The Effect And Mechanism Of Long Non-coding RNA-ANRIL In The Proliferation Of Hypoxic Pulmonary Artery Smooth Muscle Cells

Pulmonary arterial hypertension is a progressive syndrome based on diverse aetiologies.When pulmonary arterial hypertension occurs,there is a persistent increase in pulmonary vascular resistance.The pathological features of pulmonary arterial hypertension include abnormal proliferation of pulmonary vascular smooth muscle cells and pulmonary vascular remodeling.Hypoxia is the main cause of pulmonary arterial hypertension.It directly induces contraction and proliferation of pulmonary artery smooth muscle cells,and the end result is pulmonary vascular remodeling.Recent studies have shown that long non-coding RNAs play important roles in a variety of biological processes,including cell proliferation and apoptosis,and the occurrence and progression of eardiovascular disease.Studies have shown that long non-coding RNA ANRIL promotes the proliferation of vascular smooth muscle cells and the formation of atherosclerotic plaques.Therefore,we speculate that ANRIL may existin pulmonary artery smooth muscle cells and play a regulatory role.In this study,we detected the expression of ANRIL in normoxic and hypoxic human pulmonary artery smooth muscle cells by qPCR.Small interfering RNA was designed to knock down the expression of ANRIL and the cell viability was detected by MTT assay.Under the condition of knocking down ANRIL,the expression of PCNA protein was detected by Western blot and the proliferation of cells by CCK-8 assay.Furthermore,the expression of proliferation-associated nuclear antigen KI-67 was observed by immunofluorescence assay to determine the effect of ANRIL on the proliferation of human pulnonary artery smooth muscle cells.Flow cytometry was used to analyze the effect of ANRIL on cell cycle to explore the effect of ANRIL on the cell cycle of human pulmonary artery smooth muscle cells,and the expressions of Cyclin A,D,E were determined by Western blot.Knock down the expression of ANRIL under normoxia and hypoxia to investigate the effect of ANRIL silencing on the migration of hypoxia-induced human pulmonary artery smooth muscle cells.The scratch-wound assay was used to detect the distance of the migration of human pulmonary artery smooth muscle cells,and the transwell assay was used to detect the number of the migration of human pulmonary artery smooth muscle cells.The results of these experiments showed that ANRIL is expressed in human pulmonary artery smooth muscle cells.The expression of ANRIL was significantly down-regulated when the cells were given hypoxic modeling(P<0.01).After knocking down the expression of ANRIL with small interfering RNA,the cell viability was increased(P<0.01),indicating that the expression of ANRIL had an effect on human pulmonary artery smooth muscle cells.In addition,knocking down ANRIL also aggravated the proliferation of human pulmonary artery smooth muscle cells by CCK-8(P<0.01).The expression of PCNA increased(P<0.01),and the expression of proliferation-related protein KI-67 was up-regulated by fluorescence microscopy.Then we examined the cell cycle,and found the silence of ANRIL could increase the percentage of human pulmonary artery smooth muscle cells in G2/M+S phase(P<0.05)to accelerate the cell cycle progression.At the same time,the expression of cyclin A,D and E were also changed.When ANRIL was down-regulated,the expression of cyclins increased(cyclin A,P<0.01;cyclin D,E,P<0.001).This further validated the effect of ANRIL on the cell cycle.Moreover,the migration distance and migration number of human pulmonaiy artery smooth muscle cells also increased(migration distance,P<0.05;migration number,P<0.01).This study found for the first time that ANRIL had an effect on cell cycle,and also could regulate the proliferation and migration of human pulmonary artery smooth muscle cells under hypoxia,which suggested that ANRIL may be a key regulator of human pulmonary artery smooth muscle cells.This study provides a research basis for the future development of gene therapy hypoxic pulmonary arterial hypertension.