Screening And Study On The Function Of Differential Expression Of Plasma MiRNA In Congenital Heart Disease With Secondary Pulmonary Hypertension

BackgroundPulmonary arterial hypertension(PAH)is a severe disorder clinically defined by mean pulmonary arterial pressure of at least 25 mmHg at rest.The lack of specific noninvasive diagnostic techniques and medications of secondary pulmonary arterial hypertension(PAH),a common complication of congenital heart disease(CHD),what directly and seriously affects the prognosis of surgical operation along with interventional therapy of patients and is also an crucial factor of patients/their death in perioperative period.For patients with pulmonary arterial hypertension related to congenital systemic-to-pulmonary shunts,the key issue about prognosis is our early diagnosis and positive intervention before the appearance of clinical symptoms.Briefly,miRNAs molecules of about 21-23 nucleotides in length are highly conserved and noncoding RNA,which exists widely in plants,microorganisms and mammals,in the occurrence and development of many diseases,such as cardiovascular disease and cancer.MicroRNAs regulate gene expression by interacting with the 3’UTR regions of specific mRNA targets and inhibiting translation.It is also predicted that on an average,a single miRNA can have more than 100 targets.Recent studies have implicated an important role for microRNAs(miRNAs)in various cellular processes,including cell differentiation,proliferation and apoptosis in pulmonary artery smooth muscle cells(PASMCs)and pulmonary artery endothelial cells PAECs.MicroRNA-143(miR-143)and miR-145 have been shown to regulate proliferation and differentiation of pulmonary artery smooth muscle cells(PASMCs),thus linking the function of miRNAs to SMC plasticity and fate determination.Recently,miRNAs have been widely implicated in both healthy and pathological processes of vascular remodeling,such as wound healing development of pulmonary vessels,and tumor angiogenesis,and in the proliferation apoptosis imbalance of cancer.Interestingly,alteration of miRNA expression has been widely found and recognized by the scientific community as a critical actor in PAH establishment.Recent studies have shown that circulating microRNAs are a potential biomarker in various types of PAH.Currently,the plasma miRNA is a hot spot in research of molecular biology as well as translational medicine.However,the circulating miRNA and PAH pathogenesis and target is not clear.We intend to find new biomarkers for disease and targets for drug treatment through the research on characteristic different expression of miRNAs which induced by pulmonary arterial hypertension related to congenital systemic-to-pulmonary shunts of CHD.The nested case study was used in this experiment.ObjectiveThe aim of this study was to investigate the feasibility of using plasma microRNAs as novel serological biomarkers for VSD-PAH,and we assessed the role of miR-16 in VSD-PAH.Method1 We respectively selected 6 cases of simple ventricular septal defect(VSD)and secondary PAH of VSD to analyze the expression of plasma miRNAs with significant differences between two groups by gene chips;2 We respectively selected 100 cases of simple VSD and secondary PAH of VSD to verify the level of screened and specific miRNAs in this 200 patients through stem-loop method and real-time PCR technology.The expression levels of selected microRNAs were normalized to Caenorhabditis elegans microRNA(Cel-miR-39).The RNA data were not in conformity with the normal distribution,so use nonparametric tests(Mann Whitney U test for statistical difference,to median +interquartile range expression of each group of data distribution.3 Correlation of clinical data analysis:selected 100 cases in each group,and the patient’s name,sex,age,clinical examination indexes of information were recorded detaily,the validation of differential miRNAs and the clinical indexes correlation was analysis by Pearson correlation analysis.4 Study on the effects of miRNA-16:In this study,we investigated the role of miR-16 in HPASMC proliferation and migration.by transfected with mimics or inhibitor,5.miRNA-16 target gene prediction and validation:using miRNA target gene prediction database targetscan,pictar and miRBase