The Effects And Mechanisms Of Hsa＿circ＿0000284 On Coronary Heart Disease

Chapter 1Expression profiles of circRNA in peripheral blood leukocytes from patients with CHDBackground Coronary heart disease（CHD）is one of the leading causes of death in humans.Understanding the causes of coronary artery disease is important for early detection and treatment and thus lowers mortality for this disorder.Many studies have indicated that circRNAs are related to the pathogenesis of coronary artery disease.Very recently,hsa＿circ＿0000284 and hsa＿circ＿0001445 have been found to be associated with the development of CHD.However,there are no reports on their roles in the pathogenesis and development of CHD.Objectives To evaluate circRNA expression in human peripheral blood leukocytes（PBLs）from CHD patients and analyze its association with clinical parameters in an attempt to explore the roles of circRNA in the pathogenesis of CHD.Methods This study collected 126 blood samples from healthy humans and 94 blood samples from CHD patients,including a healthy group aged 60.75±8.81 years and a CHD group aged 65.96±9.78 years.RNA was extracted from PBLs,and q RT-PCR was used to analyze the expression of hsa＿circ＿0000284 and hsa＿circ＿0001445 in PBLs between the two research groups.At the same time,clinical parameters were collected,and T-test,Spearman,Pearson analysis,logistic regression analysis,and receiver operator characteristic curve（ROC）for correlation analysis were used to evaluate the association of these parameters with the level of hsa＿circ＿0000284 and hsa＿circ＿0001445 expression.Results（1）The study found downregulated expression of hsa＿circ＿0000284 and hsa＿circ＿0001445 in human peripheral blood leukocytes in CHD patients compared with normal controls,[0.161±0.059 and 0.218±0.079]and[0.640±0.254 and 1.079±0.453],respectively.This difference was statistically significant（P<0.001）.（2）Analyzing the correlation between hsa＿circ＿0000284 and hsa＿circ＿0001445 expression and clinical parameters,they correlated with age[（r=-0.258,P<0.001）and（r=-0.290,P<0.001）],number of neutrophils[（r=-0.185,P<0.05）and（r=-0.137,P<0.05）],hemoglobin concentration in erythrocytes[（r=0.159,P<0.05）and（r=0.181,P<0.05）],blood TG[（r=0.283,P<0.001）and（r=0.249,P<0.001）],HDL-C[（r=0.292,P<0.001）and（r=0.284,P<0.001）]and LDL-C[（r=0.305,P<0.001）and（r=0.293,P<0.001）].（3）Analyzing logistic regression,the expression of hsa＿circ＿0000284 and hsa＿circ＿0001445 act as an independent influencing factors for CHD（P<0.05）.（4）Analyzing the ROC curve,hsa＿circ＿0000284 showed a sensitivity of 64.3%and a specificity of 66%,and hsa＿circ＿0001445 had a sensitivity of 67.5%and a specificity of 76.6%.Conclusion Downregulation of hsa＿circ＿0000284 and hsa＿circ＿0001445are linked to the development of CHD,which warrants further investigation.Chapter 2Construction of the hsa＿circ＿0000284/miRNA/mRNA interaction networkBackground The development of genome sequencing technology has greatly affected the field of medical research.The available high-throughput sequencing data have been increasing exponentially.Therefore,the use of many online databases to analyze data in research is receiving great concern.Based on the results from chapter one,especially the close relationship of hsa＿circ＿0000284 with CHD,in this chapter,we used a bioinformatics algorithm to predict the function of hsa＿circ＿0000284 in the interaction network of hsa＿circ＿0000284/miRNA/mRNA in an attempt to understand the potential roles of hsa＿circ＿0000284 in the pathogenesis and development of CHD.Objectives To predict the hsa＿circ＿0000284/miRNA/mRNA interaction network in CHD.To analyze the potential role of hsa＿circ＿0000284 in CHD.Methods（1）Via the circ Bank and circ Interactome online databases,we predicted the miRNA expression and function of hsa＿circ＿0000284.At the same time,from the GEO database,the GSE61741 dataset related to miRNA differential expression was searched in CHD and analyzed by using R software.Then,the miRNA similarity of the 3 datasets was analyzed.