Resarch On The Mechanism Of Targeting Regulation Of ADAMTS1 By MiRNA-142-5p In Pulmonary Arterial Hypertension

Background and objectivePulmonary arterial hypertension(PAH)refers to a series of clinical diseases characterized by increased pulmonary artery pressure,contraction of pulmonary arterioles,vascular remodeling and thrombosis,including pathophysiological and hemodynamic changes.Hemodynamic Diagnostic Criteria for PAH: In static state,the mean pulmonary artery pressure(mPAP)detected by right cardiac catheterization is equal or more than25 mmHg,and the pulmonary artery wedge pressure(PAWP)is equal or less than 15 mmHg and pulmonary vascular resistance is more than 3 Wood units.In 2018,China updated the consensus of experts on diagnosis and treatment of pulmonary hypertension in the 2007 edition,issued the?Guidelines for Diagnosis and Treatment of Pulmonary Hypertension in China?,and provided a new basis for diagnosis and treatment of pulmonary hypertension in China.Experts at home and abroad have made remarkable progress in screening methods,gene diagnosis and targeted drug therapy ofpulmonary arterial hypertension through their respective efforts,but epidemiological surveys show that the natural survival rate of pulmonary hypertension is only 2.8 years.Even patients who are given targeted drugs such as Ambrisentan(endothelin receptor antagonist,Ietairis),Sildenafil(phosphodiesterase inhibitor-5)can only survive for 3.6 years.The 5-year survival rate of Idiopathic pulmonary arterial hypertension(IPAH)and hereditary pulmonary arterial hypertension was only 20.8% after routine treatment.In recent years,a large number of studies have shown that a variety of microRNAs(miRNAs)are involved in the occurrence and development of various cardiovascular diseases,including pulmonary arterial hypertension,which makes microRNAs gradually become a research hot spot.Method:A large number of animal experiments and scientific studies have shown that ADAMTS1 is closely related to the occurrence and development of pulmonary arterial hypertension.The application of bioinformatics software(microRNAs.org,TargetScan,etc.)to predict the targeted regulation of microRNAs in ADAMTS1.The levels of ADAMTS1 in plasma of 16 blank control subjects and 60 patients with pulmonary arterial hypertension were detected by enzyme-linked immuno sorbent assay(ELISA).Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression of 6 predictive targetmicroRNAs in plasma of 16 blank control subjects and 60 patients with pulmonary arterial hypertension,and to determine whether there was correlation.MicroRNA-142-5p(miR-142-5p/ miRNA-142-5p)is selected as our research subject.Then we chose monocrotaline(MCT)inducing to establish a pulmonary hypertension rat model,and cultured primary pulmonary artery smooth muscle cells(PASMCs),sub-cultured and preserved them.MiR-142-5p mimic and miR-142-5p inhibitor were transfected into PASMCs by Lipofectine 2000.The expression of miR-142-5p was detected by qRT-PCR.CCK-8 assay was used to detect the effect of miR-142-5p on the proliferation of PASMCs.Flow cytometry(FCM)was used to detect the effect of miR-142-5p on cell cycle of PASMCs.Bioinformatics software was used to predict that the target gene of miR-142-5p was ADAMTS1.Using molecular cloning technology,pmirGLO-ADAMTS1 3'-UTR vector and mutant were constructed.HEK293 cells were cultured and co-transfected with miR-142-5p and pmirGLO-ADAMTS1 3'-UTR or mutant.The fluorescence intensity of HEK293 cells in different transfection groups was detected by double luciferase reporter gene assay,and the relationship between them was clarified.PASMCs were cultured.MiR-142-5p mimic and miR-142-5p inhibitor were transfected into PASMCs by the same Lipofectine 2000.The expression of ADAMTS1 mRNA and protein levels were detected by qRT-PCR and Western blot respectively.The expression plasmid ofpcDNA3.1-ADAMTS1 was constructed and co-transfected with pcDNA3.1-ADAMTS1 and miR-142-5p mimic into PASMCs.The expression of ADAMTS1 was detected by Western blot assay.CCK-8 assay was used to detect the effect of co-transfection of ADAMTS1 and miR-142-5p on the proliferation of PASMCs.Flow cytometry(FCM)was used to detect the effect of co-transfection of ADAMTS1 and miR-142-5p on the cell cycle of PASMCs.Result:ELISA results showed that the expression of ADAMTS1 in pulmonary arterial hypertension group was significantly lower than that in control group.The results of qRT-PCR showed that the expression of miRNA-181b-5p and let-7e-5p in plasma was too low;the expression of miRNA-181a-5p and miRNA-181c-5p in plasma was not significantly different from that in normal subjects;the expression of miRNA-142-5p and let-7b-5p in plasma of pulmonary arterial hypertension patients was significantly increased,and there were statistical differences.Compared with the two groups,the expression of miR-142-5p was significantly higher and the difference was significant.The expression of miR-142-5p in plasma of patients with pulmonary hypertension increased by 127%compared with that of control group.CCK-8 results showed that miR-142-5p promoted the proliferation of PASMCs after overexpression of miR-142-5p in PASMCs.FCM results showed that over-expression ofmiR-142-5p reduced the percentage of G0/G1 phase cells from 66.93% to58.64%,while the percentage of S phase cells increased from 22.01% to38.64%.A number of studies have shown that ADAMTS1 is closely related to pulmonary arterial hypertension.Bioinformatics software also predicted that one of the target genes of miR-142-5p that might be involved in the development of pulmonary arterial hypertension was ADAMTS1.The results of double luciferase reporter gene experiment showed that luciferase activity in ADAMTS1 3'-UTR region could be reduced by miR-142-5p.Overexpression of miR-142-5p in PASMCs reduced ADAMTS1 gene expression by 47% and protein expression by 31%.After co-transfection of miR-142-5p mimic and pcDNA3.1-ADAMTS1 plasmid in PASMCs,the expression level of ADAMTS1 protein increased by 152%.CCK-8 showed that co-transfection of ADAMTS1 and miR-142-5p could inhibit the proliferation of PASMCs.Compared with the control group,the percentage of G0/G1 phase cells in PASMCs increased from 61.18% to 73.04% after co-transfection of ADAMTS1 and miR-142-5p,while the percentage of S phase cells decreased from 34.67% to 21.13%.Conclusion:The results showed that the expression of miR-142-5p was significantly increased in the plasma of patients with pulmonary arterial hypertension,while the expression of ADAMTS1 was significantly decreased in the plasma of patients with pulmonary hypertension.Afteroverexpression of miR-142-5p in PASMCs,miR-142-5p promotes the proliferation and cycle transition of PASMCs by down-regulating the expression of target gene ADAMTS1.These results suggest that in the pathogenesis and development of pulmonary arterial hypertension,miR-142-5p may participate in the proliferation of PASMCs by down-regulating the expression of ADAMTS1.The level of miR-142-5p may provide a new target for clinical diagnosis of PAH,and the regulation of its downstream target may provide a new theoretical basis for the treatment of pulmonary arterial hypertension.