Study Of Aberrantly Expressed Non-coding RNAs Involved In The Pathogenesis Of Mitochondrial Myopathy And Potential Clinical Implications

Mitochondrial diseases are heterogeneous group with varying clinical features,such as the most prevalent mtDNA A3243G mutation,the well-recognized phenotype of which is MELAS syndrome.Although there is increasing clarification of the primary aberrant cellular processes responsible for the respiratory chain dysfunction,the underlying mechanisms of many mitochondrial disorders are still not fully understood.In addition,comparatively little has been accomplished to date in the field of mitochondrial epigenetics.Researchers in the field of mitochondrial biology are increasingly incorporating these concepts into their studies and discovering a "gold mine" of regulatory interactions between mitochondrial function and ncRNAs biology.An emerging body of research points to close interactions between miRNA and mitochondria.Several studies have documented the localization of various miRNAs and their processing machinery in mitochondria,and that uncoupling of mitochondria decreased cellular miRNA functional efficiency.Mitochondria-associated miRNAs,IncRNAs and their regulatory networks need to be specifically and thoroughly investigated in mitochondrial disease.In the present study we aimed to arouse the attention of ncRNAs regulation in studying mitochondrial myopathy,which might make contributions to the unveiling of the complex mechanisms underlying mitochondrial myopathy and,possibly,new tools applicable to clinical practice.Through high-throughput technology,qRT-PCR and bioinformatics analyses,we performed,for the first time,one systematic and integrative analysis of the potential ncRNA-mRNA regulatory networks in muscle biopsies between MELAS patients with mtDNA A3243G mutation and controls.We found that the deregulated ncRNAs caused by mtDNAA3243G mutation formed complex regulation networks and participated in immune system,signal transduction,translation,muscle contraction and other pathways.Among these dysregulated ncRNAs,we found miR-27b-3p reduced in muscle and serum of MELAS patients with A3243G mutation or non-A3243G mutation,which was negatively associated with lactate,NMADS and the mutation load in muscle,had the better diagnosis value for MELAS than lactate.These results will enrich the genome-wide analysis of RNA molecules with potential cross-talk,provide the rationale for further confirmation studies in larger prospective cohorts and contribute to further systematic studies of mitochondrial disorders.PART ONEStudy of aberrantly expresstion of ncRNAs involved in the pathogenesis of mitochondrial myopathyProfiling the distinct miRNAs,IncRNAs and mRNAs caused by mtDNA A3243G mutation in muscle biopsies.Firstly,the genome wide miRNA,IncRNA and mRNA expression profiles in muscle biopsies were detected in discovery phase(including 20 MELAS patients with A3243G mutation and 20 matched controls).By microarry profiling,there were 31 and 41 miRNAs significantly up-and down-regulated in MELAS vs.controls,respectively.By sequencing profiling,57 and 52 IncRNAs were identified significantly up-and down-regulated by A3 243 G mutation,respectively.Meanwhile,categorization of IncRNAs indicated that more antisense and intergenic than intronic and processed transcripts were dysregulated.Using the same sequencing profiling with IncRNAs,we found 167 and 162 mRNAs up-and down-regulated in muscle in MELAS patients vs.controls.In addition,mRNA profiles were compared to that of mitochondrial myopathy with TK2 mutation.When compared with normal subjects,differential gene expression profiling in muscle tissue was identified between TK2 and mtDNA A3243G mutation.Interestingly,41 dysregulated mRNAs were common between two kinds of mitchondrial myopathy,while others seemed to be specific to the given phenotype.This suggested that different kinds of mutation just caused partly common pathogenesis of mitochondrial myopathy.Corresponding to the above features,we screened out subsets of 6 miRNAs(including miR-6089、miR-27b-3p、miR-214-3p、miR-150-5p、let-7e-5p and miR-145-5p),5 lncRNAs(including LINC01405、SNHG12、RP11-403P17.4、CTC-260E6.6 and RP11-357D18.1)and 6 mRNAs(including PDK4、CDKN1A、ATP2A2、SOD3、DDIT4 and EEF1A1)for qRT-PCR confirmation.The findings were all consistent with results from our high-throughput data.PART TWOStudy of the interaction networks of aberrant ncRNAs involved in the pathogenesis of mitochondrial myopathyThe miRNA,mRNA and lncRNA regulatory networks in MELAS with mtDNA A3243G mutation.To explore biologic function of significantly dysregulated ncRNAs with fold change>2 that were screened out from the discovery