The Role Of MiR-98 In The Pathogenesis Of Hypoxia-induced Pulmonary Hypertension And Its Preliminary Study As A Marker For Early Diagnosis Of Pulmonary Hypertension

Pulmonary hypertension(PH)is a physiological pathological disorder that involves many clinical manifestations in the clinic and eventually which leads to cardiovascular and respiratory diseases.According to reports,the current prevalence of PH in the world is about 97 people per million people,and the mortality rate is about 5-10 people per 100,000 people.Hypoxia-induced pulmonary hypertension(HPH)is pulmonary hypertension due to hypoxia caused by lung disease and other pathological factors.It is clinically high in interstitial lung disease and chronic obstructive pulmonary disease.The development of the disease caused by the PH is often accompanied by deterioration of the patient's exercise capacity,resulting in increased hypoxia and shortened survival time.Therefore,the in-depth study of the mechanism of development of HP has important clinical significance.MicroRNA,also known as MicroRNA,is a small,non-coding RNA that is widely present in the body and is approximately 22 bases in length.Numerous studies have confirmed that factors associated with pulmonary hypertension,such as hypoxia induction and inflammatory damage,both can regulate the expression level of microRNA.Recent studies have found that microRNA can be used as an upstream signaling molecule to regulate the biological function of cells and play an important role in the remodeling of PH pulmonary blood vessels,and more and more research shows that microRNA is expected to become an effective target in diagnosis and treatment of PH.Our group has searched and analyzed the microRNAs that are highly likely to play a key role in the development of pulmonary hypertension through online database and the published microRNA chipset database of the pulmonary hypertension,and finally selected miR-98 as our research object;Quantitative PCR assays revealed that the expression of miR-98 in the lungs of HPH rats was significantly downregulated.The effects of PAIs on the proliferation and apoptosis of PASMCs were studied in rat primary pulmonary artery smooth muscle cells(PASMCs),and online data and targets were used.The gene prediction software was used to predict and verify the target gene.The target gene of miR-98 was ALK1.The expression of ALK1 was significantly up-regulated in the lung tissue of HPH rat model.Furthermore,miR-98 mimic could be significantly improved by tail vein injection.HPH rat pulmonary hypertension symptoms.We also analyzed the expression of miR-98 in the blood of patients with pulmonary arterial hypertension.The results showed that the level of miR-98 in the blood of patients with pulmonary hypertension was significantly reduced.The conclusions of this study provide new insights into the pathogenesis of HPH and provide potential targets for the clinical diagnosis and treatment of HPH.Part ?:The expression of miR-98 in hypoxia-induced pulmonary artery smooth muscle cells and its functions in cell proliferation and apoptosisObjective:1.Detection of miR-98 expression in lung tissue of HPH rat model;2.To investigate the expression of miR-98 in PASMCs in hypoxic condition and its effect on the proliferation and apoptosis of PASMCs.Methods:1.First,the rat model of HPH was induced by feeding at low oxygen concentration.The right ventricular pressure(RVSP),right ventricular hypertrophy index(RVHI)and pathological HE staining were used to determine whether the HPH animal model was successfully constructed.2.The expression of miR-98 in the lung tissue of rats with HPH and its normoxic control group was detected by real-time fluorescence quantitative PCR.3.The rat primary PASMCs were extracted and cultured by enzyme digestion and digestion.The purity and morphology of the cells were identified by immunofluorescence staining.The expression level of miR-98 in PASMCs treated with low oxygen concentration was detected by real-time PCR.M miR-98-mimic and miR-98-inhibitor were over-expressed or inhibited respectively miR-98.Cells transfected with miR-NC were used as negative control group.The effect of miR-98 on proliferation and apoptosis of PASMCs was detected by CCK-8,western blot and flow cytometry respectively.Results:1.Real-time fluorescence quantitative PCR showed that miR-98 expression was significantly down-regulated in rat pulmonary