The Changes And The Significance Of Serum MiRNAs And HO-1in Patients With Chronic Heart Failure

Introduction:Heart failure is the end of all kinds of heart diseases. In recent years, although the treatment of heart failure has progressed significantly, the mortality remains high, especially lacking of clear and effective treatment for the end-stage heart failure. Recent studies have shown that miRNAs are up regulated in the process of chronic heart failure. MiRNAs can stably exist in circulating blood, which has important clinical value for its convenient, quick and non-invasive detection. MicroRNAs (miRNAs) may become new diagnostic markers, or therapeutic targetes for new drug therapy. However, its role is not fully understood, and currently there are few reports on the changes of miRNAs in patients with heart failure.One of the mechanisms of heart failure is myocardial cells apoptosis. HO-1/CO-bilirubin system is known to have antioxidant and anti-apoptotic functions.It has been suggested that this system may play a protective role in the pathophysiology of heart failure. However, there is limited research on it in different stages of heart failure. Therefore, we propose to study the regulation of gene expression and the changes in neuroendocrine with the relationship of cardiac function by analyzing the expression of serum miRNAs and HO-1in patients with chronic heart failure.Thesis I The changes of serum miRNAs in patients with chronic heart failure and the relationship with heart function Background and Objective:MiRNAs are endogenous18-24nucleotides small RNAs, which depress protein expression by binding to the3’non-coding region of the target mRNA. At present, it is believed that most of the protein-coding genes have their targeted miRNAs correspondingly. MiRNAs are highly conserved and are stabled in serum, and they play important roles in the regulation of various physiological and pathophysiological processes. Some researches abroad have shown that the expression of miRNAs are upregulated in animal models with chronic heart failure, and the expression of serum miRNAs in patients with chronic heart failure are also increased. However, the related researches are limited. The stability and application value of miRNAs as biomarker is still unclear. The aim of this study was to determine the changes of serum miRNAs in patients with chronic heart failure by detecting the expression of miRNAs use quantitative real-time polymerase chain reaction (qRT-PCR) technology and study the relationship between miRNA expression and heart function.Methods:One hundred and fourty-seven patients with chronic heart failure were enrolled in the chronic heart failure group. Patients’ cardiac function were evaluated according to the American New York Heart Association (NYHA) classification. Patients were divided into four subgroups according to their NYHA class:subgroup Ⅰ (35cases of NYHA Class Ⅰ), subgroup Ⅱ(38cases of NYHA Class Ⅱ), subgroup Ⅲ(34cases of NYHA Class Ⅲ), subgroup Ⅳ (40cases of NYHA Class Ⅳ). Thirty healthy volunteers were enrolled in the control group. All patients’blood samples were collected the morning after hospitalization. The expression of serum miR-181c-5p, miR-214-3p, and miR-423-5p were measured according to qRT-PCR kit instructions, using miRNeasy Serum/Plasma Spike-In Control (C. elegans miR-39miRNA mimic) as the standard reference. Concentration of serum hs-cTnT was measured using Roche Cobas E601immune analyzer, and NT-proBNP level was measured using electrochemiluminescence immunoassay method. Total bilirubin, direct bilirubin, indirect bilirubin, blood lipid, blood glucose, aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, and creatinine were measured by full automatic biochemical analyzer; free triiodothyronine and free thyroxine were measured using chemiluminescence method. Echocardiography was performed to measure the following cardiac parameters:the left atrial diameter (LAd), left ventricular end diastolic diameter (LVEDd), and left ventricular ejection fraction (LVEF) using Dual-plane Simpson’s method, PHILIPS IE33color Doppler flow imaging.Results:1. The expression of miRNAs in each group①The expression of miR-423-5p in heart failure group was significantly higher than that of control group (5.41±0.78vs4.97±0.56, P<0.05). The expression of miR-423-5p in heart failure subgroup IV was significantly different from heart failure subgroup Ⅰ, Ⅱ, and Ⅲ (6.21±0.67,5.05±0.49,5.11±0.53,5.19±0.51, P<0.05). There are no significant difference between heart failure subgroup Ⅰ, Ⅱ, Ⅲ group and the control group (P>0.05).②There were no significant difference between the expression of miR-181c-5p in heart failure group and control group (1.67±0.39vs1.61±0.42). Also of heart failure subgroup Ⅰ,Ⅱ, Ⅲ,Ⅳ(1.68±0.38,1.63±0.40,1.74±0.39,1.75±0.43)(P>0.05).