| [Background] Acute Stanford type A dissecting aneurysm is a kind of life-threatening cardiovascular disease, which is with a higher morbidity and a higher mortality rate. The statistical data shows that in the first 48 hours after the acute Stanford type A dissecting aneurysm happens, the mortality rate reaches 50%-68%. With the time passing by, the mortality rate will continuously increase by average 1% per hour. Without timely treatment, approximately 90% of the patients will die in 3 months. However, the etiological mechanism of the acute aortic dissection is very complex, which involves many capital factors, while the specific etiological mechanism is not clear yet. Nowadays the rapid development of molecular biological techniques and the appearance of biochip technology supply powerful methods to reveal the pathogenesis of the disease at the genetic level, promoting medical science to transit from the "system, blood vessel, tissue and cell levels" (phase 2 medical science) to the "DNA, RNA, protein, and their interactions level" (phase 3 medical science). This research is aimed to discuss the pathogenesis of acute Stanford type A aortic dissecting aneurysm at the LncRNA and mRNA expression levels.PART I:The Blood Vessels Structural Characteristics of Stanford A Type Aortic Dissection[Objective] To investigate the blood vessels structural characteristics of Stanford A type aortic dissection and normal human ascending aorta.[Methods] HE staining and Vigesion staining were included to investigate the blood vessels structural characteristics of Stanford A type aortic dissection and normal human aorta. Five Stanford A type aortic dissection tissues and five normal ascending aorta tissues were included in the study.[Results] HE and VG staining show that the vascular wall of normal ascending aortae consists of intima, tunica media and adventitia, the elastic fiber and collagen fiber are compact and in order. Stanford A type aortic dissection ascending aortae usually do not have complete there layers of intima, tunica media and adventitia in gross observation. HE and VG staining show conspicuous collapse of elastic and collagen fibers,thinner, inflammatory cells infitration and blood cells.[Conclusions] Compared with normal ascending aortae, Stanford A type aortic dissection ascending aortae show conspicuous collapse of elastic and collagen fibers, thinner, and inflammatory cells infitration.PARTⅡ:Research of the LncRNA/mRNA expression spectrum in the Stanford type A aortic dissection [Objective] The experiment of this part is to analyze the LncRNA and mRNA expression levels in the human acute Stanford type A dissecting aneurysms and normal aortic vascular vessel wall tissues with the Arraystar human LncRNA/mRNA expression spectrum biochip technique. [Methods] Each 5 vascular vessel Wall tissue specimens were taken from the specimen library of the ascending aortic vascular tissues of patients with acute Stanford type A dissecting aneurysms who accepted operative treatments in our hospital and the specimen library of normal ascending aortic vascular tissues of donor hearts used in heart transplant operations separately.NanoDrop ND-1000 was used to test the RNA expression levels in the 10 specimens incorporated in the experiment, and the quality of the specimens was ensured to comply with the requirements of the biochip test experiment.Arraystar Human LncRNA/mRNA V3.0 expression spectrum biochips(8 x 60K,Arraystar)were used to test the LncRNA and mRNA expression levels in the tissue specimens. Agilent Feature Extraction(version 11.O.1.1) software was used to extract the information Contained in the microarray images obtained. GeneSpring GX(Agilent Technologies, v12.0)software was used to further screen the obtained original expression information of LncRNA and mRNA. [Results](1)The 10 selected specimens of the ascending aortic vascular tvessel wall issues of patients with acute Stanford type A dissecting aneurysms and normal ascending aortic vascular vessel wall tissues all complied with the requirements of LnGRNA/mRNA expression spectrum biochips.The RNA OD260/280 ratios in the 5 type A aortic dissection vascular vessel wall tissues(TAAD 1,TAAD 2,TAAD 3, TAAD 4,TAAD 5)were 1.91,1.89,1.92,1.84 and 1.91 respectively,and those in the 5 normal ascending aortic vascular tissue specimens(N1,N2,N3,N4,N5)were 1.91, 1.92,1.88,1.91, and 1.91 respectively.All the OD 260/280 ratios were greater than 1.8 and between 1.8 and 2.1,meeting the experimental requirements.Cy 3 fluorescent;dyes were used to mark the RNA in the tissue specimens, and the specific mark efficiencies of TAAD1, TAAD2, TAAD3, TAAD4 and TAAD5 were 21.1,21.4, 21.8,22.2 and 21.3 respectively, and those of N1,N2, N3, N4 and N5 were 21.4,21.6, 20.5,20.4 and 23.6 respectively, they all meeting the experimental requirements. (2) The LncRNA/mRNA expression spectrum biochip initial data results after filtering low expression messages and standardizing showed that:in the above-mentioned tissue specimens, the total number of detectable LncRNAs was 29,198, and the detectable mRNA target genes were 22,959 in total. The quality assessment of standard data showed that:the Box-plot analyzed the average expression level of IncRNAs and mRNAs in each group was in very closely level, and about 50% data met high consistency. Scatter diagram indicated that there were a large number of differentially expressed(up or down) LncRNAs and mRNAs in the aortic dissection tissues comparing with normal aortic tissues. [Conclusions] The results of this part research showed our specimens of the ascending aortic vascular tvessel wall issues of patients with acute Stanford type A dissecting aneurysms and normal