arteries of the HPH model.2.The enzyme combined digestion method can obtain higher purity rat primary PASMCs.3.miR-98 was significantly down-regulated in hypoxia-induced rat PASMCs.4.miR-98 inhibits the proliferation of rat PASMCs in hypoxic environment.5.miR-98 promotes apoptosis of rat PASMCs in hypoxic conditions.Conclusions:miR-98 was significantly down-regulated in lung tissue of hypoxic-induced HPH rat models and regulated PASMCs proliferation and apoptosis.Part ?:The mechanism of miR-98 in regulating the proliferation and apoptosis of pulmonary artery smooth muscle cellsObjective:1.Predict the possible target genes of miR-98 and perform relevant validation.2.Wheather miR-98 mimic could improve the symptoms of pulmonary hypertension in HPH rat models via tail vein injection.Methods:1.Predict possible target genes of miR-98 through bioinformatics website and transfect miR-98-mimic into rat primary PASMCs,and detect the target gene expression level of miR-98 by fluorescence quantitative PCR;2.Analyzed the expression level of ALK1,a possible target gene of miR-98 in lung tissue of HPH rats by westerm Blot;3.The pgl-promoter + ALK1 3' UTR lentiviral plasmid was constructed and transfected into HEK293 cells together with miR-98-mimic and PRL plasmids.The dual luciferase reporter gene was used to detect the fluorescence of miR-98-mimic after transfection.4.miR-98-mimic was transfected in PASMCs and the expression of ALK1 was detected by Westerm Blot.5.After transfected with miR-98-mimic by PASMCs,ALK1 was over-expressed.Cell proliferation was detected by CCK8.Cell apoptosis was detected by flow cytometry.6.Construct a rat model of HPH and inject miR-98 mimic though the tail vein to detect the effect of miR-98 on HPH treatment by detecting RVSP,calculating RVHI,pathological HE staining,immunohistochemistry,and masons staining.Results:1.The expression of ALK1 in the lungs of HPH rats was significantly increased and it was a possible target gene of miR-98.2.In rat PASMCs,miR-98 can directly bind to ALK1 3'UTR and inhibit ALK1 expression.3.In rat PASMCs,overexpression of ALK1 can inhibit apoptosis induced by over-expression of miR-98;4.Hypoxia inhibits the expression of miR-98 and activates the ALK1 pathway in the development of HPH.5.The tail vein injection of miR-98 mimic can effectively inhibit the activation of ALK1 pathway;6.The tail vein injection of miR-98 mimic can effectively reduce pulmonary artery pressure in HPH rats;7.The tail vein injection of miR-98 mimic can effectively reduce pulmonary vascular proliferation and collagen deposition in HPH model rats.Conclusions:ALK1 is a target gene of miR-98.Injecting miR-98 mimic by tail vein injection significantly inhibits the expression of ALK1 and improves the symptoms of pulmonary hypertension in rats.Part ?:The serum Level miR-98 and its predictive diagnosis in patients with pulmonary hypertension Objective:To detect and analyze the correlation between the expression of miR-98 in patients'blood and the pulmonary artery pressure and pulmonary vascular resistance measured by ultrasonic cardiogram test.To investigate the relationship between the development of miR-98 and the occurrence and development of pulmonary hypertension,and to investigate the relationship between miR-98 and PAH.Methods:We collected patients who were hospitalized between January 2016 and June 2017.All patients underwent ultrasonic cardiogram and arterial blood gas analysis.According to the guidelines for pulmonary hypertension diagnosis published by ESC in 2015,the patient's mean pulmonary artery pressure(mPAP)? 35 mmHg by ultrasonic cardiogram test,patients were divided into two groups,15 cases of pulmonary arterial pressure increase group and 15 cases of normal pulmonary artery pressure.Total RNA was extracted by Trizol method and cDNA was obtained by reverse transcription.The expression level of miR-98 was detected by real-time fluorescence quantitative PCR,and the correlation between miR-98 expression and pulmonary artery pressure was analyzed.Results:1.The levels of miR-98 in the blood of patients with pulmonary hypertension were significantly reduced.2.The relative levels of miR-98 in the blood were significantly negatively correlated with pulmonary artery pressure.Conclusions:The relative levels of miR-98 in the blood provide a possible target for clinical diagnosis,evaluation,and prognosis evaluation of pulmonary hypertension...