HIF-1Alpha-induced Up-regulation Of MiR-9Contributes To Phenotypic Modulation In Pulmonary Artery Smooth Muscle Cells During Hypoxia

Background and ObjectiveChronic hypoxia-induced pulmonary hypertension is called hypoxic pulmonaryhypertension (HPH), which plays a key role in the development of high altitude heartdisease and chronic pulmonary heart disease such as chronic obstructive pulmonary disease.HPH is a severe disease characterized by pulmonary vasoconstriction and vascularremodeling, leading to increased vascular resistance and right ventricular dysfunction.Thickening of the media occurs consistently at all levels of the pulmonary arterial tree,especially in small pulmonary arteries, and there is extension of new smooth muscle into thepartially muscular and non-muscular peripheral arteries. The changes of vascular narrowingmay lead to a progressive increase in pulmonary vascular resistance and right ventricleafterload, which in turn induce right ventricular hypertrophy, or even cause the rightventricular failure and death. HPH is believed to be the major pathophysiological process ofmany cardiovascular diseases. Therefore, it is worthy to elucidate the pathogenesis of HPHfor clinical significance of prevention and treatment in cardiovascular diseases.Pulmonary arterial smooth muscle cells (PASMCs) are the cellular components of thenormal pulmonary arterial wall that provides structural integrity and regulates the diameterby contracting and relaxing dynamically in response to vasoactive stimuli. Hypertrophy andhyperplasia of PASMCs are important pathological features during hypoxic pulmonaryvascular structural remodeling. Unlike the skeletal and cardiac muscle cells, vascularsmooth muscle cells (VSMCs) are non-terminally differentiated cells and retain remarkableplasticity. The differentiated VSMCs are characterized by specific contractile proteins, ionchannels, and cell surface receptors that regulate the contractile process and are thus termedcontractile phenotype cells. VSMCs can modulate their phenotype from contractile featureto proliferative feature under certain environmental cues. The phenotype-switched VSMCs always exhibit increased proliferation, migration and matrix synthesis, characterized bydecreased expression of contractile marker proteins, including smooth muscle (SM) α-actin(SM-α-actin), SM-myosin heavy chain (SM-MHC), h1-calponin (calponin1) and SM22α.Previous studies suggested that phenotypic switch of VSMCs is the initial step of somemolecular events such as proliferation and migration and is the pathophysiological basis incardiovascular diseases. Recently, several studies have demonstrated that PASMCs caninduce a phenotype switch from contractile to proliferative activity and induce excessiveproliferation when exposed to hypoxia. These changes represent the majorpathophysiological characteristics in HPH. However, the mechanisms involved in theseevents are poorly understood.MicroRNA (miRNA) is a class of small endogenous non-coding RNA moleculesranging from21-23nucleotides in length that controls gene expression inpost-transcriptional level by targeting mRNAs for translational repression or cleavage. It isestimated that miRNA can directly regulate at least30%of the genes in the human genomeand are therefore believed to be involved in regulating almost all physiological andpathological cellular processes. Recent studies have found that several miRNA are involvedin the changes of proliferation, apoptosis, migration and other functions in pulmonary arterysmooth muscles during hypoxia and participate in HPH, however, the underlyingmechanisms remain obscure.In an initial quantitative RT-qPCR-based screen for differentially expressed miRNA,we identified miR-9as the most significantly up-regulated miRNA in primary PASMCs inresponse to hypoxia. The objective of our current study is to determine whether miR-9playsan important role in phenotypic switch of PASMCs and, if so, to determine the epigeneticregulatory mechanism for the up-regulation of miR-9in phenotypically modulated PASMCsunder hypoxia.Methods and contents:1. Collagenase digestion methods were applied for culturing primary PASMCs of rats,PASMCs were purified by differential adherence and were identified by immunofluorescence staining for SM-α-actin antibody.2. The marker gene expressions of vascular smooth muscle cells were detected byWestern Blot; and miRNA expressions were measured by quantitative RT-PCR (RT-qPCR). 