| Part IBackground and Objective:Cardiovascular diseases(CVD) are still a prevailing cause of death worldwide. The leading causes of the majority of CVD are atherosclerosis(AS) and its thr ombotic complications. In the heart, AS causes coronary artery disease(CAD). The current therapeutic strategies for CAD, including lowering plasma low-density lipoprotein(LDL), antiplatelet agents and/or anticoagulants following revascularization therapy, such as, percutaneous coronary intervention(PCI) and coronary artery bypass surgery(CABG), have proven effective, but the mortality has remained dismal. Early diagnosis of CAD may assist early interventions and improve the outcome. So an early identifi cation of patients with CAD remains an emerging need。Over the past few decades, developments in genome-wide analyses has identified that almost all of the human genome is transcribed and produces a large number of long non-coding RNAs(lnc RNAs). Lnc RNAs, ranging from 200 to over 10,000 nucleotides, lack the coding ability for protein. Many lnc RNAs have been shown to be functional and are involved in specific physiological and pathological processes through epigenetics and transcriptional or post-transcriptional regulatory mechanisms. Recently, several studies have shown that lnc RNAs are involved in the development of various types of pathological conditions, including cancers, autoimmune diseases, and neurological disorders. Some lnc RNAs are also involved in the development of cardiovascular diseases, including heart failure, cardiac hypertrophy, cardiometabolic diseases, myocardial infarction and AS. Moreover, lnc RNAs have been used as biomarkers for many diseases. For example, a prostate specific linc RNA P CA3 in urine is assayed for the detection of prostate cancer. Other biomarkers include: circulating H19 for gastric cancer, lnc RNA HULC in hepatocellular carcinoma, and lnc RNA LIPCAR in heart failure after myocardial infarction. We need to determine whethe r or not circulating lnc RNAs can also work as a specific CAD biomarker.CAD is considered as a chronic inflammatory disease that results in the formation of plaques in large and mid-sized arteries. Many studies indicate that the recruited circulating peripheral blood monocyte cells(PBMCs) which constitute 5–10% of peripheral blood leukocytes, play a pivotal role in this event by migrating towards the arterial wall, increasing the size of atherosclerotic lesions hereby leading to CAD event to happen. Therefore, our current study was initiated and designed to address the differences in lnc RNAs of the PBMCs between CAD patients and control subjects, to study its diagnostic value as a biomarker of CADs.Methods:Chapter I:The study recruited 502 patients admitted to the Department of Cardiology, Daping Hospital(Chongqing, China) from February 2013 to May 2014, for clinically diagnosed or suspected CAD, and were subsequently analyzed. Their clinical and demographic features were the primary parameters used to assess their risk of CAD. Lnc RNA were screened by global transcriptome profiling of monocytes and plasma based on Arraystar, Human Lnc RNA array, version 2.0 gene chip and verified by quantitative PCR(q PCR) in the same monocyte samples(15 male CAD patients and 15 male controls, respectively). And we examined the expression of lnc RNAs by q PCR, and ROC analyses in another cohort(20 CAD patients and 20 control patients). For further confirmation of Coro Marker expression and investigation of the potential rel ationship between Coro Marker expression and the risk of CAD, we conducted a case-control study. The cohort consisted of 211 cases(CAD patients) and 171 controls(control subjects), who were divided into the training(161cases and 121 controls) and test(5 0 cases and 50 controls) sets by sampling randomly with uniform distribution each time, and repeated for 100 times, then constructed a diagnostic model by Fisher criteria according to the training set, and validated in the test set. Further, performed ROC curve and risk factors analysis on the corhorts(case-control study cohort, training set and test set), also tested the Coro Marker levels in PBMCs from patients with other cardiovascular diseases. Lastly, the diagnostic value of the lnc RNA was assessed in another independent cohort of 50 CAD patients and 40 control subjects, respectively.Chapter II:According to the results of above gene chip from monocytes, we screened the different genes, and performed GO analysis, Pathway analysis, lnc RNA-m RNA coexpression net work, and functional enrichment analysis of genes correlated with the signature of Coro Marker. Human THP-1 cells were transfected using Nucleofector? Technology(Lonza group Ltd, Basel, Switzerland) with Coro Marker-specific si RNA(100n M) or non-silencing si RNA(100n M) for negative control. The efficiency of si RNA transfection was tested by q PCR. The function of Coro Marker in cell proliferation was investigated using Cell Counting Kit-8 detection. Culture medium was collected after transfection for 48 h and analyzed the concentration of IL-1β, IL-6 and tumor necrosis factor(TNF)α using an enzyme-linked immunosorbent assay(ELISA) kit(Boster, Wuhan, China) following