The Study Of The Correlation Of MiRNA-29a-3p And Left Ventricular Hypertrophy Induced By Hypertension

Hypertension is a major public health concern and one of the most important modifiable cardiovascular(CV) risk factors,responsible for up to half of all cardiovascular deaths. It is also a powerful risk factor for incident heart failure(HF), with both preserved and reduced ejection fraction,conditions that are increasing in prevalence in parallel with the aging population. Most importantly, treating hypertension can effectively prevent its associated complications,even in the elderly.Hypertensive heart disease(HHD) defines the complex and diverse perturbations of cardiac structure and function occurring secondary to hypertension. It is frequently characterized by left ventricle hypertrophy(LVH), left atrial enlargement(LAE), and left ventricular(LV) systolic and diastolic dysfunction, which are themselves potent risk factors for heart failure and atrial arrhythmias.LVH, in particular, is a hallmark of HHD. The pathogenesis underlying LVH is likely multifactorial. In the face of abnormal loading conditions,particularly chronically elevated afterload, LVH allows for normalization of LV wall stress and preservation of LV mechanical function. Despite its presumptive adaptive nature, LVH correlates with the overall CV risk of the hypertensive patient and is one of the most robust and validated prognostic markers in hypertension. Beyond LV mass alone, assessment of LV shape,or geometry, provides additional information regarding the cardiac response to hypertension. In a seminal paper published more than fifty years ago,Linzbach described specific ventricular morphologic adaptations to different hemodynamic stimuli and their functional correlates. Left ventricular hypertrophy is mainly characterized by myocardial cell hypertrophy and myocardial extracellular matrix remodeling.The collagen in the extracellularmatrix components accounted for 85%,which are mainly made up of the type I and type III collagen. Collagen synthesis is mainly affected by gene m RNA transcription regulation,those degradation is regulated by matrix metalloproteinases(MMPs).Studies have shown concentrations of MMP- 9and left ventricular end-diastolic volume in the process of left ventricular remodeling, are negatively related to the left ventricular ejection fraction.So MMP- 9 can reflect the degree of left ventricular remodeling.β-MHC is by Myh7 gene encoding and is a slow reaction of atpase, the gene expression is dominant in fetal and suppressed after birth.When the heart load increases, the expression of β-MHC will increase, at the same time which can be reduced along with decreases of cardiac function.Even in the process of the development of heart disease,the expression will become the main form.Studies have shown that the mi RNA-208 a can lead to myocardial hypertrophy by increasing the expression of β-MHC which is associated with myocardial fibrosis.Another study showed that plasma ANP in patients with hypertension were significantly higher than that of normal people, and both annulare hypertrophy and centripetal hypertrophy, the plasma ANP levels were significantly higher than that of patients without myocardial hypertrophy, so ANP concentrations may be used as a reference index to determine left ventricular hypertrophy in essential hypertension patients.Micro RNAs are small, non-coding RNA molecules ap-proximately 22 nucleotides in length that act as post-transcriptional regulators of gene expression. Commonly, although not exclusively, through binding to complimentary sequences in the 3’ untranslated region(UTR) of target messenger RNA(m RNA), micro RNAs induce translational repression or m RNA degradation, the exact mechanisms of which remain unclear. A crucial feature of micro RNA biology is that a single micro RNA can target multiple genes, and a single gene can be targeted by multiple micro RNAs.Thus, micro RNAs represent a complex regulatory network that controls both physiological and pathological processes fundamental to a wide varietyof cardiovascular diseases, including hypertension. The occurrence and development of myocardial hypertrophy are accompanied by a large number of abnormal expression of mi RNAs.In recent years, there has been a series of studies reported that the abnormal expression of mi RNAs.Sayed, Curcio,etc, their study have found the mi RNA-1, the mi RNA-133 express in myocardial hypertrophy