Differential Expression Profiles And Functional Analysis Of LongNon-Coding RNAs In Children With Dilated Cardiomyopathy

BackgroundDilated cardiomyopathy is an unexplained primary myocardial disease,its main characteristics are left or right ventricle or bilateral ventricular enlargement,accompanied by ventricular systolic dysfunction,with or without congestive heart failure,and expansion and dysfunction caused by non-hemodynamic reasons.Dilated cardiomyopathy(DCM)is the main cause of progressive refractory heart failure which necessitates heart failure and can lead to death at any stage.The annual incidence of DCM in children accounts for approximately 50%of pediatric cardiomyopathies(0.57/100,000)and is the most common type of pediatric cardiomyopathy.The mortality rate of children with DCM is high,and most children with this condition die from heart failure.However,the pathogenesis of DCM remains unclear yet.The existence of family aggregation in DCM suggests that genetics play a certain role in its pathogenesis.In recent years,literature has confirmed that familial DCM accounts for 20%～50%of the total number of DCM cases.Further,an increasing amount of gene mutations have been found to be highly related to DCM,of which autosomal dominant inheritance accounts for 90%of all gene mutations including 16 genes,which in general this may only account for a small part of the genetic cause.Another hypothesis supported by previous studies suggests that failure to eliminate pathogens or activation of chronic autoimmune mechanisms against cardiac antibodies after subacute and chronic viral myocarditis may finally foster the conversion to DCM.Therefore,further clarification of its etiology and pathogenesis is very important for clinical and scientific research.Long non-coding RNA(lncRNA)are usually defined as transcripts greater than 200 nucleotides in length of noncoding RNA(ncRNA)which is a type of RNA that can be transcribed from the genome,but not translated into protein.They can perform a variety of molecular functions such as signaling,decoy,guidance,and scaffolding by interaction with DNA,RNA,or proteins.It has been reported that lncRNA is indeed regulated in human cardiac diseases.Additionally,there is increasing evidence that lncRNA play a vital role in vascular biology and cardiovascular diseases,such as myocardial infarction,cardiac hypertrophy and fibrosis,atherosclerosis,angiogenesis,and vascular remodeling.In addition,the lncRNA Microarray Chip is an effective tool for high-throughput analysis of lncRNA expression and it has been used to study the expression profiles of lncRNA in many kinds of diseases.However,data related to DCM in children is sparse.ObjectiveTo explore the differential expression of lncRNA and mRNA in peripheral blood leukocytes of DCM and healthy children,and to screen DCM specific lncRNA,and study whether there are expression differences in different left ventricular function in children with different left ventricular function.To discover new DCM biomarkers and provide clues for the study of pathogenesis of DCM.MethodA total of 25 DCM and 25 normal peripheral blood leukocytes were collected,and three DCM samples(D1,D2,D3)with classic clinical symptoms and three normal samples(C1,C2,C3)were chosen for microarray analysis.The chosed samples were sent to Shanghai Sinomics Corporation(Shanghai,China)then used to generate biotinylated cRNA targets for the Sino Human ceRNA array V3.0.Then the differential expressed lncRNA and mRNA are analyzed,including GO and KEGG pathway analysis,lncRNA cis/trans target gene prediction,lncRNA-mRNA co-expression network,and lncRNA-miRNA-mRNA interaction network.Result1.The Microarray Analysis detected a total of 75,589 lncRNAs in 3 DCM samples and 3 control samples.In summary,there are a total of 874 lncRNAs that are differentially expressed in patients with DCM as compared to the control group(fold change>2,P<0.05),of which 369 are up-regulated and 505 are down-regulated.Moreover,there are 641 differentially expressed mRNAs between the DCM group and control group in all,which include 482 up-regulated and 159 down-regulated mRNAs.2.Through the Go enrichment analysis,among the three different gene functions of lncRNAs,we selected the top 20 go items of enrichment factors,in which the molecular function is biological pathway.These items include IgG binding,immunoglobulin