（2）Through online databases and miRTar Base,miRWalk2 predicted the regulatory function of miRNA/mRNA.Simultaneously,from the GEO database,the GSE56885 dataset related to mRNA expression in CHD was searched and analyzed by using R software.Then,the mRNA homogeneity of the 3 datasets was analyzed.（3）Cytoscape software was used to predict the interactive network hsa＿circ＿0000284/miRNA/mRNA in CHD.（4）R software was used to analyze the biological information of hsa＿circ＿0000284 in GO and KEGG.（5）R software was used to predict the protein interaction network.Results（1）Through predictions from an online miRNA database and analysis of miRNA datasets in CHD,hsa＿circ＿0000284 was linked to 15miRNAs,including hsa-miR-382-5p,hsa-miR-508-3p,hsa-miR-1283,hsa-miR-338-3p,hsa-miR-653-5p,hsa-miR-1178-3p,hsa-miR-1250-5p,hsa-miR-558,hsa-miR-637,hsa-miR-1290,hsa-miR-1825,hsa-miR-330-5p,hsa-miR-375-3p,hsa-miR-199b-5p,and hsa-miR-302d-5p.（2）Through prediction from the online database of mRNA and analysis of mRNA datasets in CHD,15 miRNAs can regulate 219 mRNAs.（3）The network interaction hsa＿circ＿0000284/miRNA/mRNA in CHD was predicted.（4）The results showed 276 terms in biological processes,66 terms for cell composition and 74terms for molecular function of the GO terms.The results of KEGG mainly focused on the cell aging pathway hsa04218＿Con,the signaling pathway hsa04068＿Fox O,longevity regulating pathway-multiple species,and the longevity regulator hsa04211＿Con.The protein-protein interaction network achieved 3 modules.Conclusion The interaction network of hsa＿circ＿0000284/miRNA/mRNA was constructed with the genes that associated mainly with the regulation of aging,longevity pathways and cellular processes,further suggesting their roles in the development of CHD.Chapter 3Expression of hsa＿circ＿0000284 in human umbilical vein endothelial cells under oxidative stress and inflammatory injuryBackground Endothelial cells play an important role in the regulation of the homeostasis of blood vessels.EA-hy926 is a human umbilical vein cell fusion line that is widely used in vascular-related research.Objectives Using hydrogen peroxide（H₂O₂）and the inflammatory factor TNF-αto stimulate human umbilical vein endothelial cells to establish model cells of inflammatory damage and oxidative stress in vitro,the expression of hsa＿circ＿0000284 was measured,and its potential roles in the processes of oxidative stress and inflammatory insult were evaluated.At the same time,the factors that change the expression of hsa＿circ＿0000284 are discussed.Methods（1）EA-hy926 cells were treated with 400μmol/L H₂O₂,800μmol/L H₂O₂,1000μmol/L H₂O₂ and,100 ng/m L TNF-α,and 200 ng/m L TNF-αfor 1,2,6,10 and 14 hours.Cellular morphological changes were observed carefully by inverted microscopy.CCK-8 method was used to detect cell survival rate,ROS Assay Kit detects cell reactive oxygen species（ROS）,β-galactosidase staining method evaluates cell senescence rate,cell cycle.（2）RNA was extracted,and q RT-PCR was applied to evaluate hsa＿circ＿0000284expression in inflammatory and oxidative stress damage models.（3）CRISPR/Cas9 technology was used to study hsa＿circ＿0000284 expression changes.Results（1）EA-hy926 cells were compromised after stimulation with H₂O₂ for different periods of time.The cell survival rate decreased significantly,and the survival rate of the cells decreased gradually over the prolonged treatment period;the cell structure changed abnormally.When EA-hy926 cells were stimulated with TNF-αfor different periods of time,the cell structure did not appear to change,and the increased rate of cell death with time of stimulation was not as significant as that in the H₂O₂ model.H₂O₂ caused cell oxidative stress at 1 h,2 h,6 h,10 h,and 14 h,cell viability was