model.Overexpression of micrornas-133 can inhibit protein synthesis and myocyte hypertrophy induced by endothelin-1 or phenylephrine stimulation, at the same time it can inhibit the expression of myocardial hypertrophy gene as ANF, β-MHC. The members of mi RNA-29 family, including mi RNA-29a-3p,mi R-29 b, mi R-29 c, and several others,play an important role in the pathological fibrosis of many organs. Studies have shown that transforming growth factorβ(TGFβ)/Smad signaling pathway regulates the expression of MMP through modulation of mi R-29,which in turn contributes to renal fibrosis. In systemic sclerosis,mi RNA-29a-3p has been evidenced to modulate collagen synthesis. A depletion of mi R-29 resulting in promoted collagen synthesis and renal fibrosis has also been reported. While an over expression of mi R-29 b has been associated with reduced collagen synthesis, a decreased serum level of mi RNA-29a-3p has been related to hepatic fibrosis. In hypertensive hearts and cultured cardiac fibroblasts, angiotensin II has been shown to cause Smad3-dependent cardiac fibrosis through mi RNA-29a-3p down regulation.On the contrary, the elevated serum levels ofmi RNA-29a-3p have recently been correlated with cardiac hypertrophy and fibrosis. We detect the plasma mi RNA expression profiles in hypertension patients with left ventricular hypertrophy and hypertension patients and healthy controls by Ion Torrent semiconductor high-throughput sequencing technologies and SYBRGreen I real-time fluorescent PCR technology and screen differentially expresssion of mi RNAs from hypertensive left ventricular hypertrophy. We compare and confirm that mi RNA-29a-3p belongs to the mi RNAs those difference are significant. At the same time, we forecast target genes and gene function analysis of mi RNA-29a-3p.To take correlation study in mi RNA-29a-3p,MMP-9, type I and type III collagen and reveal the relationship of mi RNA-29a-3p and hypertensive left ventricular hypertrophy and myocardial fibrosis. Finally, by animal experiment and Western blotting method,we improve the degree of myocardial hypertrophy by inhibiting the increase of mi RNA-29a-3p expression suppression of ANP and beta MHC protein levels.The technical routes in our study:firstly,we collected plasma samples from left ventricular hypertrophy and hypertension patients and healthy controls and build three plasma sample library. We obtained the mi RNA expression of three groups through high-throughput sequencing and screened differentially expressed mi RNAs from the patients of hypertension with left ventricular hypertrophy. To compare and confirm that mi RNA-29a-3p belongs to one of differentially expressed obviously mi RNAs.Then,we took the gene function analysis of mi RNA-29a-3p, predicted its target genes,conducted GO analysis and Pathway analysis.Secondly, we obtained the relative expression in the above three groups of large populations by using SYBR Green I real-time fluorescent PCR technology and took correlation analysis between the index of myocardial fibrosis and MMP-9,type I collagen and type III collagen.Third, Through the establishment of TAC mice model and Method of Western blotting, We reveal that inhibition of mi RNA-29a-3p expression can inhibit the increase of ventricular hypertrophy index-protein levels of ANP andβ-MHC.We provide new molecular targets of treatment for hypertension ventricular hypertrophy and remodeling.Part one Expression profiling of plasma mi RNA in Hypertension patients with left ventricular hypertrophy and gene function analysis of mi RNA-29a-3pObjective:To screen differential mi RNA profiling in plasma from hypertension patients with left ventricular hypertrophy and gene function analysis of mi RNA-29a-3p.Methods:1 In this study,We set up three experimental groups: hypertensive left ventricular hypertrophy, pure hypertension group and healthy control group,enrolled 20 subjects in each group and established three plasma samples.We detected expression profile of mi RNA in the three groups and screened differential expression of mi RNAs.At last,we predicted target genes and took GO analysis and Pathway analysis.2 Software SPSS 13.0 was used for the statistical analyses.Data is expressed as mean ± SD. ANOVA was used for comparison among different groups. SNK test was used for comparison between two groups.3 By using the Fast-QC software,we took overall assessment of the quality of the sequencing data and selected the