binding,regulation of interferon-beta production,interferon-beta production,antiporter activity,positive regulation of osteoblast differentiation,regulation of Notch signaling pathway,regulation of osteoblast differentiation,protease binding,regulation of interferon-gamma production,regulation of type I interferon production,activation of cysteine-type endopeptidase activity,type I interferon production,interferon-gamma production,bone mineralization,extrinsic component of plasma membrane,organic anion transmembrane transporter activity,phosphatase binding,response to alkaloid,zymogen activation and other biological processes.3.In KEGG pathway analysis,biological metabolic pathways are mainly divided into three levels,in which we selected the top 30 rich KEGG pathways in the immune system and signaling pathways.Among them,we are more interested in "NF-κB signaling pathway","Toll-like receptor signaling pathway","PPAR signaling pathway","MAPK signaling pathway","Apoptosis","Nod-like receptor signaling pathway" and so on,because they may be related to DCM.Finally,we screened some differentially expressed genes in these pathways for future experiments.4.We predicted the target genes of lncRNA from the cis and trans modes of action.The results prove that lncRNA may be involved in the regulation of transcription level in the "NF-κB signaling pathway","Toll-like receptor signaling pathway" and "apoptosis" pathways.5.We constructed the lncRNA-mRNA co-expression network.We have identified 120 lncRNAs and 22 mRNAs altogether in the pathways mentioned above(Pearson’s coefficient>0.98).The lncRNA-mRNA network consisted of 142 net nodes and 149 connections of which each lncRNA was linked to 1～3 mRNAs,and each mRNA was linked to 1～18 lncRNAs.The results suggest that TLR1,TLR2,TLR3,TLR4,TLR6,IL-6,IL1R1,IL1B,IL1RAP,ACS14,CASP1,CASP5,MAP2K6 and TAB3 play an important role in children with DCM.6.We used Cytoscape 3.6.0 to observe the interaction of lncRNAs and their potential complementary miRNAs,and predicted the top five miRNAs with the highest binding potential of each lncRNA.We selected the differentially expressed lncRNAs according to the function of the differentially expressed mRNA in the selected pathway,which may be regulated by lncRNAs that can competitively bind to the corresponding miRNAs.It is predicted that and NONHSAT215378.1 may interact with hsa-miR-4485-3p and negatively regulate mRNA FBXO32.Using this information,we constructed a ceRNA network using Cytoscape.7.We screened 9 lncRNAs,of which 5 were up-regulated and 4 were down regulated.After QRT PCR analysis,the nine lncRNAs were amplified separately.However,compared with the control group,the results of NONHSAT 242978.1 and ENST0000637940 in DCM group were contrary to the trend of microarray data.Among the other seven lncRNAs,three were up-regulated(ENST0000560465,NONHSAT252242.1,ENST0000457996)and four were down regulated(NONHSAT175499.1,ENST0000596816,NONHSAT215378.1,NONHSAT137060.2)with statistical significance(p<0.05).The accuracy of microarray data was verified.8.In order to explore whether lncRNAs in peripheral blood leukocytes of DCM children with improved left ventricular function and acute DCM children also have differential expression after treatment,we also verified whether these lncRNAs have differential expression in DCM patients with poor cardiac function and DCM patients with improved left ventricular function.The results showed that only NONHSAT252242.1,ENST0000596816,NONHSAT215378.1 and NONHSAT137060.2 were significantly different from those in acute DCM group(p<0.05,multiple of difference>2).This finding suggests that the expression of these molecules may be related not only to the diagnosis of DCM,but also to the severity of DCM,and can be used as biomarkers to measure the recovery of DCM in the future.Conclusion1.In the peripheral blood of children with DCM,there are differences in the expression of lncRNA and mRNA,which may be involved in the occurrence and development of children with dilated cardiomyopathy.2.In pediatric DCM,lncRNA may play a role mainly by regulating the relevant signal pathways of the immune system.3.The lncRNA NONHSAT252242.1,ENST00000596816,NONHSAT215378.1,NONHSAT137060.2 in the peripheral blood of children with DCM may be a biomarker for the diagnosis of DCM in children,which initially reflects the development of the disease.