Role Of LncRNA MALAT1 As A CeRNA(Competitive Endogenous RNA) In The Regulation Of MiR-124-3p.1/KLF5 In The Development Of Pulmonary Hypertension

Objectves: Pulmonary arterial hypertension(PAH)is a highly malignant cardiovascular disease characterized by vascular proliferation as the main pathological feature.There is currently no specific cure for PAH,and treatment goals are limited to delays or prevent progression.Although more progress has been made in the treatment of PAH in recent years,it is still an incurable disease.Therefore,finding the cause of PAH has important clinical significance.Long non-coding RNA(LncRNA)is a rising star in non-coding RNA following microRNA,and plays an important role in the progression of various cardiovascular diseases such as vascular remodeling.Metastasis-associated lung adenocarinoma transcript 1,MALAT1,is a highly conserved LncRNA that is expressed in tumor tissues in many patients.Recently,a large number of studies have shown that MALAT1 is involved in the regulation of vascular endothelial cell dysfunction and hypoxic pulmonary artery smooth muscle cell proliferation,but MALAT1 as a competitive endogenous sponge RNA(ceRNA)together with microRNA regulates the pathogenesis of PAH vascular remodeling has not been reported.Therefore,this study aimed to investigate the pathogenesis of PAH from the perspective of MALAT1 regulation of microRNA activity.Materials and methods: HE staining technique was used to analyze the thickness of human PAH vessel wall;Western blotting and Real-time PCR were used to determine the effect of MALAT1 gene silencing on the proliferation of human pulmonary artery smooth muscle cells(HPASMCs)and the expression of MALAT1 downstream target genes;CCK8 kit,flow Techniques such as cell analysis verified the effect of MALAT1 on proliferation of pulmonary artery smooth muscle cells;bioinformatics software was used to analyze miR-124-3p.1 response elements in the MALAT1 nucleic acid sequence.Further studies have found that zinc lipoprotein transcription factor(Kruppel-1ike factor 5,KLF5)can be used as a new target of miR-124-3p.1 to mediate pulmonary vascular remodeling.The dual luciferase reporter gene,RNA-binding protein immunoprecipitation(RIP)assay,gene transfection,molecular biology and other techniques clarified that MALAT1/miR-124-3p.1 regulates the expression of KLF5 and regulates pulmonary vascular remodeling.Results: MALAT1 is up-regulated in pulmonary vessels and HPASMCs in patients with PAH.Further studies revealed that knockdown of MALAT1 in HPASMCs inhibited the expression of cell cycle-associated proteins such as proliferating cell nuclear antigen(PCNA),Cyclin A and Cyclin E,and ultimately inhibited the proliferation and migration of HPASMCs;overexpression of MALAT1,PCNA,Cyclin Increased expression of cell cycle-associated proteins such as A and Cyclin E promoted proliferation and migration of HPASMCs.In addition,we also found that the target of miR-124-3p.1 is present in the nucleic acid sequenceof MALAT1,and the wild-type LncRNA MALAT1(h LncRNAs MALAT1)reporter vector was co-transfected with miR-124-3p.1 mimics by luciferase assay.The luciferase activity was significantly decreased.After the vector binding site was mutated,the inhibitory effect of miR-124-3p.1 on luciferase activity disappeared,suggesting that MALAT1 interacts with miR-124-3p.1 as a competitive endogenous ceRNA.The RNA binding protein immunoprecipitation(RIP)assay more strongly demonstrates the binding of MALAT1 to hsa-miR-124-3p.1.Western Blotting showed that the expression of KLF5 was increased in pulmonary vascular tissue and hypoxic HPASMCs in PAH patients,and the expression of KLF5 was decreased after transfection of miR-124-3p.1 mimics.After interferencewith MALAT1,the gene and protein expression of KLF5 in HPASMCs also decreased significantly.Similarly,inhibition of the expression of has-miR-124-3p.1 also resulted in a significant decrease in KLF5 expression in HPASMCs.Thus,we conclude that the MALAT1/hsa-miR-124-3p.1/KLF5 axis plays an important role in the development of pulmonary hypertension.Conclusions: We found that the MALAT1/hsa-miR-124-3p.1/KLF5 axis is involved in the regulation of PAH development and provides a new target for the treatment of PAH.On one hand,hsa-miR-124-3p.1 binds to the 3'-UTR of KLF5 m RNA and regulates KLF5 after transcription;on the other hand,MALAT1 can inhibit the expression of hsa-miR-124-3p.1 and promote the proliferation of smooth muscle cells.And migration.Silencing MALAT1 up-regulated hsa-miR-124-3p,which in turn inhibited the expression of KLF5.This study not only reveals the possible mechanism of PAH development,but also hopes to open up new ideas for the clinical diagnosis and treatment of PAH.