differences of mi RNAs TPM value by using the algorithm of DEGSeq. Significant differences of standards were:Fold Change>1.5or<0.667,P<0.05,FDR<0.05.We predicted all target genes of mi RNA-29a-3p through internationally recognized target gene prediction database mi RNAbase and Target Scan. Then we took GO annotation of target genes according to the database and got all the GO target genes. By adopting Fisher test,we calculated the significance level of each gene. By correcting the results of multiple hypothesis testing,we got fault discriminate rate(FDR) and screened out significant GO including rich target genes. Secondly, We noted patheway of target genes according to the KEGG database and got all the pathways those genes involved in. By using the Fisher test according to the geometric distribution,to calculated the significance level(P-Value) of each pathway. we got fault discriminate rate(FDR) and screened out significant Pathway of target genes.Results:1 Basic characteristics of these volunteers in this study: Healthy subjects(n=20) including13male(65.0%) and 7female(35.0%),hypertension subjects(n=20) including12male(60.0%) and 8 female(40.0%),hypertensive left ventricular hypertrophy subjects(n=20) including13 male(65.0%) and 7female(35.0%),on an average age of 65.80±7.68, 64.50±8.10, 63.50±6.03 years, were selected. Clinical data included age,sex,smoking status,blood glucose and body mass index distribution,all data were not statistically significant in the three groups. But compared with healthy controls, significant difference was found in hypertension group and hypertension left ventricular hypertrophy, with statistical significance, no significant differences, there is no significant differences and no statistical significance compared with two groups in blood pressure including SBP,DBP and hypertension progression. There were significant difference and statistically significant in the aspect of LVMI from two comparisons between the three groups. The above datas show that the results can be comparative. 2 Ion Torrent semiconductor high-throughput sequencing results of three groups: For the convenience of expression,S1 as samples of healthy controls,S2 as hypertension disease control sample,S3 as hypertensive left ventricular hypertrophy disease samples. 2.1 Differential expression analysis:According to the scheme design,we screened different mi RNAs in S2 vs S1, S3 vs S1 and S3 vs S2. Using the algorithm of DEGSeq,we screened TPM of differentially expressed 313 mi RNA.Significant differences of the standards for: Fold change>1.5 or < 0.667,P-value< 0.05, FDR< 0.05. There are 59 differentially expressed micrornas in S2 vs S1,including 28 up regulation and 31 down regulation. There are 87 differentially expressed micrornas in S3 vs S1,including 66 up regulation and21 down regulation. There are 75 differentially expressed micrornas in S3 vs S1,including 55 up regulation and 20 down regulation. 2.2 The expression of mi RNA-29a-3p was differential, but not significantly in S2 vs S1, fold change 1.253332; The expression of mi RNA-29a-3p were differential and significantly in S2 vs S1 and S3 vs S2, fold change are2.6910523 and 2.147118894. This suggests that there is a correlation between mi RNA-29a-3p and left ventricular hypertrophy induced by hypertension. 3 Analysis of target genes of mi RNA-29a-3p: 3.1 The result of predicting target genes of mi RNA-29a-3p: We obtained 884 target genes of mi RNA-29a-3p through internationally recognized target gene prediction database mi RNAbase and Target Scan. Among theses target genes, thescore is combined score values which reflects the combination degree of mi RNA-29a-3p and its target genes. The higher the score, the higher the accuracy of the target gene prediction. 3.2 GO Analysis: We got the target genes according to the database and took the GO annotation through the gene function analysis, we obtain the significant GO and its containing genes and carry on the analysis from three aspects,Respectively cluding:biochemical process(BP);molecular functions(MF); cellular component(CC). It can be seen that the top five GO are extracellular matrix organization, collagen catabolic process, extracellular matrix disassembly,homophilic cell adhesion via plasma be adhesion molecules, collagen fibril organization,its(-log2P) values are: 19.42798892, 18.94812724, 17.45448077, 18.94812724, 17.45448077. 