ascending aortic vascular vessel wall tissues all complied with the requirements of LncRNA/mRNA expression spectrum biochips. The specimens got high consistency within group and between groups. There were a large number of differentially expressed (up and down)LncRNAs and mRNAs in the aortic tissues of the patients with sporadic acute Stanford type A aortic dissecting aneurysms without clear etiological mechanisms (such as, Marfan syndrome, acute trauma, etc). The LncRNA could regulate the genes at epigenetic level, transcriptional level and post-transcriptional level, while the differentially expressed mRNA genes could participate in the causing the abnormities in the structure of aortic vascular vessel walls.dyes were used to mark the RNA in the tissue specimens, and the specific mark efficiencies of TAAD1, TAAD2, TAAD3, TAAD4 and TAAD5 were 21.1,21.4, 21.8,22.2 and 21.3 respectively, and those of N1,N2, N3, N4 and N5 were 21.4,21.6, 20.5,20.4 and 23.6 respectively, they all meeting the experimental requirements. (2) The LncRNA/mRNA expression spectrum biochip initial data results after filtering low expression messages and standardizing showed that:in the above-mentioned tissue specimens, the total number of detectable LncRNAs was 29,198, and the detectable mRNA target genes were 22,959 in total. The quality assessment of standard data showed that:the Box-plot analyzed the average expression level of IncRNAs and mRNAs in each group was in very closely level, and about 50% data met high consistency. Scatter diagram indicated that there were a large number of differentially expressed(up or down) LncRNAs and mRNAs in the aortic dissection tissues comparing with normal aortic tissues. [Conclusions] The results of this part research showed our specimens of the ascending aortic vascular tvessel wall issues of patients with acute Stanford type A dissecting aneurysms and normal ascending aortic vascular vessel wall tissues all complied with the requirements of LncRNA/mRNA expression spectrum biochips. The specimens got high consistency within group and between groups. There were a large number of differentially expressed (up and down)LncRNAs and mRNAs in the aortic tissues of the patients with sporadic acute Stanford type A aortic dissecting aneurysms without clear etiological mechanisms (such as, Marfan syndrome, acute trauma, etc). The LncRNA could regulate the genes at epigenetic level, transcriptional level and post-transcriptional level, while the differentially expressed mRNA genes could participate in the causing the abnormities in the structure of aortic vascular vessel walls.PART Ⅲ:Bioinformatics analysis of the LncRNA/mRNA expression spectrum in the Stanford type A aortic dissection [Objective] We intended to use bio-informatics methods to further analyze the LncRNAs and mRNAs which were differentially expressed in the LncRNA/mRNA expression spectrum chips of acute Stanford type A dissection vascular vessel wall tissues, and to discuss the molecular mechanism of the pathogenesis of acute Stanford type A dissection. [Methods] (1) Two Class Diff method was used to select the different IncRNAs and mRNAs. Volcano Plots and hierarchical cluster diagram showed the expression of those different genes in the acute Stanford type A dissection vascular vessel wall tissues. (2) GenomeStudio Gene Expression Module v1.0 software and gene enrichment analysis method were used to further analyze the differentially expressed mRNAs. Gene ontologyical analysis was made for the differential genes that were screened out, including biological process (BP), cellular component (CC) and molecular function (MF). KEGG database was used to analyze the biological signaling pathways of the differentially expressed genes. (3) Agilent GeneSpring GX v12.0 software and gene enrichment analysis method were used to further analyze the differentially expressed LncRNAs. Home-made scripts were used to make classification and subgroup analysis of the LncRNAs detected by the chips. [Results] (1) Volcano Plots was used to analyze the detected LncRNAs, and the number of obtained differentially expressed genes was 1791, of which the genes whose expressions were up-regulated by TAAD Vs N added up to 636, while those whose expressions were down-regulated by TAAD Vs N added up to 1155; Volcano Plots was used to analyze the detected mRNA, and the number of obtained differentially expressed genes was 2057, of which the genes whose expressions were up-regulated by TAAD Vs N added up to 1103, while those whose expressions were down-regulated by TAAD Vs N added up to 954. (2) According to the gene ontological analysis results, among the mRNAs genes which were up-regulated by;TAAD Vs N, the number of the genes participating in biological processes (immune responses, inflammatory reactions, etc) was 800, the number of the genes participating in encoding cell components was 78, and the number of the genes participating in exercising the cellular functions was 113; while among the mRNAs genes which were down-regulated by TAAD Vs N, the number of the genes participating in biological processes (immune responses, inflammatory reactions, etc) was 531, the number of the genes participating in encoding cell components was 73 and tthe number of the genes participating in exercising the cellular functions was 77. The different geng message pathway analysis found that the up-regulated expressed mRNAs participated in 52 message pathway in TAAD group and the down-regulated expressed mRNAs participated in 32 message pathway in TAAD group. The enrichment score showed the abnormities of genes at transcriptional level appeared in the adhesion plaque pathway of VSMCs(vascular smooth muscle cells), smooth muscle contraction-related pathways, actin cytoskeleton-related pathways and extracellular matrix receptor interaction pathways. And these pathways were closely related with aortic dissection.