significantly reduced compared with untreated group（P<0.05）and significantly increased the level of ROS in the cells in each group of EA-hy926 cells at 2 h,6 h,10 h and 14 h.As the concentration of H₂O₂ increased,the level of intracellular ROS increased gradually,and the difference in ROS levels was statistically significant.Significance（P<0.05）.When TNF-αwas used to stimulate EA-hy926 cells at10 h and 14 h,cell viability was significantly reduced compared with untreated group（P<0.05）.TNF-αstimulated cellular inflammatory factors,which significantly increased the level of intracellular ROS at 2 h,6 h,10 h,and 14 h,and the difference in ROS levels was statistically significant（P<0.05）.Treating cells with H₂O₂（1000μmol/L）and TNF-α（200 ng/m L）for 6 hours,H₂O₂increased in the S phase components of the cell cycle（P<0.05）and TNF-αdecreased in the S phase components of the cell cycle（P<0.05）.The rate of senescent cells in the H₂O₂ and TNF-αcell groups was significantly increased（P<0.05）.（2）hsa＿circ＿0000284 expression increased in both groups after 2 hours of stimulation,and this difference was statistically significant（P<0.05）.Then,it decreased sharply at 6 h,and there was a statistically significant difference compared to 2 h.hsa＿circ＿0000284 expression increased after 14 hours of stimulation,and the difference was statistically significant（P<0.001）.（3）The hsa＿circ＿0000284 expression level in EA-hy926 cells was significantly reduced after the Alu Sq2 element was knocked out.Conclusion Oxidative stress and inflammatory injury lead to damage to endothelial cells and changed expression of hsa＿circ＿0000284,suggesting the potential roles of this circRNA in the pathogenesis of CHD.At the same time,the Alu Sq2 element is a regulator of hsa＿circ＿0000284 expression.Chapter 4The influence of hsa＿circ＿0000284 on cell biological functionsBackground Gene overexpression and silencing are common biological techniques to test the function of a given gene.In this chapter,lentivirus transfection technology was used to overexpress and silence hsa＿circ＿0000284in EA-hy926 cells to speculate the possible roles of this circRNA in the pathogenesis of CHD.Objectives To explore the effects and related mechanisms of hsa＿circ＿0000284 overexpression and silencing in EA-hy926 cells.Methods（1）Culture of EA-hy926 cells;lentivirus was used as a vector for stable silencing and overexpression of hsa＿circ＿0000284;72 hours after transmission,hsa＿circ＿0000284 expression was observed in cells under an inverted fluorescence microscope and verified by q RT-PCR assay.（2）CCK-8was performed to detect cell proliferation,flow cytometry to detect the cell cycle and apoptosis,β-galactosidase staining method evaluates cell senescence rate and to speculate about the role of hsa＿circ＿0000284 in the biological process of EA-hy926 cells.Results After the overexpression of hsa＿circ＿0000284 in EA-hy926cells,the expression of hsa＿circ＿0000284 was significantly increased（P<0.001）.After the silencing of hsa＿circ＿0000284 in cells,the expression of hsa＿circ＿0000284 was significantly decreased（P<0.05）.The CCK-8experiment showed that the proliferation rate of EA-hy926 cells in the circ＿0000284 overexpression group was significantly higher than that in the control group,and this difference was statistically significant（P<0.001）.The proliferation of EA-hy926 cells in the hsa＿circ＿0000284 silencing group（Sh RNA-circle＿0000284）was decreased（P<0.05）.The cell flow meter was used to detect the effect of hsa＿circ＿0000284 on the cell cycle distribution of EA-hy926:Compared with the control group,the ratio of cells in the G0/G1phase of EA-hy926 decreased in hsa＿circ＿0000284 overexpression group,and the ratio of the S phase increased;the S phase cells is reduced in hsa＿circ＿0000284 silence group（P<0.05）.For the flow cytometric method,it was found that overexpression of hsa＿circ＿0000284 can reduce the