Study Of Transcriptomics Of Acute Ischemic Stroke With Phlegm Syndrome

ObjectivesPhlegm was one of the main syndromes of acute ischemic stroke,but there was less understanding of the biological mechanism of acute ischemic stroke.And it was of great significance to improve the diagnosis and treatment of the disease by looking for relevant markers of acute ischemic stroke with phlegm syndrome and analyzing the different differences and possible mechanisms.In this study,high-throughput transcriptome sequencing techniques were used to screen microRNAs(miRNAs)and messenger RNA(mRNAs)expression profiles for acute ischemic stroke with phlegm syndrome,and to screen miRNAs and mRNAs that could identify phlegm syndrome of acute ischemic stroke.In this way,the specific genes and molecular mechanisms of acute ischemic stroke were preliminarily understood,and the objectification basis of acute ischemic stroke was provided.Methods1.Five peripheral blood samples of patients with ischemic stroke phlegm syndrome,non-phlegm syndrome and non-stroke control were collected,and the expression profiles of mRNA and miRNA in serum were detected by high-throughput sequencing method,and the differentially expressed genes were analyzed.KEGG and GO gene function enrichment were performed to analyze the possible mechanism of action of differential genes associated with phlegm syndrome evidence in acute ischemic stroke2.Forty patients with acute ischemic stroke(20 patients with phlegm syndrome and 20 non-phlegm syndrome patients)and 20 non-stroke control patients were continuously collected,and the expression of some differential genes screened by high-throughput sequencing was determined in serum by real-time quantitative polymerase chain reaction(qPCR)method,and the expression differences and significance of genes in different syndromes were analyzed.3.A model of acute ischemic stroke with phlegm syndrome was constructed by high-fat diet rat combined with ligation of bilateral common carotid arteries,and the expression levels of some differential genes related to phlegm syndrome in the published literature and some differential gene expressions in clinical sequencing screening and verification were verified by reverse transcription-polymerase chain reaction(RT-PCR)method.Results1.High-throughput sequencing was used to construct differential miRNA and mRNA expression profiles of acute ischemic stroke,phlegm syndrome and non-phlegm syndrome.(1)Compared with phlegm syndrome and non-phlegm syndrome,a total of 2126 differential mRNAs were screened out.The KEGG analysis of the differentially expressed mRNAs of the two were mainly expressed in ECM-receptor interaction,cell cycle,small cell lung cancer,PI3K-Akt signaling pathway,pathways of cancer and other pathways.74 differential miRNAs were screened out.The main differential miRNA KEGG was enriched in Vascular smooth muscle contraction,PI3K-Akt signaling pathway,Antigen processing and presentation,Glutamatergic synapse,Antifolate resistance and other pathways.(2)Compared with the phlegm syndrome and the non-stroke control group,a total of 2078 differential mRNAs were screened out.The KEGG analysis of the differentially expressed mRNAs of the two were mainly expressed in the interaction of ECM-receptor interaction、PI3K-Akt signaling pathway、small cell lung cancer pathway.25 differential miRNAs were screened out.The main differential miRNA KEGG was enriched in cell adhesion molecules(CAM),basal cell carcinoma,and Rapl signaling pathways.(3)Compared with the non-phlegm syndrome and the non-stroke control group,192 differential mRNAs were screened out.The KEGG analysis of the differentially expressed mRNAs of the two were mainly expressed in Arrhythmogenic right ventricular cardiomyopathy、Glutathione metabolism、Pyrimidine metabolism、Arachidonic acid metabolism pathway,etc.29 differential miRNAs were screened out.The main differential miRNAs KEGG were enriched in cGMP-PKG signaling pathway,vascular smooth muscle contraction,and Rapl signaling pathway.2.Inthe quantitative PCR validation of clinical samples,compared phlegm syndrome with with non-stroke control groups,the expression differences in FAM83G,NCAPG2,miR-181a-5p,miR-1287-5p,miR-221-3p,let-7b-5p,miR-196b-5p,miR-93-5p were statistically significant(P<0.05).Compared phlegm syndrome with non-phlegm syndrome of acute ischemic stroke,there were statistically significant differences in the expression of CAMP,NCAPG2,miR-181a-5p,miR-584-5p,miR-221-3p,let-7b-5p,miR-96-5p,miR-196b-Sp and miR-93-5p(P<0.05).3.Compared with the sham surgery group,rats in the phlegm syndrome model group had statistical differences in body weight,triglycerides(TG),and low-density lipoprotein(LDL)(P<0.05).The expression of KLRC2,SPRED1,TRAF6,ARG1,and LY96 mRNA in the peripheral blood of rats in the phlegm syndrome model group was down-regulated,and the expression of MMP9,FAM83G,NCAPG2,sICAM1,TLR4,and TLR8 mRNA was upregulated,and the differences between the two groups were significant(P<0.05);the differences between MGLL,CCR7,VEGFA,EEF1A1,and CAMP mRNA expression groups were not significant and not significant(P>0.05).Conclusions1.There are differential expression profiles of mRNA and miRNA between acute ischemic stroke with phlegm syndrome and non-phlegm syndrome,acute ischemic stroke with phlegm syndrome and non-stroke control.2.Through bioinformatics pathway analysis,phlegm syndrome was related to pathways with abnormal functions such as extracellular matrix receptor interactions and cell adhesion molecules.After the establishment and bioinformatics analysis of miRNA and mRNA expression profiles in patients with acute ischemic stroke with phlegm syndrome,it is helpful to reveal the molecular mechanism of ischemic stroke syndrome from the perspective of transcriptome molecules.3.FAM83G、NCAPG2、miR-1287-5p might be potential candidate markers to differentiate acute ischemic stroke patients with phlegm syndrome and non-stroke control people.CAMP、NCAPG2、miR-584-5p,miR-96-5p might be used as a potential candidate marker for differentiating acute ischemic stroke patients with phlegm syndrome and non-phlegm syndrome.4.In rat models with acute ischemic stroke with phlegm syndrome,NCAPG2,FAM83G,and KLRC2 were likely to be potential biomarkers for phlegm syndrome of acute ischemic stroke.