(3) The LncRNA/mRNA expression spectrum chip results showed that:In the above-mentioned tissue specimens, the LncRNAs detected were 29,198 in total. The subgroup analysis showed that:the LncRNAs playing the roles of enhancers were 1,781, the number of encoding genes around enhancers was 81, the LncRNAs with HOX cluster sequences were 108, the number of LincRNAs was 11,885, and the number of encoding genes near LincRNAs was 459. The number of LncRNAs that have different expressions in Stanford type A aortic dissection tissues (TAAD) and in normal aortic tissues (N) was 1791, of which the genes whose expressions were up-regulated by TAAD Vs N added up to 636, while those whose expressions were down-regulated by TAAD Vs N added up to 1155. [Conclusions] The gene ontological analysis showed that many genes in the Stanford type A aortic dissection tissues participated in the cell biological functions, the exertion of functions of cell components and molecules and other processes by their up-regulated or down-regulated expressions. In addition, the biological pathway;analysis found that the abnormities of genes at transcriptional level appeared in the adhesion plaque pathway of VSMCs(vascular smooth muscle cells), smooth muscle contraction-related pathways, actin cytoskeleton-related pathways and extracellular matrix receptor interaction pathways. The association analysis of LncRNAs and gene expressions prompted that the LncRNAs participated in the gene epigenetic modifications, regulations at transcription level and post-transcription level, and played important regulating effects on the expressions of the genes. Our results prompted that the LncRNA-mRNA-gene expression network participated in the regulating of the exertion of the functions of vascular smooth tmuscle cells in the aortic tunica media. The dysfunction of smooth muscle cells could be the key in the occurrence and development of acute aortic dissections.;analysis found that the abnormities of genes at transcriptional level appeared in the adhesion plaque pathway of VSMCs(vascular smooth muscle cells), smooth muscle contraction-related pathways, actin cytoskeleton-related pathways and extracellular matrix receptor interaction pathways. The association analysis of LncRNAs and gene expressions prompted that the LncRNAs participated in the gene epigenetic modifications, regulations at transcription level and post-transcription level, and played important regulating effects on the expressions of the genes. Our results prompted that the LncRNA-mRNA-gene expression network participated in the regulating of the exertion of the functions of vascular smooth tmuscle cells in the aortic tunica media. The dysfunction of smooth muscle cells could be the key in the occurrence and development of acute aortic dissections.PART Ⅳ:Correlation analysis and verification of key different expressed LncRNAs and mRNAs in Stanford Type A aortic dissection [Objective] Some key different expressed LncRNAs and mRNAs in Stanford Type A aortic dissecting aneurysm were taken correlation analysis and verification in this part of research. [Methods] LncRNAs and mRNAs were screened on this basis of 4 signal pathways associated with Stanford Type A aortic dissecting aneurysm, including pathways involved in interaction between cytokines and cytokine receptor, vascular smooth muscle contraction, cortical actin cytoskeleton and adhesion plaque. The expression level of selected lncRNAs and mRNAs were verified by real-time quantitative PCR. Using CNC Network Map to establish the relationship between LncRNA and mRNA, and build the genetic regulatory network. [Results] 23 differentially expressed mRNAs and lncRNAs were found involved in pathways associated with interaction between cytokines and cytokine receptor.31 differentially expressed mRNAs and lncRNAs were found involved in pathways associated with vascular smooth muscle contraction.46 differentially expressed mRNAs and lncRNAs were found involved in pathways associated with development of cortical actin cytoskeleton. And 37 differentially expressed mRNAs and lncRNAs were found involved in pathways associated with development of adhesion plaque. The genes that we were interested in were TNFRSF10A, IL6, IFNGR2, MYLK, ACTA2, CAV1 and COL4A3. The results of Q-PCR showed highly consistence with that of biochips.CNC Network Map analysis showed the role of differentially expressed mRNAs and lncRNAs in the function of vascular smooth muscle cells. [Conclusions] The results of Q-PCR showed highly consistence with that of biochips. Position correlation analysis and CNC Network Map analysis showed the role of differentially expressed mRNAs and lncRNAs in the function of vascular smooth muscle cells, since that the dysfunction of smooth muscle cells was supposed to be of the most important factor in the occurrence and development of acute Stanford Type A;aortic dissection.According to the position relationship and the expression level relevance of IncRNAs and mRNAs, we selected IL-6,LOC541472(uc010kun.2). COL4A3,AC097662.2(ENST00000396588),MYLK,MYLK-AS1(ENST0000048516 2), CAV1 and AC006159.5(ENST00000452009) to further functional analysis.;aortic dissection.According to the position relationship and the expression level relevance of IncRNAs and mRNAs, we selected IL-6,LOC541472(uc010kun.2). COL4A3,AC097662.2(ENST00000396588),MYLK,MYLK-AS1(ENST0000048516 2), CAV1 and AC006159.5(ENST00000452009) to further functional analysis. |
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