3. To explore the roles of miR-9in normoxia, PASMCs were transfected miR-9mimics.To explore the roles of miR-9under hypoxia, PASMCs were transfected with miR-9inhibitor. The protein expressions of marker genes were measured by Western Blot and theproliferation ability was detected by EdU assay.4. To investigate the role of HIF-1α in miR-9expression under hypoxic conditions,PASMCs were transfected with HIF-1α-specific siRNA before miR-9expression wasdetected by RT-qPCR.5. RT-qPCR was performed to determine the expressions of three primary transcripts ofmiR-9under hypoxic conditions and HIF-1α-specific siRNA were applied to investigate therole of HIF-1α in the differential expressions of three primary transcripts of miR-9underhypoxia.6. The putative HIF-1binding motifs (5′-RCGTG-3′) located within the5-KB regionsupstream of TSSs of miR-9-1, miR-9-2and miR-9-3loci were predicted with the MAPPERdatabase. ChIP assays were performed to determine whether HIF-1α was recruited to theputative binding sites under hypoxia.7. To investigate the functional consequences of each binding sites, wild type anddeletion mutant reporters were constructed and dual-luciferase reporter assays wereperformed.8. To investigate miR-9expression in small pulmonary arteries of HPH, the HPHmodel of rats was established and then the miR-9expression in pulmonary arterial sectionwas detected by RT-qPCR.Results:1. After several times of purification for PASMCs by differential adherence, theSM-α-actin-positive cells can reach95%.2. Hypoxia treatment down-regulated several VSMCs-specific genes at protein levels,including SM-MHC, SM-α-actin, calponin1and SM22α, compared with the normoxiacontrol group. Cells exposed to hypoxia for48h showed an increased EdU positivePASMCs. Among the candidate miRNAs in HPH, miR-9showed the highest increase(5.3-fold) after24h of hypoxia and was maintained at above4-fold increase after48h ofhypoxia.3. Compared with the negative control group，transfection with the miR-9inhibitor significantly increased the expression of contractile phenotypic marker genes at proteinlevels and reduced the percentage of EdU positive PASMCs after48h of hypoxia exposure.By contrast, miR-9mimic, but not mimic control, significantly reduced VSMC markergenes at protein levels and increased the percentage of EdU positive PASMCs in normoxia.4. Transfection of HIF-1α specific siRNA significantly reduced miR-9expressionunder hypoxia.5. After48h hypoxia exposure, both pri-miR-9-1and pri-miR-9-3significantlyelevated, while pri-miR-9-2had no significant difference. In addition, silencing HIF-1αnearly abolished the hypoxia-induced increase in pri-miR-9-1and pri-miR-9-3comparedwith scrambled siRNA group.6. Using in silico analysis, we found the presence of putative HIF-1binding motifs(5′-RCGTG-3′) located within the5-KB regions upstream of TSSs of both miR-9-1andmiR-9-3loci. The miR-9-1locus contains three putative HIF-1binding sites, located at-1292/-1279(R1),-1008/-995(R2) and-874/-861(R3), respectively, and miR-9-3locus hasa putative HIF-1binding site located at-43/-28(R). The HIF-1α enrichment increased in allthe three HIF-1motifs upstream of miR-9-1after24h and48h of hypoxia; among them,miR-9-1R1showed the most significant increase. HIF-1α enrichment upstream of miR-9-3also increased after24h and48h of hypoxia exposure.7. The functional consequences of HIF-1binding sites were assessed by dual luciferasereporter assays. For miR-9-1, hypoxia exposure (1%O2) or transfection of HIF-1α vectorenhanced the luciferase activity of wild-type reporter Luc-miR-9-1(WT) to2.1-fold or1.8-fold, respectively, but there were no significant difference on the luciferase activity ofdeletion mutant reporter Luc-miR-9-1(M3). Conversely, depletion of HIF-1α using siRNAfollowed by hypoxia exposure attenuated the luciferase activity of wild-type reporter (WT)but had no effect on the deletion mutant reporter (M3). For miR-9-3, the luciferase activityof wild-type reporter Luc-miR-9-3was increased2.7-fold by hypoxia exposure and wasincreased1.9-fold by transfection of HIF-1α vector. However, neither hypoxia nor HIF-1αoverexpression had any effect on the luciferase activity of the deletion mutant reporterLuc-miR-9-3M (M). Conversely, siRNA-mediated depletion of HIF-1α followed byhypoxia exposure attenuated the luciferase activity of wild-type reporter (WT) but not ofdeletion mutant reporter (M). 8. Rats were exposed to5000m simulated high altitude in hypobaric chamber toestablish the HPH model. Compared with normoxic control group, the miR-9expression ofchronic hypoxia group in pulmonary arterial section increased significantly.Conclusion:1. miR-9is involved in hypoxia-induced phenotypic modulation in PASMCs of rats.2. The up-regulation of miR-9under hypoxia mainly originates from the transcriptionalactivation of miR-9-1and miR-9-3.3. The hypoxia-induced upregulation of miR-9expression is regulated by HIF-1.In summary, HIF-1-miR-9regulatory pathway is an important regulatory mechanismof hypoxia-induced phenotypic modulation in PASMCs of rats; miR-9could be a potentialtherapeutic target for prevention and treatment of HPH.