhibited.The m RNA and protein expressions of HB-EGF and CCND1 in hypoxia group were significantly higher than those in normoxia group.After transfection of HB-EGF si RNA and CCND1 si RNA into PAMSCs cultured under hypoxia(1% O2),the expression of HB-EGF m RNA in hypoxia + HB-EGF si RNA group was significantly inhibited compared with the other four groups,while the expression of HB-EGF m RNA in hypoxia group,hypoxia + NC si RNA group and hypoxia + CCND1 si RNA group was similar.CCND1 m RNA expression in hypoxia + HB-EGF si RNA group and hypoxia + CCND1 si RNA group was significantly inhibited compared with hypoxia group and hypoxia + NC si RNA group.Conclusion:The proliferation of rat PASMCs is active under hypoxia.HB-EGF can promote the abnormal proliferation of rat PASMCs by regulating CCND1 under hypoxia.