Research On The Effects Of MiR-23a In The Pathogenesis Of Hypoxic Pulmonary Hypertensionon

Long-term chronic hypoxia can cause pulmonary hypertension,and increased pulmonary arterial pressure leads to right heart failure subsequently.Studies have reported that hypoxic pulmonary hypertension(HPH)is also the key link of onset of high altitude heart disease.Understanding the pathogenetic mechanism of HPH is the key for prevention and treatment of chronic hypoxia pulmonary disease and high altitude heart disease.One of the major pathological change characteristics of HPH is hypoxic pulmonary vasoconstriction and pulmonary vascular structural remodeling.Hypertrophy and proliferation of PASMCs,increased extracellular matrix synthesis,hypertrophy of muscular tunica media vasorum,and presence of smooth muscle-like cells in non-muscular blood vessels during the process of pulmonary vascular remodeling can lead to thicker pulmonary arteriole walls and narrower inner diameter,increased pulmonary vascular resistance and pulmonary arterial pressure.Long-term consistent pulmonary arterial hypertension may lead to compensatory hypertrophy of the right ventricle and even right heart failure.Abnormal proliferation,apoptosis and migration of PASMCs are the most important characteristics of pulmonary vascular structural remodeling.Previous studies often focused on the mechanism of changes in anoxia and anoxia-induced changes in related bioactive molecules in regulating such functions as proliferation apoptosis and migration.Results obtained from recent studies revealed that anoxia can significantly downregulate the expression of PASMCs contractile phenotype marker proteins.However,the specific mechanism of this down-regulation is still unclear.Therefore,in-depth research on the molecular mechanism of phenotypic transformation of PASMCs may help us to explain the internal mechanism involve in pulmonary arterial structural remodeling during theprocess of hypoxic pulmonary hypertension.Micro RNAs(miRNAs)are small endogenous non-coding single stranded RNA molecules with regulatory capacities.It is estimated that mi RNA can regulate the expression of about one third of the human genes,which tend to play critical roles in some normal physiological processes or specific pathological and physiological processes.The action mechanism of mi RNAs can be explained in the following two aspects.Firstly,it degrades the mi RNA by directly binding to 3′-UTR of its target mi RNA.Secondly,it regulates the expression through incomplete complementary pairing and inhibition of mRNA translation.Several studies have been shown that mi RNAs participate in multiple physiological processes such as cellular metabolism,differentiation,apoptosis and proliferation.Also it is involved in regulation of many other pathological processes such as tumorigenesis,inflammation resolution and vascular proliferation.Part one: Mechanism underlying the involvement of miR-23 a in hypoxiainduced pulmonary arterial smooth muscle phenotypetransformation.Objective: To investigate the role of mi R-23 a in hypoxia induced pulmonary artery smooth muscle expression and the level of miR-23 a expression in hypoxia.Methods: The collagenase digestion method was used to culture of the rats’ primary PASMCs.SM-MHC,SM-α-actin,Calponin-1,and SM22αprotein expression levels were evaluated using western blot after the PASMCs were subjected to anoxia treatment(3% of O2).Transfection with miR-23 a mimics were conducted when PASMCs were under normoxia and anoxia conditions.EdU staining was used to detect the proliferative activity of PASMCs.Results:1 Effect of anoxia on the general characteristics of rat PASMCs and screening of differentiated mi RNAPrimary rat PASMCs cells were growing on the culture flask’s wall in anextended form,most cells were fusiform,the cytoplasm was rich and cytoplasmic density was high.Sheaf-like parallel arrangement was observed,also peak-valley distribution with PASMCs characteristics was observed.After immunofluorescent staining using SM-α-actin antibody,we observed bright green filamentous fluorescence in the cytoplasm.The positive rate of SM-α-actin was over 95% on average.MiR-23 a,mi R-25,miR-93,and mi R-106 expression levels at 24 h after anoxia(H24h)were compared with those in the normoxia control group.The increase in miR-23 a expression was the most significant.It was 5.1 times higher than that in the normoxia control group(N24h).No significant changes in the expression of miR-143 and mi R-145 were observed.The expression level of miR-23 a at 48 h after anoxia was detected.The result indicated that the expression level of miR-23 a at 48 h after anoxia(H48h)was about 3.8 times higher than that in the normoxia control group(N48h).2 Effect of anoxia on the expression level of contractile phenotype marker protein in the PASMCs and the proliferative activity of PASMCs.The PASMCs in the normoxia control group and anoxia(3%O2,24 h or48h)treatment group were subjected to SM-MHC,SM-α-actin,Calponin-1,and SM22α detection.The result indicated that the expression levels of SM-MHC,SM-α-actin,Calponin-1,and SM22α in the H24 h group were significantly