3.3 Pathway-Analysis: Pathway-Analysis is a method of detecting significantly different gene Pathway according to the gene annotation database. We put the target genes of mi RNA-29a-3p as the research object. By analyzing genetic signaling pathways,we obtained significant Pathway and its genes. It can be seen that the top five Pathway are protein digestion and absorption,ECM-receptor interaction,PI3K-Akt signaling pathway,focal adhesion,small cell lung cancer,the(-log2P) values are::23.17177941、21.10576753、20.66932209、20.28737691、15.27204612。Part two Correlation stduy between mi RNA-29a-3p and the degree of myocardial fibrosis in hypertension patients with left ventric-ular hypertrophyObjective: To explore the correlation between mi RNA-29a-3p and the degree of myocardial fibrosis in hypertension patients with left ventricular hypertrophy.Method:1 Patients with hypertension or hypertension with left ventricular hypertrophy were included in the present study. The mi RNA-29a-3p expression in serum was measured by SYBR Green I RT-PCR; matrixmetallo proteinases-9(MMP-9), procollagen I and III in serum were detected by enzyme linked immunosorbent assay(ELISA).2 Software SPSS 13.0 was used for the statistical analyses. The main statistical indexes were adopted normality test Data is expressed as mean±SD. ANOVA was used for comparison among different groups. SNK test was used for comparison between two groups. Pearson coefficients were employed for analyzing the correlation between two variables.Results: 1 Basic characteristics of these volunteers in this study:Healthy subjects(n=50) including 28 male(56.0%) and 22 female(44.0%),hypertension subjects(n=80) including 42 male(52.5%) and 38female(47.5%), hypertensive left ventricular hypertrophy subjects(n=70)including 39 male(55.7%) and 31 female(44.3%),on an average age of62.01±7.80, 63.20±8.20, 61.50±8.03 years,were selected. Clinical data included age,sex,smoking status, blood glucose and body mass index distribution,all data were not statistically significant in the three groups. But compared with healthy controls, significant difference was found in hypertension group and hypertension left ventricular hypertrophy, with statistical significance, no significant differences, there is no significant differences and no statistical significance compared with two groups in blood pressure including SBP,DBP and hypertension progression. There were significant difference and statistically significant in the aspect of LVMI from two comparisons between the three groups. The above datas show that the results can be comparative. 2 The serum level of mi RNA-29a-3p in three groups: The serum levels of mi RNA-29a-3p were found to be 1.72 ± 0.79, 3.52 ± 1.21, and 5.37 ± 1.56 for control,hypertensive and hypertensive with left ventricular hypertrophy groups,respectively. Compared to the controls, the hypertensive patients with and without left ventricular hypertrophy showed significantly higher serum levels of mi RNA-29a-3p(P<0.05). 3 Index of serum myocardial fibrosis in three groups: The results demonstrate that compared to the control group,hypertensive patients with and without ventricular hypertrophy showedsignificantly increased serum levels of PINP, PIIINP, and MMP-9(P<0.05).Also, the serum levels of these proteins were significantly higher in the hypertensive patients with ventricular hypertrophy than in those with hypertension alone(P<0.05). 4 Correlation analysis between mi RNA-29a-3p and above 3 indexs of serum myocardial fibrosis demonstrated that serum mi RNA-29a-3p levels were positively correlated with serum PINP,PIIINP, and MMP-9 levels. Correlation coefficients were 0.58,0.45 and0.66,respectively, which were statistically significant, P<0.05.Part three Blockade of mi RNA-29a-3p reduced the pressure overload-induced ventricular hypertrophy and myocardial fibrosisObjective: To explore the improvement of ventricular hypertrophy induced by pressure overload in mice by inhibiting the expression of mi RNA-29a-3p.Method:1 Thirty 11 to 12-week-old C57BL/6 male mice, each weighing about25 g, were purchased from Vital River Laboratory Animal Technology Co.Ltd. Mice were randomly divided into three groups of 10 each: sham,TAC+antagomir vehicle and TAC+antagomi RNA-29a-3p.Transverse aortic constriction(TAC) model in mice was established, 7 days after surgery, the mice were randomly received intraperitoneal injection with antagomir negative control or antagomi RNA-29a-3p for three times.4 weeks after surgery, the