the manufacturer’s instructions, respectively.Results:Chapter I:The baseline characteristics of the microarray cohort of subjects differed significantly only in their antiplatelet therapy between the two groups. According to the arraystar, only 5 lnc RNAs were successfully screened among the 51 up-regulated lnc RNAs with general agreement between these PBMCs and plasma sources of lnc-RNA, they were AC100865.1, IL21R-AS1, BAT5, AC107016.1 and RP11-203B9.4. Then, validated by q PCR in the PBMCs of the microarray cohort, it demonstrated an approximate 3.013-fold increase for AC100865.1, a 2.524-fold increase for IL21R-AS1 and a 2.217-fold increase for BAT5 expression in CAD patients compared to control subjects, while no significant difference for AC107016.1 and RP11-203B9.4 between the two groups. To further independently validate the expressions of AC100865.1, IL21RAS1 and BAT5 in a second set of PBMC samples by q PCR from 20 CAD patients and 20 controls, respectively, the results showed an around 2.807-fold increase for AC100865.1, a 2.212-fold increase for IL21R-AS1, and a 2.007-fold increase for BAT5 expression in CAD patients as compared to the controls. Also, ROC curve analysis was performed in the same population and the AUC was 0.940 for AC100865.1, 0.825 for IL21R-AS1 and only 0.818 for BAT5, respectively. Taken together, these results indicated that AC100865.1 in PBMCs might be a good candidate biomarker to predict CAD in patients. The diagnostic accuracy of Coro Marker was analized in a larger cohort(211 CAD cases and 171 controls). According to the diagnostic model constructed by Fisher criteria, the optimal discriminant function, which corresponded to the 15 th training set, as the final discriminant factor, was defined as follow: f =-3.052 x + 4.649. Where ―x‖ denotes the expression level of Coro Marker and a patient was classified as ?CAD‘ if x > =1.523, or as ?contrl‘ if x < 1.523. The characteristics of these samples differed significantly in age, arterial hypertension, AT1 receptor blocker, calcium channel blocker, beta-blocker use, and antiplatelet therapy, and patients who are current smokers, former smokers and never smoked, respectively, but not in other clinical and pathological f actors. Then validated in the test set, repeated 100 times, the average correction classification number was 32 and 48 in cases and controls, respectively. The corresponding average sensitivity and specificity were 64% and 80%, respectively. And for the 100 models, the average diagnostic accuracy was 0.81, the best and the worst one was 0.89 and 0.74, respectively. The average diagnostic accuracy for CAD group and control group was 0.65 and 0.97, respectively, also the best one was 0.82 and 1, the worst one was 0.5 and 0.9, respectively. The optimal correction classification number(15th time) was 40 and 49 in cases and controls, respectively. The corresponding optimal sensitivity and specificity was 80% and 98%, respectively. The total diagnostic accuracy w as 0.89, and in CAD and control group it was 0.80 and 0.98, respectively. Meanwhile, the AUC was 0.920, 95% CI(0.892-0.947) for the original set, 0.905, 95% CI(0.869-0.940) for the training set and 0.960, 95% CI(0.923-0.997) for test set, respectively. In a prospective study, we found the sensitivity and specificity of Coro Marker were 76% and 92.5%, respectively, and its correction classification number was 38 and 37 in cases and controls, respectively. And it was independence of Coro Marker on CAD risk factors and other cardiovascular diseases but Coro Marker was a risk factor for CAD.Chapter II:According to the PBMC arraystar, 556 different m RNAs were screened. By GO analysis, It indicated that the upregulated genes in CAD group were mainly involv ed in the signal transduction, small molecule metabolic process, transmembrane transport and other cell physiological function, while the downregulated genes were clustered significantly in small molecule metabolic process, inflammatory response, lipopolysaccharide-mediated signaling pathway and lipid metabolic process, and so on. And the results from pathway analysis showed that the upregulated genes in CAD group were mainly taking part in Jak-STAT signaling pathway, vascμlar smooth muscle contraction, leukocyte transendothelial migration, metabolic pathways and other cell signal transduction pathway. And the downregulation genes were largely involved in rheumatoid arthritis, chemokine signaling pathway, Toll-like receptor signaling pathway, antigen processing and presentation, metabolic pathways, apoptosis and others. Then, the correlation between the PBMC Coro Marker and m RNAs was studied from the same microarray, it demonstrated that 35 protein coding gene expressions were positively correlated(Pearson correlation coefficient ≥ 0.990 and FDR ≤ 7.61E-05) with Coro Marker, while 33 genes were negative with Coro Marker(Pearson correlation coefficient <-0.900, FDR ≤9.83E-05). Those positively correlated genes were clustered most significantly in small molecule metabolic and signal transduction processes in the GO biological process enrichment analysis. While the negatively correlated genes were clustered with innate immune response, cytokine-mediated signaling and apoptotic process-mediated signaling pathways. What‘s more, no significant changes in levels of pro-inflammatory cytokines IL-1β, IL-6 and TNFα in THP-1 culture medium samples were observed in the negative control groups as compared with the control groups, however, in the transfection with Coro Marker-specific si RNA groups these cytokine levels in culture medium were reduced significantly. Also si RNA suppression of Coro Marker in THP-1 cell lines reduced cell viability significantly.Conclusion:In this study,(1), The high expression level of lnc RNA(Coro Marker) was identif ied in PBMCs of CAD patients.