Circulating microRNA expression profiling and bioinformatics analysis of patients with coronary artery disease by RNA sequencing

Abstract Background MicroRNAs play a vital role in coronary artery disease. Abnormal expression of microRNAs has been found to be associated with the occurrence of CAD. Methods We identified significantly differentially expressed microRNAs in plasma between 40 patients with CAD and 10 controls with NCA using RNA sequencing. The differentially expressed microRNAs were analyzed for Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Results Fifty cDNA libraries were constructed and sequenced, and a total of 1871.82 M raw reads were obtained, and 2135 microRNAs were found. Compared to the expressed microRNAs of NCA controls, 159 microRNAs were differentially expressed in CAD patients, including 119 upregulated microRNAs and 40 downregulated microRNAs. The top 10 upregulated miRNAs were miR‐144‐3p, miR‐34a‐5p, miR‐15b‐3p, miR‐22‐3p, miR‐29b‐3p, miR‐1270, miR‐6891‐5p, miR‐106a‐5p, miR‐15b‐5p, and hsa‐miR‐499b‐3p. The top ten downregulated miRNAs were miR‐4437, miR‐6842‐3p, miR‐4664‐3p, miR‐671‐3p, miR‐219a‐1‐3p, miR‐7848‐3p, miR‐664a‐3p, miR‐1284, miR‐361‐3p, and miR‐6780a‐5p. The target genes of differentially expressed microRNAs were related to many basic biological terms, such as biological process, cellular component, and molecular function. According to the KEGG pathway analysis, the most enriched pathways of the differentially expressed microRNAs were endocytosis, focal adhesion, axon guidance, and so on. Furthermore, six upregulated and two downregulated microRNAs were detected by qRT‐PCR (Quantitative Real‐time PCR) and ROC analysis for diagnosing CAD. Conclusion The results suggest that the expression levels of some microRNAs may play a vital role in the physiological and pathological course of CAD. Our study may provide useful information for the diagnosis and treatment of CAD.


| INTRODUC TI ON
Coronary artery disease (CAD) is a common heart disease. It is a myocardial malfunction and structural lesion caused by coronary artery stenosis and poor blood supply. The occurrence of CAD is closely related to the degree and number of coronary artery stenoses. Hypertension, diabetes, obesity, smoking, and drinking are the main factors that induce this disease. [1][2][3] Clinically, it is often divided intostableangina(SA)andacutecoronarysyndrome(ACS).ACScan bedividedintounstableangina(UA),acutenon-ST-segmentelevationmyocardialinfarction(NSTEMI)andacuteST-segmentelevation myocardial infarction (STEMI), according to severity. STEMI and NSTEMIaretogetherknownasacutemyocardialinfarction(AMI). 4,5 Coronary artery disease is one of the most fatal diseases in the world. 6,7 Atherosclerosisplaysapivotalroleintheoccurrenceand progression of CAD. It is strongly associated with arterial stenosis because of the accumulation of atheromatous plaque, adipopexis, andextracellularmatrix.Coronarythrombosisoftenleadstoacute coronary events. Inflammation is involved in various stages of the formationanddevelopmentofatherogenesisplaque,andinflammatory factors produced in this process are predicted to be potential markers of CAD. They play a key role in coordinating the interactions of the cells during the development of atherogenesis. [8][9][10][11] Inflammatory mediators involved in the function of some cells, including endothelial cells, macrophages, and T cells. They stimulate some signalling pathways in the onset and progression of atherosclerosis. 12,13 TheycanberegulatedbymicroRNAs(miRNAs),whichare smallnon-codingRNAsthathaveemergedasimportantregulators in atherosclerosis. [14][15][16][17][18] MicroRNAsareaclassofsingle-strand,non-codingRNAswith a length of 18-22 bp that play a vital regulatory role in different cell development processes by downregulating the expression of their target genes. 19,20 TheanalysisoftheexpressionofmicroRNA in blood, tissue, or cell samples provides important information for the study of the biological functions of these molecules. In recent years, researchers have developed many methods to detect the differences in the expression of microRNAs in some physiological and pathological processes, and they have found that the abnor-malexpressionofmicroRNAsisconnectedwiththeoccurrenceof cancer, 21 nervous disorders, 22,23 diabetes, 24 and heart diseases. [25][26][27] Many studies have shown that microRNAs have some important regulatory effects in the cardiovascular system, including the heart, inflammatory response, angiogenesis, and metabolism. 20,[28][29][30] AbnormalexpressionofmicroRNAshasbeenfoundtoberelatedto theoccurrenceofCAD.

