| ObjectiveScreening for rapid and effective diagnostic markers is an crucial means for implementing precise treatment strategies to improve the detection rate of obstructive sleep apnea(OSA).Small extracellular vesicles(sEVs)are nanoscale vesicles secreted by living cells,rich in proteins,lipids and nucleic acid,carrying information about the source cells.It is considered to be the promising biomarkers for precise diagnosis and targeted therapy of diseases.Long non-coding RNAs(lncRNAs)are stably expressed in sEVs,which extensively involved in many important biological processes and have good diagnostic and prognostic value for diseases.Therefore,exploring the possibility of sEV-lncRNAs as non-invasive diagnostic biomarkers can bring new opportunities to basic research and clinical treatment of OSA.In this research,high-throughput sequencing(HTS)was used to analyze the expression of plasma sEV-lncRNAs in non-OSA individuals with or without hypertensive(HTN)and correlated them with clinical features.We aim to screen out the biomarkers of OSA and through which we have a further understanding about the molecular biology mechanism of OSA.MethodsThis was an observational research.Plasma samples were collected from non-OSA,OSA individuals with or without HTN,and sEVs were extracted.The morphology of sEVs was identified by transmission electron microscopy and Nanosight nanoparticle analysis,the characteristic protein expression was detected by Western blot.At the screening stage,transcriptome deep sequencing was performed by lncRNA sequencing(lncRNA-seq)technology,and bioinformatic analysis of differentially expressed lncRNA profiles was performed.Three differentially expressed lncRNAs were verified by droplet digital PCR(ddPCR),and their diagnostic value was determined by receiver operating characteristic(ROC)analysis.In the clinical evaluation phase,a clinical cohort include 41 participants was further established to analyze the association between the diagnostic value of lncRNA marker and clinical characteristics,their diagnostic efficacy with ROC curves were discussed.Meanwhile,its stability as a biomarker was also evaluated.Finally,MiRanda software was used to predict the binding sites of lncRNA-regulated miRNAs,and the possible mechanism was speculated.Results1.Ultracentrifugation was selected as the primary method for the extraction of three groups of plasma sEVs because of its advantages in reducing the contaminating of proteins and the stability of the products obtained.2.A total of 1987 differentially expressed lncRNAs(1008 up-regulated,979 downregulated),444 mRNAs(223 up-regulated,221 down-regulated)were obtained,and they probably reflected the different stages of OSA.A large number of target genes of lncRNAs were significantly enriched in mTOR,WNT,glutamatergic synapses,branched-chain amino acids and other pathways.3.Detection of expression levels of ENST00000592016、ENST00000442889、ENST00000319701.It was found that ENST00000592016 can better identify NC and OSA samples(AUC=0.8460,95%CI:0.72-0.97,P=0.0002),ENST00000592016 expression increased with the severity of OSA,BMI(r=0.522),TS90%(r=0.490),ODI(r=0.505),and AHI(r=0.564)showed the same ENST0000059201 was positively correlated and LSpO2(r=0.486)was negatively correlated.Compared to CRP and HbA1c,ENST00000592016 showed a better diagnostic efficiency.4.It is presumed that ENST00000592016 present in plasma is mainly encapsulated in sEVs.5.We constructed a network diagram of lncRNA-miRNA-mRNA regulation pattern with ENST00000592016 as the core.ENST00000592016 may affect PI3KAkt,MAPK and TNF pathways by regulating microRNA(miRNA)expression.Perhaps it influenced the development of OSA in terms of apoptosis,metastasis,metabolism,and secretion.ConclusionIn this study,the differential expression of lncRNA in plasma sEVs was obtained between non-OSA and individuals with or without HTN.It was confirmed that sEVENST00000592016 has high sensitivity,specificity and stability for OSA diagnosis and is expected to be developed as a biomarker for OSA diagnosis. |
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