③There were no significant difference between the expression of miR-214-3p in heart failure group and control group (1.86±0.44vs1.86±0.38). Also of heart failure subgroup Ⅰ,Ⅱ, Ⅲ,Ⅳ(1.87±0.37,1.98±0.41,2±0.39,2.03±0.42)(P>0.05).2. Correlation analysis of the expression of miR-423-5p and NT-proBNP concentration, hs-cTnT concentration, LAd, LVEDd, LVEF. There was a positive correlation between expression of miR-423-5p and NT-proBNP, hs-cTnT (r=0.562,0.543, P<0.01)(Spearman). No strong correlation about LAd, LVEDd (r=0.290,0.268, P<0.01)(Pearson). No strong correlation about LVEF (r=0.271, P<0.05)(Spearman).3.Area under ROC curve of miR-423-5p: AUC=0.714(P<0.01).Conclusion: 1. The expression of miR-423-5p increased in patients with end-stage chronic heart failure, and it can well reflect the heart function. But there were no changes in expression of miR-423-5p in early-stage heart failure.2. The expression of miR-181and miR-214in patients with chronic heart failure has no change. Thesis Ⅱ The changes and the significance of HO-1in patients with chronic heart failureBackground and Objective:Heme oxygenase1(heme oxygenase-1, HO-1) is the key enzyme of heme metabolism process. It can catalyze heme decomposition into carbon monoxide (carbon monoxide, CO), ferrous ion (Fe2+) and biliverdin, which will further generate bilirubin. Recent studies have shown that HO-1/CO bilirubin system has antioxidant, anti-inflammatory, anti-apoptotic, and vasodilatory effects. It also plays a significant role in the protection of the cardiovascular system. But the relationship between the HO-1/CO bilirubin system and chronic heart failure remains to be elucidated. The aim of this study was to find out the changes of HO-1in patients with chronic heart failure by detecting the expression of them using ELISA technology and study their relationship with heart function.Methods：One hundred and fourty-seven patients with chronic heart failure were enrolled in the chronic heart failure group. Patients’ cardiac function were evaluated according to the American New York Heart Association (NYHA) classification. Patients were divided into four subgroups according to their NYHA class:subgroup Ⅰ (35cases of NYHA Class Ⅰ), subgroup Ⅱ (38cases of NYHA Class Ⅱ), subgroup Ⅲ(34cases of NYHA Class Ⅲ), subgroup Ⅳ (40cases of NYHA Class Ⅳ). Thirty healthy volunteers were enrolled in the control group. All patients’blood samples were collected the morning after hospitalization. Detected the expression of serum HO-1according to ELISA kit instructions. Concentration of serum hs-cTnT was measured using Roche Cobas E601immune analyzer, and NT-proBNP level was measured using electrochemiluminescence immunoassay method. Total bilirubin, direct bilirubin, indirect bilirubin, blood lipid, blood glucose, aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, and creatinine were measured by full automatic biochemical analyzer; free triiodothyronine and free thyroxine were measured using chemiluminescence method. Echocardiography was performed to measure the following cardiac parameters: the left atrial diameter (LAd), left ventricular end diastolic diameter (LVEDd), and left ventricular ejection fraction (LVEF) using Dual-plane Simpson’s method, PHILIPS IE33color Doppler flow imaging.Results:1. The comparison of HO-1(ng/mL) in each group The expression of HO-1in heart failure group was significantly higher than that of control group (19.1±3.6vs15.9±4, P<0.05). The differences between heart failure subgroup IV and subgroups Ⅰ, Ⅱ, Ⅲ were statistically significant (24.7±3.7,15.8±3.9,16.7±3.5,20.3±3.3respectively, P<0.05). The difference between heart failure subgroup Ⅲ and subgroups Ⅰ, Ⅱare statistically significant (20.3±3.3,15.8±3.9,16.7±3.5respectively, P<0.05). There was no statistical significance between subgroupsⅠ, Ⅱ and control group (15.8±3.9,16.7±3.5,15.9±4respectively, P>0.05).2. The comparison of TBIL, DBIL, IBIL (mol/L) concentration in each group±The concentration of TBIL in heart failure group was significantly higher than control group (27.3±6.5vs17.4±5.4, P<0.05). The differences between heart failure subgroup IV and subgroups Ⅰ, Ⅱ, Ⅲ were statistically significant (37.7±4.5,18.6±5.3,20.1±5.5,28.7±4.2respectively, P<0.05). There was statistical significance between heart failure subgroupⅢand subgroupsⅠ, Ⅱ(28.7±4.2,18.6±5.3,20.1±5.5respectively, PO.05). There was no statistical significance between subgroups Ⅰ, Ⅱand control group (18.6± 5.3,20.1±5.5,17.4±5.4respectively, p>0.05).②The concentration of DBIL in heart failure group was significantly higher than that of control group (7.1±3.2vs4±1.9, P<0.05). There was statistical significance between heart failure subgroup IV and subgroupsⅠ, Ⅱ, Ⅲ (8.2±2,4.2±2,4.8±1.9,6.9±1.6respectively, P<0.05). There was statistical significance between subgroup Ⅲ and subgroups Ⅰ, Ⅱ (6.9±1.6,4.2±2,4.8±1.9respectively, P<0.05). No statistical significance between subgroupsⅠ, Ⅱand control (4.2±2,4.8±1.9,4.0±1.9, P>0.05).③The concentration of IBIL in heart failure group was significantly higher than that of control group (20.2±7.5vs14±4.8, P<0.05). There was statistical significance between heart failure subgroup Ⅳ and subgroups Ⅰ, Ⅱ, Ⅲ (29.5±4.7,14.4±4.5,15.3±5,21.8±4.4, P<0.05). There was statistical significance between heart failure subgroupⅢand subgroupsⅠ, Ⅱ (21.8±4.4,14.4±4.5,15.3±5, P<0.05). There was no statistical significance between subgroups Ⅰ, Ⅱ and control group (14.4±4.5,15.3±5,14±4.8, P>0.05).3. Correlation analysis of HO-1and TBIL, DBIL, IBIL, NT-proBNP, hs-cTnT concentration, LVEF, LAd, LVEDdThere was a positively correlation between the concentration of HO-1and TBIL, DBIL, IBIL (Pearson)(r=0.572,0.562,0.518, P<0.0\). Positively correlation with the NT-proBNP (Spearman)(r=0.559,P<0.01). Negatively correlated with LVEF (Spearman)(r=-0.600,P<0.01). No strong correlation with hs-cTnT (Spearman)(r=0.480, P<0.01). No strong correlation with LAd, LVEDd (Pearson)(r=0.342,0.342, P<0.01).Conclusion:The concentration of HO-1and bilirubin increased with the development of heart failure in patients with chronic heart failure.