SRF Regulates CTGF Involved In Hypoxia-induced Abnormal Proliferation Of Rat Pulmonary Artery Smooth Muscle Cells

Objective: To investigate whether serum response factor(SRF)regulates connective tissue growth factor(CTGF)involved in inducing pulmonary artery smooth muscle cell proliferation in rats under hypoxic conditions.Methods: Healthy Sprague-Dawley rats were collected,and PASMCs were isolated from the rats for primary culture and identification.Continue to culture for 3generations,and follow-up experiments can be performed.Rat PASMCs cultured at1%,2%,5%,10%,21% oxygen concentration,respectively,and observe cell proliferation after 24 h,48h,and 72 h,and then to establish the optimal hypoxic concentration in the experiment.PASMCs were cultured under normoxic and optimal low oxygen concentrations,and the expression of SRF and CTGF in both groups was detected.SRF-siRNA and CTGF-siRNA sequences were designed for rats.The experiments were divided into 5 groups: normoxic group,hypoxia group,hypoxia +NC-siRNA group,hypoxia + SRF-siRNA group,hypoxia + CTGF-siRNA group.Quantitative real-time PCR was used to detect the expression of SRF m RNA and CTGF m RNA in each group.The expression of SRF protein and CTGF protein in each group were measured by Western-blot method.The MTT method was used to detect the proliferation activity of PASMCs in 5 groups(normoxic group,hypoxia,hypoxia + NC-siRNA,hypoxia + SRF-siRNA,hypoxia + CTGF-siRNA).Results: After 72 hours of observation,it was found that there was a significant difference in cell proliferation under different concentrations of oxygen.The 1% O2 group had the highest proliferation activity,and the optimal low oxygen concentration was set to 1% O2.The expressions of SRF and CTGF m RNA and protein in the hypoxic(1% O2)group were significantly higher than those in the normal oxygengroup.Under hypoxia(1% O2)conditions,SRF-siRNA transfected rat PASMCs in SRF-siRNA group and CTGF-siRNA transfected rat PASMCs in CTGF-siRNA group.Real-time quantitative PCR showed that the expression of SRF m RNA in the SRF-siRNA group was significantly suppressed compared with other groups,while the expression of SRF m RNA was similar in the hypoxic group,hypoxia +NC-siRNA,and CTGF-siRNA groups.The expression of CTGF m RNA in the SRF-siRNA group and CTGF-siRNA group was significantly inhibited compared with the hypoxia control group and hypoxia + NC-siRNA.This shows that SRF regulates the expression of CTGF.The MTT assay found that the proliferation activity of PASMCs was highest in the hypoxic group and the hypoxia + NC-siRNA group,and the proliferation activity of PASMCs in the hypoxia + SRF-siRNA group and the hypoxia + CTGF-siRNA group was significantly affected compared with the hypoxia.Conclusion: This experiment verified that rat PASMCs proliferate actively under hypoxic conditions and that SRF promotes abnormal proliferation of PASMCs by regulating CTGF under hypoxic conditions.