MiRNA Omics Analysis And Functional Study Of Human Pulmonary Artery Endothelial Cells Induced By Hypoxia

Background: Pulmonary arterial hypertension(PAH)is a fatal cardiovascular disease characterized by persistent increases in pulmonary artery pressure and vascular resistance.Various factors such as hypoxia can cause pulmonary artery endothelial cell(PAEC)dysfunction,triggering the proliferation of pulmonary artery smooth muscle cells(PASMC)and pulmonary vascular remodeling,leading to the formation of PAH.MicroRNAs(miRNAs)are a class of highly conserved small non-coding RNAs that negatively regulate the expression of target genes at the post-transcriptional level.Numerous studies have shown that abnormal expression of miRNAs is closely related to the occurrence and development of various diseases.With the further research,mature miRNAs are also found in the nucleus and exosomes.Nuclear miRNAs play different regulatory roles due to their specific localization,such as activation and silencing of transcriptional genes,and inhibition of the maturation of other non-coding RNA.Nuclear miRNAs have also been shown to be associated with a variety of diseases,but no research has been reported in the PAH field.Exosome miRNAs,as a signaling molecule,are involved in cellular communication and enable long-range targeted regulation.They have also been shown to be involved in the development of various diseases including PAH and have been studied as a potential diagnostic marker and treatment.Objective: To perform the miRNAs profiling of cells,nuclei and exosomes of human pulmonary artery endothelial cells(HPAEC)under normoxic/hypoxic conditions and to identify the differentially-expressed miRNAs and the miRNAs enriched in HPAEC nuclei.The preliminary function of the HPAEC nuclear miRNAs will also be studied.Methods and Results: Using the highly sensitive and specific S-Poly(T)Plus miRNA detection technology developed by our laboratory,650 miRNAs in HPAEC under hypoxic and normoxic conditions were detected.After removing some miRNAs with poor amplification,the expression profiles of the remaining 568 miRNAs in the hypoxic/normoxic treated HPAEC were mapped.From the preliminary screening results,miRNAs with a foldchange≥1.5 and a Ct value≤30 were selected for individually detection in other 4 groups of hypoxic/normoxic HPAEC samples.10 miRNAs are down-regulated at least two-fold in hypoxia HPAEC :miR-139-3p、miR-323a-5p、miR-572、miR-596、miR-663a、miR-1343-5p、miR-1913、miR-3196、miR-3960 和 miR-4497;3 miRNAs are up-regulated at least two-fold in hypoxia HPAEC: miR-210-3p,miR-424-5p and miR-503-5p.For the screening of miRNAs in HPAEC nuclei,the nuclei were isolated from HPAEC and the nuclear RNA was confirmed to be basically free of cytoplasmic RNA.Using the S-Poly(T)Plus miRNA detection technology,the same 660 miRNAs were detected in HPAEC nuclei.The ΔCt value of the miRNA in the HPAEC nucleus is compared with the ΔCt value in the total cells to determine how much it is enriched in the nucleus.Those miRNAs whose ΔCt value in the nuclear RNA equal to or less than the ΔCt value in the total RNA are selected for individually detection to finally obtain 6 miRNAs that may be enriched in the nucleus: miR-92b-5p,miR-1246,miR-1273 f,miR-3960,miR-4466 and miR-4508.A preliminary functional analysis of these nuclear miRNAs revealed that the overexpression of miR-1246 and miR-3960 can promote the angiogenic capacity of HPAEC,and miR-1246 can also promotes the migration of HPAEC.The animal experiment shows that miR-92b-5p is significantly down-regulated in the pulmonary artery tissues of MCT-induced PAH rats.The high-throughput sequencing of miRNAs in exosomes of hypoxic HPAEC was performed,and the expression profiles of 702 miRNAs were mapped.It was found that miR-1246 was not only enriched in the HPAEC nucleus but also expressed in its exosomes;miR-210-3p was up-regulated in both hypoxic HPAEC cells and exosomes.Conclusion: This study performed miRNA expression profiling of cell 、 nucleus and exosomes of human pulmonary artery endothelial cells(HPAEC)under normoxic/hypoxic conditions.A total of 13 differentially expressed miRNAs with a fold change≥2 in hypoxic HPAEC were identified,including 10 downregulated miRNAs: miR-139-3p,miR-323a-5p,miR-572,miR-596,miR-663 a,miR-1343-5p,miR-1913,miR-3196,miR-3960,miR-4497;and 3 upregulated miRNAs:miR-210-3p,miR-424-5p and miR-503-5p,besides,miR-210-3p was also up-regulated in exosomes of hypoxic HPAEC;6 miRNAs enriched in the nucleus of HPAEC were identified: miR-92b-5p,miR-1246,miR-1273 f,miR-3960,miR-4466 andmiR-4508,miR-1246 was also enriched in the exosomes of HPAEC.miR-1246 and miR-3960 can promote the angiogenic ability of HPAEC,and miR-1246 can also promote the cell migration.The expression level of miR-92b-5p was significantly down-regulated in the pulmonary artery tissue of MCT-induced PAH rats.