lower than those in the normoxia control group(P<0.05).The level of contractile phenotype marker protein in the PASMCs at 48 h after anoxia treatment(H48h)was significantly lower than that in the control group(N48h).The proliferative activity of the primary PASMCs were subjected to normoxia treatment and those subjected to 3% O2 anaerobic treatment were detected with the EdU staining method.The positive rate of EdU staining at48h(H48h)after anoxia treatment was lower than that in the normoxia control group,indicating that the anoxia treatment could significantly strengthen the proliferative activity of PASMCs.3 Role of mi R-23 a in anoxia-induced down-regulation of the expression of contractile phenotype marker protein in PASMCsContractile marker proteins were significantly up-regulated after transfection with the miR-23 a inhibitor.Expression of the contractile phenotype marker gene protein in PASMCs in the transfection mimic control group(mimic ctrl)demonstrated no significant changes compared to that in the blank control(vehicle)group.The expression levels of the contractile marker proteins in the PASMCs at 48 h after transfection with mi R-23 a mimic were down-regulated to different extents compared with those in the transfection mimic control group.4 Effect of mi R-23 a on promoting anoxia-induced proliferative activity of PASMCs.The positive rate of the staining result of PASMCs transfected with mi R-23 a inhibitor under anoxia was detected.It was found that the positive rate of the Ed U in PASMCs in the transfection inhibitor control group(inhibitor ctrl)had no significant difference compared with that in the blank control(vehicle)group.The positive rate of the EdU staining result of the PASMCs in the transfection inhibitor control group(inhibitor ctrl)at 48 h after transfection with mi R-23 a inhibitor decreased significantly compared with that in the inhibitor control group.It indicated that miR-23 a played a role in promoting anoxia-induced proliferative activity of PASMCs.The positive rate of the EdU staining result of PASMCs in the transfection mimic control(mimic ctrl)under normoxia,had no significant difference compared with that in the blank control group(vehicle).The positive rate of the EdU staining result of PASMCs in the transfection mimic control(mimic ctrl)at 48 h after transfection with miR-23 a mimic increased significantly compared with that in the mimic control group.It indicated that over-expression of miR-23 a under normoxia could also lead to increased proliferation of PASMCs.Summary: The contractile phenotype marker protein in the PASMCs decreased after anoxia treatment which indicating that the anoxia treatment could significantly strengthen the proliferative activity of PASMCs.The expression level of miR-23 a at 48 h after anoxia(H48h)was higher than that in the normoxia control group.It indicated that miR-23 a played a role inpromoting anoxia-induced proliferative activity of PASMCs.Part two: Mechanism underlying hypoxia-induced up-regulation of miR23a expression in PASMCsObjective: To investigate the regulatory mechanism of miR-23 a expression in hypoxia and its pathophysiological significance.Methods: Cells were transfected with HIF-1α specific siRNA under anoxia condition.RT-qPCR was used to detect miR-23 a expression in PASMCs.Ch IP method was employed to verify the binding sites of HIF-1α.The dual-luciferase reporter gene was used to study the role of HIF-1and its binding sites.Results:1 Role of HIF-1α in anoxia-induced up-regulation of the miR-23 a expressionThe expression level of miR-23 a in PASMCs transfected with the HIF-1αspecific siRNA under anoxia decreased significantly.It indicated that the up-regulation of mi R-23 a under anoxia was regulated by HIF-1α.The HIF-1αprotein level and the mi R-23 a expression level in the PASMCs under normoxia in the EDHB(500μM)24h(EDHB 24h)group increased significantly compared with those in the control group.The expression level of the HIF-1α protein was significantly higher than that in the control group(Vehicle 48h)and the expression level of mi R-23 a was significantly higher than that in the control group(Vehicle 48h)at 48 h after addition of EDHB 48h(EDHB 48h).Changes in content of pri-miR-23a-1,pri-miR-23a-2,and pri-miR-23a-3 reflected the transcriptional activity of miR-23a-1,miR-23a-2,and miR-23a-3.When PASMCs were cultured under anoxia and transfected with HIF-1αspecific siRNA,the expression levels of pri-miR-23a-1 and pri-miR-23a-3 decreased significantly,indicating that HIF-1α participated in transcriptional activation effect of miR-23a-1 and miR-23a-3.2 Detecting bonding of HIF-1α within the upstream 5KB region of the transcription start site of miR-23a-1 and mi R-23a-3The bonding of the three prediction sites(R1,R2,and R3)within theupstream 5KB regulatory 5’-UTR of miR-23a-1 in PASMCs to HIF-1α at 24h(H24h)and 48h(H48h)after anoxia increased significant compared with that before anoxia(N group).The enrichment level of HIF-1α in R1 was the highest.Bonding of HIF-1α to a binding site(R)within 5’-UTR of miR-23a-3in the anoxia 24 h group(H24h)and the anoxia 48 h group(H48h)increased significantly