| Sample collection and processing
Two samples were taken before the coronary angiography surgery, onefortheextractionofwhole-bloodRNAandoneforthedetection of BNP and cTnI. Peripheral blood samples (6 mL) were taken fromtheanteriorelbowveinusingEDTAanticoagulanttubes,mixed gently up and down for 10 times, saved at 4°C, and subjected to plasma separation within 1 hour. The blood samples were centrifuged at 800 g for 10 minutes, and the plasma was transferred to a 1.5mLcentrifugetube(RNase-free).TotalRNAwasobtainedwith anRNeasyKit(TianGene),andRNAintegritywasassessedusingan AgilentBioanalyzer2100system(AgilentTechnologies).

| Library construction and highthroughput sequencing
After screening test samples, a library of small RNAs was con-

| Identification of differentially expressed genes
TherawreadsinFASTQformatwerefilteredtoremovetheadapter sequences, poly-N sequences, and low-quality sequences before data analysis. The remaining reads were also called "clean reads" and wereacquiredfortranscriptomeassemblyandquantification.Next, the index of the reference genome was built using Bowtie v2.0.6, andpaired-endcleanreadsweremappedtothereferencegenome using TopHat v2.0.9. Transcriptome assemblies were generated

| Gene ontology and KEGG enrichment analysis
Gene ontology analysis was carried out to determine the potential biological process terms of the differentially expressed genes in GO annotations. 31 Pathway analysis was utilized to find the significant pathways of the differentially expressed genes according to the KEGG database. 32 Fisher's exact test was applied to evaluate whether the GO terms or the KEGG pathways were enriched amongthedifferentiallyexpressedgenes,andP<.05wassetasthe threshold for.

| Quantitative real-time PCR
ToverifythereliabilityoftheRNAsequencingdata,somemicroR-NAswithdifferentialexpressionweredetectedbyqRT-PCR.Total RNA in the plasma was extracted using an RNeasy Kit (TianGene) and was reverse-transcribed to cDNA. qPCR was performed using

| Differentially expressed microRNAs in plasma
MicroRNAs were analyzed with strict data quality control, and a total of 2135 microRNAs were found. To systematically study the levelofmicroRNAexpressionassociatedwithCAD,40patientswith CADand10NCAcontrolswereanalyzedinthisstudy.Thediffer-encesbetweentheCADpatientsandtheNCAcontrolsareshown in Figure 2. The blue and red in the picture indicate that the relative expressionwasreducedandraised,respectively.

| Gene ontology and KEGG pathway analyses
Gene ontology and KEGG pathway analysis showed that genes were sorted by hierarchical categories according to biological process, cellular component, and molecular function.
AmongtheidentifiedmRNAs,19243geneswererelatedtobiological function ( Figure 6). The enriched GO terms of these potential targets included many biological events. The most enriched path-

| ROC analysis
ROC analysis was done to evaluate the diagnostic value of some differentially expressed microRNAs that were randomly selected for differentiatingbetweenCADpatientsandNCAcontrols.MicroRNAs

| D ISCUSS I ON
Heart disease is the main cause of death for both males and females and is a great burden to some countries, which includes the cost of health care services, medications, and lost productivity. [33][34][35][36] Before vessel obstruction, the vascular wall has a plaque for a long time, and several cytokines are involved in all stages of the formation and progressionofatherogenicplaque,whichprovidesapossibilityfor the prediction and early diagnosis of cardiovascular adverse events.

| CON CLUS IONS
This study comprehensively identified and analyzed microRNA expression in CAD patients using RNA sequencing. The results F I G U R E 6 GOanalysisofdifferentiallyexpressedmicroRNAscoveringthreedomains:biologicalprocess,cellularcomponent,and molecularfunction.X-axis:GOtermsofbiologicalprocess,cellularcomponent,andmolecularfunction.Thegreencolumnindicates biologicalprocess,theredcolumnindicatescellularcomponent,andthebluecolumnindicatesmolecularfunction.Y-axisontheleft:number ofgenes(microRNAs) F I G U R E 7 Pathway analysis of differentiallyexpressedmicroRNAs. Pathway analysis is a functional analysis mapping genes to KEGG pathways and other pathway databases. The lower the P value, the more significant the pathway association

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from thecorrespondingauthoruponrequest.