premature and delayed apoptosis of EA-hy926 cells,thus affecting cell apoptosis of EA-hy926.The difference was statistically significant（P<0.05）;silencing hsa＿circ＿0000284 had no effect on EA-hy926 apoptosis.At the same time,the rate of senescent cells in silencing hsa＿circ＿0000284 cell groups was significantly increased（P<0.05）.Conclusion Upregulation of hsa＿circ＿0000284 expression significantly promoted the proliferation of EA-hy926 cells and influenced the cell cycle distribution and apoptosis of EA-hy926 cells.The rate of senescent cells in silencing hsa＿circ＿0000284 cell groups was significantly increased.These results imply that the downregulation of hsa＿circ＿0000284 in CHD patients may lead to damaged vascular endothelial cells and apoptosis,thereby promoting the development of CHD.Chapter 5hsa circ 0000284 targets ETS1 by regulating miR-338-3pBackground Bioinformatics analyses indicate that hsa＿circ＿0000284 is closely related to the hsa-miR-338-3p and ETS1 genes.Objective To explore the expression levels of hsa-miR-338-3p and ETS1in patients with CHD and HUVECs to further understand the association among hsa＿circ＿0000284,hsa-miR-338-3p and ETS1 and their roles in the development of CHD.Methods（1）q RT-PCR was used to analyze the expression level of hsa-miR-338-3p in peripheral blood leukocytes between the two study groups.（2）The Pearson correlation method was used to analyze the expression levels of hsa＿circ＿0000284 and hsa-miR-338-3p.（3）We used overexpression and silencing of hsa＿circ＿0000284 in EA-hy926 cells to verify the change in hsa-miR-338-3p expression.（4）FISH technology was used to explore the location of hsa＿circ＿0000284 and hsa-miR-338-3p in the cell.（5）A dual luciferase assay verified the interaction between hsa＿circ＿0000284 and hsa-miR-338-3p.（6）Analyzing the expression of ETS1 in peripheral blood leukocytes and correlation analysis with hsa-miR-338-3p and hsa＿circ＿0000284.（7）Transfection of micro RNA mimics and inhibitors.（8）A dual luciferase assay verified the interaction between hsa-miR-338-3p and ETS1.（9）Western blot detection of protein expression.Results（1）The expression level of hsa-miR-338-3p in peripheral blood leukocytes in the CHD group was significantly increased,P<0.001.（2）There is an inverse correlation between the expression levels of hsa＿circ＿0000284 and hsa-miR-338-3p.（3）The expression level of hsa＿circ＿0000284 can affect hsa-miR-338-3p.（4）The positions of hsa＿circ＿0000284 and hsa-miR-338-3p are in the cytoplasm.（5）A dual luciferase assay showed that hsa＿circ＿0000284regulates the expression of a luciferase construct that contains the 3′UTR of hsa-miR-338-3p（P<0.05）.（6）The expression level of ETS1 in peripheral leukocytes was downregulated and inversely correlated with the expression of hsa-miR-338-3p（P=0.001,r=-0.235）and positively correlated with the expression of hsa＿circ＿0000284（P=0.036,r=0.141）.（7）Changes in expression levels of hsa＿circ＿0000284 and hsa-miR-338-3p influence expression of the ETS1 gene.（8）The dual luciferase experiment showed that hsa-miR-338-3p can regulate the expression of 3’UTR luciferase with ETS1（P<0.05）.（9）After EA-hy926 cells were transfected with sh RNA-circ＿0000284,ETS1 protein expression decreased,and the difference was statistically significant（P<0.05）.When sh RNA-circ＿0000284 and hsa-miR-338-3p inhibitor were cotransfected,the expression of ETS1 protein reversely increased（P<0.05）.After hsa-miR-338-3p mimics transfection in cells,the expression of ETS1 protein decreased with that of the control group,and the difference was statistically significant（P<0.05）.When hsa-miR-338-3p mimics and Overexpression-circ＿0000284 were cotransfected,the expression of ETS1protein expression increased（P<0.05）.Conclusion hsa＿circ＿0000284 targeted and negatively regulated hsa-miR-338-3p,which may regulate ETS1 expression and play a critical role in the pathogenesis of CHD.