(2), A novel diagnostic model constructed by Coro Marker predicts CAD patients effectively.(3), Coro Marker plays a pivotal role in the pathogenesis of CAD by pro-inflammation and proliferation in PBMCs.Part IIBackground and purpose:In the first part of the study, the results demonstrated that monocyte Coro Marker could be used as a novel biomarker for early diagnosis of CAD. And Coro Marker pl ayed pro-inflammation and proliferation roles in monocytes in the development of CAD.While we found that high abundance of monocyte Coro Marker did not parallel with that expression in the plasma of CAD patients. It suggests that only a small portion of th e plasma Coro Marker comes from monocytes, and there is another source of Coro Marker. Recent studies show that the arterial endothelial cell is a secretion organ, and plays a vital role in maintaining vascular homeostasis through secretion of a variety of substances. Preliminary results suggest that Coro Marker express highly in HUVEC, then, we reasonably speculate that endothelial dysfunction due to a variety of risk factors, causes endothelial cells secreting lots of Coro Marker into the blood, and finally leads to the development of CAD.The purpose of this study is to investigate the expression of Coro Marker in arterial endothelial cells of CAD patients and its roles in the pathogenesis of CAD and molecular mechanisms.Methods:Chapter I:To separate circulating endothelial cells(CEC) from the whole blood by percoll liquid, and to investigate the different expression of Coro Marker in CEC between CAD and controls by q PCR. Finally, to detect the secretion of inflammatory cytokines IL-1β, IL-6 and TNFα in HUVEC culture supernatants by ELISA.Chapter II:To research Coro Marker localization in endothelial cells(EC) by both Fluorescence in situ hybridization(FISH) and nucleus-cytoplasm separation, to obtain and verify the full-length sequence of Coro Marker through 3 ’ and 5’ RACE and northern blot in ECs. To predict its possible functions with bioinformatics analysis, and to verify it by Dual Luciferase Reporter Gene Assay, to research the role of mir205-5P’s direct target gene PTEN by Wester Blot and q PCR, and to detect the concentration of inflammatory cytokines IL-1β, IL-6 and TNFα in culture supernatants by ELISA.Results:Chapter I:Compared with the CEC from control patients, the expression of Coro Marker on CEC of CAD patients(5.156 ± 0.896), 95% CI(0.565,1.397) was significantly higher than that in controls(0.981 ± 0.176), 95% CI(3.036,7.275)(P < 0.05). What‘s more, in the stable transfection with Coro Marker-specific si RNA group, the concentration of inflammatory cytokines IL-1β(P = 0.0398), IL-6(P = 0.000) and TNFα(P = 0.017) in culture medium samples were reduced significantly(P < 0.05), n = 6. However, no significant changes of inflammatory cytokines IL-1β, IL-6 and TNFα in culture medium samples were observed in the negative control groups as compared with the control groups(P > 0.05).Chapter IIThe results from FISH suggested that Coro Marker labeled with cy3 mainly located in the cytoplasm of HUVEC cells, and the result from nucleus-cytoplasm separation confirmed that the expression of Coro Marker in cytoplasm was 2.5 times than that in the nucleus. On HUVEC cells, through the 3 ’and 5’RACE, respectively, a full-length sequence of Coro Marker was obtained, it was 1394 nt long, and further confirmed by Northern blot. Then, bioinformatics analysis showed Coro Marker could bind directly to micro RNA205-5P, and the results showed that the expression of micro RNA205-5P was reduced significantly in the stable transfection with Coro Marker-specific si RNA group compared with the control group and negative control group(P <0.05), what‘s more, the results from the Dual Luciferase Reporter Gene Assay showed luciferase activity increased 2 times when compared with the control group, which suggested Coro Marker could enhance the expression of mi R-205 significantly. Further, the results suggested that the target gene of micro RNA205-5P, PTEN, upregulated significantly in Coro Marker interference group, while specific si RNA of PTEN could recovery the secretion of inflammatory cytokines IL-1β, IL-6 and TNFα, at least partly.Conclusions:(1), A novel gene, Coro Marker, was identified.(2), Coro Marker mainly distributes in the cytoplasm, and regulates micro RNA205-5P promoter directly.(3), A novel signaling pathway composed of Coro Marker, mi R-205-5p and PTEN, is confirmed, which leads to the pathogenesis of CAD. |
PDF Full Text Request