phase,firstly we obtained the potential target pairs between three kinds of RNAs by bioinformatics analyses.We just focused on target regulation with contrasting expression pattern.Based on that,three mtDNA A3243G-regulated interaction networks of miRNA-mRNA，lncRNA-mRNA and miRNA-lncRNA were constructed.There were 61 miRNAs,125 mRNAs and 259 potential pairs,with contrasting expression within the miRNA-mRNA network.In regards to lncRNA-mRNA network,973 potential lncRNA-mRNA pairs consisted of 34 lncRNAs and 90 mRNAs were constructed.In addition,the miRNA-lncRNA regulating network was formed by 77 miRNA-lncRNA pairs,which consisted of 7 up-regulated miRNAs with 6 down-regulated lncRNAs and 28 down-regulated miRNAs with 19 up-regulated lncRNAs.Finally,we constructed the whole target networks of miRNA-mRNA-lncRNA and lncRNA-miRNA-mRNA,both of which were significantly dysregulated in muscle biopsies of MELAS patients vs.controls.Functional annotation was performed by Gene Ontology(GO)and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analyses,to determine the biological significance of the whole dysregulated expressed mRNAs and two subsets of mRNAs which were potentially targeted by altered miRNAs/lncRNAs with negative expression.In terms of the biological process,the common classification and significantly related categories of mRNAs,miRNAs,lncRNAs pointed to extracellular matrix organization,muscle contraction and protein binding.In addition,the significant GO items of the whole differential mRNAs still indicated the small molecule metabolic process,signal transduction and transcription.The significant GO classifications of miRNA-mRNA pairs were signal transduction,apoptotic process and development.The main associated GO classifications of lncRNA-mRNA pairs were small molecule metabolic process and muscle contraction.Correspondingly,all the three KEGG pathway analyses of mRNAs,miRNAs,lncRNAs indicated that the common significant items were immune system,infectious diseases,translation,signal transduction and cell communication.Specifically,KEGG pathway analyses of the whole differential mRNAs were associated with metabolic pathways,cytokine-cytokine receptor interaction,MAPK signaling pathway and so on.Subsequently,the pathway annotations of differential miRNAs were mainly involved in complement and coagulation cascades,RNA degradation and calcium signaling pathway.Pathway enrichment items of differential lncRNAs included metabolic pathways,muscle contraction,p53 signaling pathway and cytokine-cytokine receptor interaction.PART THREEPotential clinical implication of aberrantly expressed ncRNAs involved in the pathogenesis of mitochondrial myopathyClinical validation of candidate miRNAs and lncRNAs using an independe nt MELAS patients and controls.To test whether our findings in training set were replicable for the promising ncRNAs,an independent cohort of 54 MELA S patients and 49 age-and sex-matched controls were chosen for the validatio n of the selected ncRNAs.Muscle expressions of miR-6089N miR-27b-3p.miR-214-3p and LINC01405 were significantly down-regulated in MELAS patients vs.controls.In contrast,muscle expressions of 3 miRNAs(miR-150-5pN let-7e-5p and miR-145-5p)and 4 lncRNAs(SNHG12、RP11-403P17.4.CTC-260E6.6 and RP11-357D18.1)were significantly up-regulated、Furthermore,muscle miR-27b-3p showed the highest diagnostic accuracy among them.In addition,we co ntinued to test whether miR-27b-3p was also dysregulated in serum between 34 MELAS patients with A3243G mutation and 34 age-and sex-matched contro ls selected from the validation cohort above.Surprisingly,serum miR-27b-3p w as also down-regulated in MELAS patients vs.controls.In addition,we further validated that it was also significantly reduced in muscle and serum of 22 ME LAS patients with non-A3243G mutation compared with 34 age-and sex-matc hed controls.ConclusionThrough high-throughput technology followed by quantitative real-time polymerase chain reaction(qRT-PCR)and bioinformatics analyses,for the first time,we found that the dysregulated muscle miRNAs and lncRNAs between 20 MELAS patients with mtDNA A3243G mutation and 20 controls formed complex regulation networks and participated in immune system,signal transduction,translation,muscle contraction and other pathways in discovery and training phase.Then,selected ncRNAs were validated in muscle and serum in independent validation cohorts by qRT-PCR.Finally,ROC curve analysis indicated reduced serum miR-27b-3p had the better diagnosis value than lactate and might serve as a novel,noninvasive biomarker for MELAS.Follow-up investigation is warranted to better understand roles of ncRNAs in mitochondrial myopathy pathogenesis.