Mir-206 Mediates The Epigenetic Mechanism Of The Pulmonary Arterial Hypertension Following Intrauterine Growth Retardation Via The Phosphorylation Of Kv1.5 Changes Of Pulmonary Artery Smooth Muscle Cells

Intrauterine growth retardation(IUGR)is a deviation from an expected fetal growth pattern and is caused by multipal adverse intrauterine environments that inhibit the normal growth potential of the fetus.IUGR neonates are often having a birth weight below the tenth percentile or two standard deviations below the average weight of the corresponding gestational age called small for gestational age or SGA.According to the World Health Organization statistics,about 12 million of preterm baby were born per year in the world,and more than 10%of that were born in China.The incidence of IUGR in preterm infants is extremely high and the IUGR related disease burden has been a big challenge in neonatal medicine.A large amount of population epidemiological data analysis and animal experimental studies have indicated that the adverse intrauterine environment at early life time not only have an impact at perinatal period,but have long term or even lifetime effects on adulthood health and quality of life.This is the Fetal Origin of Adult Diseases(Barker hypothesis)or the Development Origin of Health and Disease(DOHaD).The epigenetic mechanisms are involved in these processes.Epigenetics is study of heritable changes in gene or protein expressions that are not caused by changes in the DNA sequence.In recent years,epigenetics researches are mainly focusing on DNA methylation,histone modification,chromatin remodeling,gene silencing,and RNA interference.A lot of studies have found that IUGR could cause exaggerated Chronic Hypoxic Pulmonary Arterial Hypertension(CH-PAH),pulmonary arterial remodeling and smooth muscle cell dysfunction in adulthood.The epigenetic mechanisms associated with this pathophysiological process include DNA methylation abnormalities of endothelial cell secretory proteins,changes in histone acetylation,and regulation of non-coding-RNAs.Most of the previous studies focused on the expression and function of Kv channels on pulmonary arterial smooth muscle cells,which is a classic pathway of pulmonary hypertension.In conclusion,we hypothesize that:Intrauterine growth retardation in rats with chronic hypoxic pulmonary hypertension may have an association with changes in the phosphorylation of voltage-dependent potassium channels on pulmonary artery smooth muscle cells.And epigenetic regulations could be one of the underlying mechanisms,and microRNAs may be involved in.Part1 Effects of Intrauterine Growth Retardation on Pulmonary Artery Smooth Muscle Cells and Kvl.5 Phosphorylation ModificationObjectives:In recent years,epidemiological studies and animal experiments have shown that intrauterine growth retardation may lead to adult pulmonary vascular dysfunction and increased sensitivity to hypoxia,resulting in adult hypoxic pulmonary hypertension.Previous studies have shown that voltage-dependent potassium channels on pulmonary artery smooth muscle cells play a key regulatory role in the pathophysiology of pulmonary hypertension.Many studies have shown that the activity of ion channels is affected by their related amino acid phosphorylation.The aim of this study is to investigate the pulmonary arterial hemodynamic changes and pathological changes of IUGR-induced chronic hypoxic PAH rat models and to detect the phosphorylation levels of the relevant Kv channels,and to explore the role of phosphorylation Kv channel in the disease model and its related mechanisms as well.Methods:1.Establishment of IUGR animal model:Sprague-Dawley rats were randomly assigned to IUGR group and control group.IUGR rats were produced by intrauterine malnutrition during the whole pregnancy by giving 50%of normal diet to the mother until offspring delivery.The control group was given a normal diet during pregnancy until normal offspring delivery.2.The offspring were grown to 12 weeks and followed by 2 weeks of hypoxia to establish a PAH model.3.Pulmonary artery pressure assessment and pulmonary artery remodeling of the pathological examination:by jugular vein catheterization method to measure the pulmonary artery pressure.And the degree of myofibroblasts was measured by a-SMA immunohistochemical staining.4.The expression of voltage-dependent potassium channel protein was detected by Western blot,and the mRNA level of the related ion channel was detected by real-time fluorescence quantitative PCR.5.Immunofluorescence staining and Co-Immunoprecipitation(Co-IP)were used to detect the phosphorylation of the target potassium channels at the primary cell level.Results:1.After 