Hypoxia Is Involved In Hypoxic Pulmonary Hypertension Through Inhibiting The Activation Of FGF2 By MiR-203Title

BACKGROUND:Pulmonary hypertension(PH)is an abnormal pulmonary vascular dynamics induced by many factors,which can lead to progressive increase of pulmonary vascular resistance.PH can progressively cause right ventricular hypertrophy,leading to right heart failure and finally death.The occurrence of hypoxic pulmonary hypertension(HPH)is one of the central links in the development of various diseases.HPH is common in the interstitial lung diseases and chronic obstructive pulmonary diseases.The etiology of HPH is very complex,and its mechanism may involve genetic factors,the activation of inflammatory factors and immunological runaway factors.It has been found that the main pathological changes of HPH are the enhancement of pulmonary artery contractility,pulmonary vascular remodeling and microvascular injury.Pulmonary artery smooth muscle cells are the most important cells involved in the contraction of small arteries in HPH.In recent years,the focus of PH research is to reverse pulmonary vascular remodeling and inhibit the abnormal proliferation of vascular smooth muscle cells,and promote the apoptosis of vascular smooth muscle cells.MicroRNA(microRNA or microRNA)is highly conserved in evolution.It is a single-stranded non-coding RNA containing 22 nucleotides and can alter the expression of some target genes,which is achieved through post-transcriptional regulation.MicroRNA plays an important role in many biological processes,such as cell proliferation,growth and apoptosis.It has been found that in many diseases,microRNA can even serve as a potential biological target for the diagnosis of diseases,contributing to the diagnosis and treatment of diseases.Nearly one third of human gene expression processes involve microRNAs.However,the functions of most of the microRNAs and their detailed and specific regulatory mechanisms are still waiting for further research and exploration.MicroRNA can bind with the base pairs in the 3’UTR region of target gene’s mRNA in order to regulate the target gene,and then regulate the physiological and pathological processes of human cells proliferation,apoptosis and development.Recent studies have shown that microRNA plays an important role in the formation and progress of HPH.Although many studies have done some research on how microRNAs participate in the occurrence and progress of HPH,how manymicroRNAs are involved in the pathogenesis and progress of HPH,and the specific mechanism of their involvement remains to be further studied.Recent studies have found that the expression of microRNA-203 in HPH tissue is significantly down-regulated.We inspect whether microRNA-203 is involved in the occurrence and development of HPH.OBJECTIVE:To study and analyze the expression level of microRNA-203 in HPH tissues and cells,to study its functional changes,and to explore its intrinsic molecular mechanism.METHODS:1.Construct HPH rat model and verify its success.PASMCs cells were isolated from rat models for subsequent experiments.2.The expression of microRNA-203 in PASMCs cells of hypoxic rats and normal rats was detected by qRT-PCR.EDU cell proliferation experiment,CCK8 analysis experiment and cell scratch test were carried out to verify whether microRNA-203 could regulate the proliferation and migration of PASMCs cells.3.Search the possible target gene of microRNA-203 by bioinformatics.After selecting fibroblast growth factor 2 gene as a possible target gene,we predicted the possible binding sites of miR-203 and fibroblast growth factor 2 and validated them.4.Transfection of fibroblast growth factor 2 siRNA into PASMCs cells to knock down its expression level and verify it.The lung indexes of rats in normal control group,HPH control group and HPH+FGF2 siRNA group were detected.RESULTS:1.We measured the RVSP and RVHI of HPH model rats and control rats,and found that the RVSP and RVHI of the former group were significantly higher than those of the control ones.HE staining showed that the pulmonary vascular smooth muscle layer of rats in hypoxic group was significantly thicker,the lumen was narrower,and were infiltrated by the inflammatory cells around,which indicated that the establishment of HPH rat model was successful.The results of immunofluorescence showed that rat PASMCs had high purity and could be used in the subsequent experiments.2.The expression of microRNA-203 in rat PASMCs cells treated with hypoxia and normoxia was detected by qRT-PCR.The results showed that the expression of microRNA-203 decreased significantly in PASMCs cells treated with hypoxia.The results of EDU cell proliferation assay and CCK8 assay showed that the overexpression of miR-203 decreased the proliferation of PASMCs cells,while the downregulation of the expression of miR-203 significantly increased the proliferation of PASMCs cells.In cell scratch assay,wefound that over-expression of miR-203 could decrease the migration of PASMCs,while inhibiting the migration of PASMCs could enhance the migration ability of PASMCs.3.By means of bioinformatics,we have searched for the possible target gene of miR-203 and finally selected fibroblast growth factor 2 gene as its possible target gene.Wild-type and mutant plasmids for dual luciferase reporter gene analysis were constructed.The results showed that miR-203 could target fibroblast growth factor 2 gene and bind to its3’UTR.The results of PCR and Western blot showed that the expression level of fibroblast growth factor 2 decreased after over-expression of miR-203.We concluded that miR-203 selectively binds to fibroblast growth factor 2 and reduces its expression.4.We transfected fibroblast growth factor 2 siRNA into PASMCs cells to knock down its expression level.The results of RT-PCR and Western blot showed that the expression level of fibroblast growth factor 2 in transfected cells decreased.We measured the related indexes of rats in three groups: normal control group,HPH control group and HPH+FGF2siRNA group.The results showed that the RVSP,RVHIV,WT%,(PM+FM)% and the thickness of pulmonary artery smooth muscle layer of rats in HPH+FGF2 siRNA group decreased significantly compared with the control group.We conclude that inhibiting fibroblast growth factor 2 can reduce pulmonary artery pressure in HPH rats,thus effectively improving pulmonary vascular remodeling.CONCLUSION:This study it is concluded that hypoxia participates in the formation of HPH by inhibiting the activation of fibroblast growth factor 2 by miR-203.In addition,specific inhibition of fibroblast growth factor 2 can reduce hypoxic pulmonary hypertension and improve pulmonary vascular remodeling.