compared with that in the normoxia control group(N).3 The effects of HIF-1α in transcriptional activation of miR-23a-1 and mi R-23a-3 genes with ChIP following anoxiaIntervening HIF-1α could significantly weaken the role of the luciferase activity of the enhanced wild Luc-miR-23a-1(WT)under anoxia(Figure 4G).For miR-23a-3,anoxia exposure(1% O2 concentration)and co-transfection with the plasmid with over-expression of HIF-1α could improve the luciferase activity of the wild Luc-miR-23a-3(WT)by 2.8 times and 1.8 times but had no effect on the mutant Luc-miR-23a-3(M).Intervening HIF-1α under anoxia could significantly weaken the activity of the wild Luc-miR-23a-3(WT)reporter gene.These results indicated that anoxia up-regulated the expression of miR-23a-1 and miR-23a-3,which was achieved by the transcriptional activation of HIF-1α.Summary: The expression of miR-23 a in hypoxia is related to the expression of HIF-1,and HIF-1 may be involved in the transcriptional activation of mir-23a-1 and mir-23a-3.The possible increase of miR-23 a expression in hypoxia was mainly due to the upregulation of mir-23a-1 and mir-23a-3.anoxia up-regulated the expression of mi R-23a-1 and miR-23a-3,which was achieved by the transcriptional activation of HIF-1α.Part three: Expression of mi R-23 a in the pulmonary arterioles and effects of chronic hypoxia on the mean pulmonary arterial pressure(mPAP),right ventricular mass index and pulmonary artery morphological changesObjective: To construct rat model of hypoxic pulmonary hypertension,and to explore the level of miR-23 a in PASMCs and the effect on mean pulmonary arterial pressure,right ventricular weight index,pulmonaryarteriole wall and the intima-media thickness of rats in chronic hypoxia condition.To further clarify the pathophysiological significance in animal models.Methods: Rats in CH group were exposed to 21 days of hypoxia and rats in the normoxia control group(NC group)were put in a hypobaric chamber and outside of the chamber at a simulated altitude of 5000 m respectively(Thermo Scientific,Waltham,MA).8 rats were selected from each group.Each rat was intraperitoneally injected with 10% urethane(1ml/100g)and fixed onto a platform for separating the right external jugular vein.Each rat was intravenously injected with 0.5% heparin normal saline solution(0.2ml/100 g body mass)for general anticoagulation.Right ventricle and pulmonary arterial intubations(0.45 mm ID and 0.8mmOD)were performed for 10 min.Mean pulmonary arterial pressure(mPAP)was determined with the Power lab multilead physiological recording instrument.Rats were sacrificed and thoracotomy was performed to remove the heart.The heart atrium and connective tissue were carefully separated.Right ventricular free wall was separated along the ventricular septum.The weight index of the right ventricle was calculated using the following formula: RV/(LV+S).Results:1 Effect of chronic anoxia on the mean pulmonary arterial pressure(mPAP),right ventricular weight index,and pulmonary arteriole wall,intima-medial thicknessThe mean pulmonary arterial pressure in the chronic hypoxia group(CH)increased significantly(P<0.05),approximately 2 times more than that in the control group(Table 1).It indicated that chronic hypoxia could significantly raise the pulmonary arterial pressure.The RV/(LV+S)of the rats in the chronic hypoxia group(CH)increased significantly compared with that in the normoxia control group(NC)(P<0.05).It indicated that chronic hypoxia could lead to right ventricle hypertrophy of the rat.We measured the outer diameter and canal wall thickness(outer diameter-inner diameter),and intima-medial thickness.The result indicated that the pulmonary arterioles ofthe rats in the normoxia control group(NC)had thin canal walls and large lumens with an outer diameter of 81.31±2.8 μm,canal wall thickness of10.57±2.6μm,and intima-medial thickness of 3.6±0.8 μm.The outer diameter of pulmonary arterioles in the chronic hypoxia group(CH)was 86.61±6.1μm,canal wall thickness was 21.3±4.4μm,and intima-medial thickness was8.8±1.6μm.2 miR-23 a expression of rats in the condition of chronic hypoxiaThe expression of miR-23 a in the pulmonary arterioles in the chronic hypoxia group(CH)increased significantly(P<0.05)compared with that in the normoxia control group(NC).Summary: The miR-23 a expression of rats increased significantly in the condition of chronic hypoxia.Chronic hypoxia can cause right ventricular hypertrophy in rats.Chronic hypoxia can significantly increase the thickness of pulmonary arterioles,leading to the proliferation of smooth muscle cells and remodeling of pulmonary arterioles.Conclusions:The contractile phenotype marker protein in the PASMCs decreased after anoxia treatment which indicating that the anoxia treatment could significantly strengthen the proliferative activity of PASMCs.MiR-23 a played a role in promoting anoxia-induced proliferative activity of PASMCs.It indicated that the up-regulation of miR-23 a under anoxia was regulated by HIF-1α.MiR-23 a plays an important role in the pathogenesis of hypoxic pulmonary hypertension and it is likely to be targeted for the treatment of HPH.