| Currently, increasing incidence of cardiovascular diseases has been indentified posinga great threat to people’s health. These diseases are based on pathological vascularremodeling caused by vascular dysfunction and abnormal damage repairing. Pulmonaryarterial hypertension (PAH) is a typical one. It is a type of pulmonary artery diseasecharacterized by pathological vascular proliferation and remodeling. Pulmonary vascularresistance is progressively increased, eventually leads to right heart failure or even death.Patient’s prognosis is poor once diagnosed with PAH, so far the pathological pulmonaryartery remodeling still cannot be completely reversed by modern therapy methods, butearly interventions can slow down the pathological process significantly, after all theearlier diagnosis and treatment the better prognosis. Right heart catheterization (RHC) isthe golden standard for PAH diagnosis which is invasive and may cause severecomplications. RHC also demands special equipments and good training which means itcannot be a routine examination for PAH screening. Ultrasonic cardiogram (UCG) is aneffective tool for screening PAH, but the estimates of pulmonary artery pressure, by UCG,are often inaccurate compared with RHC due to various reasons. PAH biomarkerresearches most focus on BNP and NT-proBNP which mainly for risk stratification andprognosis rather than diagnosis.MicroRNA (miRNA) is a class of small endogenously expressed noncoding RNAsthat regulate gene expression at post-transcriptional level. Recent studies have shown thatmiRNA is circulating freely in some stable forms from being digested by RNase. Thelevels of individual circulating miRNA can change significantly in diseases. So they can bepredicted as biomarkers for disease diagnosis. For example they can be biomarkers forearly diagnosis of acute myocardial infarction and heart failure. The objective of thisexperiment is to investigate the character of circulating miRNA as potential biomarker ofPAH．I. Investigate the expression levels of candidate circulating miRNA in PAH modelsObjective: To establish mice models of PAH and investigate the expression levels ofcandidate circulating miRNA preliminary.Methods:1. Modeling: PAH was induced in mice (c57) by using single abdominalmonocrotaline (MCT) injection (60mg/kg) method. Forty mice were randomized into threegroups: blank control group (control, n=12), two weeks after MCT injection group(2wk-MCT, n=12), four weeks after MCT injection group (4wk-MCT, n=16). Using threeaspects for model validation as following: First, compare the general features betweenmodel group and control group. Second, observe lung tissue slice between model groupand control group by HE staining. Third, validate right heart pathologic hypertrophy inmodeling group by observing heart slice using HE staining and detecting myocardialhypertrophy markers including atrial natriuretic peptide (ANP, Nppa), α-myosin heavychain (α-MHC, myh6) and β-myosin heavy chain (β-MHC, myh7), using qRT-PCR.2.Select candidate circulating miRNA from currently researches and preliminary works ofour laboratory and the endogenous control for data normalization.3. Validate candidatecirculating miRNA by qRT-PCR: the candidate circulating miRNA were detected by SYBRgreen fluorescence qRT-PCR.Results:1. General features of model groups were poorer than control with reducedappetite and activity, being less responsive. Pulmonary artery wall of model group wasmore thickening, compared with control, with pulmonary artery smooth muscle cellsproliferation and inflammatory cells infiltration, by HE staining. Right ventricular wall wassignificant hypertrophic with ventricular cavity expanded in4wk-MCT compared withcontrol by HE staining. The expression level of Nppa and myh7were increased (P<0.01,P<0.05), myh6was decreased (P<0.05) in myocardial tissue of the4wk-MCT groupcompared with control.2. We selected10candidate miRNA including miR-451, miR-21,miR-150, miR-126, miR-23b, miR-191, miR-204, miR-26a, miR-145and miR-210fromrecent researches for validation. MiR-16was used as the endogenous control for datanormalization.3. Compared with control（1.00±0.45）, the expression of miR-451wasdecreased in both model groups:2wk-MCT (0.50±0.28),4wk-MCT (0.52±0.38)(P<0.05).An early decreased expression of miR-451can be observed in2wk-MCT group comparedwith control (P<0.05) but no further decreasing in4wk-MCT group compared with2wk-MCT group (P>0.05). MiR-126, miR-145, miR-191, miR-21and miR-150did notchange significantly. MiR-26a, miR-23b, miR-204and miR-210were either not detected or showed too large CT values.II. Investigate the diagnostic value of circulating miR-451as biomarker of PAHObjective: To detect the expression of candidate circulating miRNA in small samplesof PAH patients, preliminary investigate the diagnostic value of circulating miR-451asbiomarker of PAH and the diagnostic value combined with ultrasonic cardiogram (UCG).Methods:1.The preoperative plasma (3～5ml) was collected from patients ofsuspected PAH who will take right heart catheterization (RHC). According to RCH,patients were divided into two groups: control group (mPAP≤20mmHg) and PAH group(mPAP≥25mmHg). We collected clinical data of enrolled patients and extracted RNA fromtwo group samples. Then we detected the candidate circulating miRNA by using qRT-PCR.2. Use receiver operating characteristic curve (ROC curve) to assess the diagnostic value ofcirculating miR-451as biomarker of PAH.3. Establish binary logistic regression model toassess the value of circulating miR-451combined with UCG for the diagnosis of PAH.Results:1. In this part of the experiment, we found that the expression of miR-451was also decreased significantly in PAH group (0.66±0.05) compared with control(1.00±0.06)(P<0.01). MiR-126, miR-21and miR-150did not show significantly changing.Mir-145, miR-191, miR-26a, miR-23b, miR-204and miR-210were either not detectable orshowed too large CT values.2. For PAH diagnosis, there was a moderate values ofmiR-451with AUC=0.767(95%CI:0.650～0.884, P<0.01). When the relative expressionof miR-451was0.789, its sensitivity and specificity were72.7%and73.3%, respectively.3. Furthermore, we assessed the value of circulating miR-451combined with UCG for thediagnosis of PAH by forward stepwise regression method. The combination of miR-451and UCG was superior with AUC=0.897(95%CI:0.822～0.972, P<0.01), showing a betterdiagnostic value than either one alone.Conclusion:1. The expression of circulating miR-451decreased in the PAH modelsinduced by MCT and decreased significantly in the early stage of the modeling.2. Theexpression of circulating miR-451aslo decreased in PAH patients.3. Circulating miR-451has a diagnostic value for PAH close to UCG. The combination of both has a superiordiagnostic value。... |
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