tor predict target genes of miR-16;luciferase report report vector,Q-PCR and Western blot was used to detect the direct regulation of the mir-16 to its key target genes;6.The molecular mechanism of the biological functions of mir-16 was study using cell growth curve experiment and cell migration experiment.Result1.Using miRNA microarray analysis,101(including miR-16)plasma miRNAs were found to be upregulated more than 3 times and 54 plasma miRNAs downregulated in VSD/PAH group.2 The results of real-time quantitative RT-PCR were consistent with those of microarray.Statistical results show that in the verification of the differences in miRNA molecules,mirK12-8,mirK12-10a,mirK12-12,mirUS25-2,mir16,mir17a,mirl9a,mir20a,mir21,mir22,mir92,mir98,Mir143,mir204 in patients with pulmonary hypertension were up-regulated and with statistical difference(P<0.05),consistent with the microarray results change trend.3 Analysis of Stepwise correlation showed that miR-16(R=,-0.538),Mir-17(R=-0.538),Mir-21(R=-0.682)expression was negatively correlated with echocardiography,LVEDd;miR-16(R=0.837),Mir-22(R=0.659),miR-92(R=0.785),miR-US25-2(R=0.581)expression was correlated with mean pulmonary arterial pressure(MPAP);miR-16(R=0.752),Mir-22(R=0.608),miR-92(R=0.701)expression was correlated with pulmonary vascular resistance before correction of drugs;Mir-17(R=0.315),the expression was positively correlated with echocardiography LA;miR-16(R= 0.836),Mir-22(R=0.713),Mir-92(R=0.817),Mir-us-25-2(R=0.603)expression index of catheter was positively related to PAP;miR-16(R= 0.649),Mir-22 R=0.550,Mir-92 R=0.623,Mir-us-25-2 R=0.445 expression was correlated with PVP;Mir-us-25-2 R=0.670 expression was correlated with heart color Doppler ultrasound RA;Mir-20a R=0.823,Mir-143 R=0.754,Mir-204(r = 0.808)expression was correlated with red blood cell distribution width(BCV).4 The experimental results show that Knockdown of miR-16 with anti-miR-16 inhibitors significantly reduced pulmonary artery smooth muscle cell(PASMC)proliferation,whereas miR-16 overexpression in normoxia enhanced cell proliferation compared with the NC transfection control group.We also found that miR-16 is essential for cell migration.5.miR-16 target gene prediction and validation:miR-16 overexpression significantly decreased RBM6 expression at both the mRNA and protein levels.Based on bioinformatics analysis of three online databases(Target Scan,Pic Tar,and miRanda),the complementary sequence of miR-16 was found in 3’-UTR of RBM6 mRNA.In contrast,RBM6 expression were induced by miR-16 inhibitor,while RBM6 was downregulated by miR-16 overexpression.To validate the transcriptional regulation of miR-16 on RBM6 expression,we cloned the RBM6 3’-UTR regions containing miR-16 binding sites or corresponding mutant sites into a luciferase reporter vector pGL4,and we performed luciferase reporter assays in 293T cells.Furthermore,miR-16 overexpression significantly reduced the luciferase activity of the RBM6 wild-type reporter plasmids compared with miR-NC,and this inhibition was not observed in RBM6 mutant reporter plasmids.These findings suggested that miR-16 can transcriptionally regulate RBM6 expression.6,Migration ability of HPASMC cells was significantly increased when miR-16 was overexpressed.Nevertheless,the Migration ability blocked by RBM6 could be partially rescued by miR-16.These results showed that the down-regulation of RBM6 protein expression can inhibit the invasion and migration of cells.In conclusion,miR-16 may down regulate expression of RBM6 cells and promote cell proliferation and metastasis.Conclusion1.The plasma levels of circulating microRNAs showed a smaller difference between VSD/PAH and VSD/NPAH.This study suggests that plasma miRNAs may be used as novel serological biomarkers for VSD/PAH.2.The expression of partial gene miRNAs from microarray data was up-regulated by using fluorescence quantitative PCR,proved that microarray data was reliable.3 Bioinformatics analysis of relationship from differentially expressed miRNAs and clinical data,It indicated that these miRNAs can be used as a potential diagnostic factor of PAH.4.MiR-16 in PAH expression was significantly up-regulated,and can promote cell proliferation and metastasis through directly regulating its target genes RBM6.