cross section area of the myocytes was measured by HE stain;the area of myocardial fibrosis was measured by masson stain;the expression of atrial natriuretic peptide(ANP) and β-MHC were detected by western blot.2 Software SPSS 13.0 was used for the statistical analyses. The main statistical indexes were adopted normality test data is expressed as mean±SD. ANOVA was used for comparison among different groups. SNK test was used for comparison between two groups. Pearson coefficients were employed for analyzing the correlation between two variables.Results: 1 Ultrasonic testing results of three groups of mice. Before the modeling, there was no significant difference of LVIDd, LVIDs, LVPWd and LVFS, no statistical significance, P> 0.05. For 4 weeks after surgery,compared with before surgery, in addition to control, four indicators have significant difference in TAC+antogomi RNA negative control group and TAC+antogomi RNA-29a-3p group, with statistical significance, P<0.01. 4weeks after surgery, compared with sham control, above 4 indicators have significant difference in TAC+antogomi RNA negative control group and TAC+antogomi RNA-29a-3p group, with statistical significance, P<0.01.Compared with TAC+antogomi RNA negative control group,above 4indicators have significant difference in TAC+antogomi RNA-29a-3p group, with statistical significance, P<0.01. 2 Histopathological detection of left ventricular myocardium in mice: 2.1 Results of HE staining: The cross-sectional area of myocardial sections was significantly increased in the TAC mice.Compared with compared with sham control, Left ventricular myocyte hypertrophy and myocardial interstitial hyperplasia were found in TAC+antogomi RNA negative control group and TAC+antogomi RNA-29a-3p group, especially which was obviously in the TAC+antogomi RNA negative control group. The degree of Left ventricular myocyte hypertrophy and myocardial interstitial hyperplasia in TAC+antogomi RNA-29a-3p group was in between sham control group and TAC+antogomi RNA negative control group.There was significant difference and statistical significance in three groups, P<0.05. 2.2 Results of Masson staining: Myocardial cell was purple or red,form neat. In the middle,there were a small blue collagen fiber. Left ventricular myocardial fibers of TAC mice arranged in disorder,part of which were fracture and blue collagen fibers significantly increased. The degree of fiber hyperplasia in TAC+antogomi RNA-29a-3p group was small than in TAC+antogomi RNA negative control group. There was significant difference and statistical significance in three groups,P<0.05.3 Results of western blot: Immunoblotting illustrated that expression levels of ANP and β-MHC protein were significantly increasedin TAC mice. In comparison, in the mice treated with antagomi RNA-29a-3p,levels of these proteins were significantly reduced(P<0.05).Conclusions:1 We obtain expression profiling of plasma mi RNA from healthy control group, hypertension group and hypertension left ventricular hypertrophy group by Ion Torrent semiconductor sequencing technology.2 Through using DEGSeq the algorithm of DEGSeq,we screened TPM value and got significant different mi RNAs.3 TO select differerntially expressed mi RNA-29a-3p as the object and predict its target genes.We took the GO annotation through the gene function analysis and obtained the significant GO and its containing genes and obtained significant Pathway and its genes by analyzing genetic signaling pathways.4 Compared to the controls and hypertensive, the serum levels of mi RNA-29a-3p,MMP-9,PINP and PIIINP were found to be highest in hypertensive with left ventricular hypertrophy groups.5 There was positive correlation between the serum levels of mi RNA-29a-3p and those including MMP-9,PINP and PIIIN in hypertensive with left ventricular hypertrophy groups.The serum levels of mi RNA-29a-3p is potential to be novel biomarker about ventricular hypertrophy and myocardial fibrosis,which is used to evaluate curative effect and prognosis.6 Antagomi RNA-29a-3p can improve the increased ventricle and cardiac function partly.7 Administration of antagomi RNA-29a-3p inhibited the pressure overload-mediated myocyte hypertrophy and myocardial fibrosis and reduced expression levels of ANP and β-MHC protein in left ventricular hypertrophy myocardial.8 Mi RNA-29a-3p is likely to be the treatment targets of hypertension venricular hypertrophy and ventricular remodeling.