2 weeks of hypoxia in 12-week-old rats,the mean pulmonary artery pressure was significantly higher in the IUGR group than that of control group,and the pulmonary arterial smooth muscle layer thickness was significantly increased.2.Following 2 weeks of hypoxia,the expression of Kvl.5 protein and KCNA5 mRNA in pulmonary arterial smooth muscle were significantly lower than those in normoxia groups and significantly decreased in IUGR-Hypoxia group compared with control-Hypoxia group.3.The expression of tyrosine phosphorylation of Kvl.5 protein in pulmonary artery smooth muscle and primary cultured pulmonary artery smooth muscle cells was higher in hypoxia groups than that in normoxia groups,and a significantly higher level was noted in IUGR group campared with control group..Conclusions:1.We have successfully established an intrauterine growth retardation rat model with an exaggerated pulmonary arterial hypertension following chronic hypoxic exposure.The expression of Kv1.5 protein in pulmonary arterial smooth muscle was significantly decreased.2.Changes in tyrosine-phosphorylation level of Kv1.5 protein in pulmonary vascular smooth muscle may be associated with hypoxic pulmonary hypertension following intrauterine growth retardation.Part 2 Epigenetic mechanism of miR-206 in intrauterinegrowth retardation induced adult pulmonary hypertensionObjectives:miRNA interference is one of the classical ways of epigenetic regulation.It is widely involved in post-transcriptional regulation of various signaling pathways.In the first part of this study,we have found that intrauterine adverse environment can cause an increased sensitivity to hypoxia insult in adulthood.Our previous study found that miR-206 may be involved in this process.This part of study aims to clarify the role and mechanism of miR-206 in the regulation of the development of PAH following IUGR.This study aims to elucidate the epigenetic regulation mechanism and to explore potential for clinical diagnosis,prevention and treatment of neonatal pulmonary hypertension.Methods:1.The expression of miR-206 in pulmonary artery smooth muscle of both IUGR and control group at 12 weeks old followed by 2-weeks normoxia or hypoxia was detected by real-time quantitative reverse transcription PCR in combination with pre-miRNA 3.0 microarray.The results were analyzed by bioinformatics analysis software.2.The expression levels of miR-206 in pulmonary artery smooth muscle tissue of IUGR or control group were detected by Fluorescence in situ hybridization(FISH)in Day 1,3 weeks,12 weeks,and 12 weeks with 2-week hypoxia.3.Primary cultured pulmonary artery smooth muscle cells were treated with miR-206-inhibitor at cellular level.CoCl2 was used to mimic the hypoxia environment.4.Bioinformatics analysis method was used to construct the function network diagram of miR-206 in pulmonary artery smooth muscle cells with the potential target molecules.Results:1.The results of Real-time quantitative reverse transcription PCR show that:The expression of miR-206 in the pulmonary arterial smooth muscle cells was significantly higher in IUGR rats than that of the control rats.The expression of miR-206 was decreased after 2 weeks of hypoxia.The expression of miR-206 in control-hypoxia and IUGR-hypoxia groups did not show significant difference.2.The results of in situ hybridization showed:The expression of miR-206 was increased in resistance pulmonary arteries of IUGR rats at 12 weeks of age(P<0.05).However,there was no significant difference in the pulmonary artery smooth muscle tissue at birth or 3-week-old in rats.The expression of miR-206 decreased following 2 weeks of hypoxia started from 12 weeks,and there was no significant difference between control-hypoxia group and IUGR-hypoxia group.3.Administration of miR-206 inhibitor to the primary cultured pulmonary artery smooth muscle cells(PASMCs)treated with COCl2 decreased miR-206 expression and cell proliferation of PASMCs in both control and IUGR group,while there were no significant difference in miR-206 expression and cell proliferation between IUGR and control PASMCs.4.Combined with multiple databases and bioinformatics analysis,the network diagram of miR-206 participate in chronic hypoxic pulmonary arterial hypertension was build,and Kvl.5 is one of the potential target molecules Conclusions:1.The increased miR-206 expression in PASMCs is associated with pulmonary artery dysfunction and CH-PAH of IUGR rats in adulthood,which implies that epigenetic regulation is one of the underlying mechanisms leading to PAH.2.miRNA array data and bioinformatic analysis suggested that miR-206 is involved in several signaling pathways related to the pulmonary vascular diseases.