| Coronary atherosclerotic heart disease(CHD) is one of the major social and public health problems harming physical and mental health of mankind in worldwide. CHD is the leading cause of morbidity and mortality in western developed country. In recent years, with the rapid development of our social economy, people’s living standard and lifespan improved greatly, working style and eating habits changed greatly, but the morbidity and mortality of coronary heart disease are also rising rapidly and showing younger trend, and gradually become one of the most serious diseases which are the fastest-growing constitutes caused death in our residents, and aggravate public health burden on our country. Reduction the incidence of coronary heart disease and mortality has become an urgent need to address the issue in public health domain.Epidemiological studies showed that active intervening the related risk factors for AS and coronary heart disease are contributed to the early prevention and CHD. Traditional risk factors for AS and CHD include age, gender, family history, smoking, high blood pressure, diabetes and metabolic syndrome, insulin resistance, hyperlipidemia, physical activity, obesity and so on. These risk factors promote AS and CHD events with separately or together, and has won full attention in clinical practice. However, the incidence and mortality of CHD are becoming more and younger in our country. A new series of risk factors, especially the inflammatory related risk factors have been found. Visible, it is impossible to achieve comprehensive and strict control the risk factors for coronary heart disease. Therefore, thorough research the pathogenesis of coronary heart disease is the key to more effective prevention coronary heart disease.Recently, the role of immune and inflammatory in the development of AS and coronary heart disease are gradually recongnized. The existing agreement showed that inflammation was a core factor for unstable plaque formation and development, the immune mechanism involved in the entire process of AS and CHD. Even some scholars thought coronary heart disease as a chronic inflammation and autoimmune diseases. Actiated T lymphocytes and antigen processed cells could be detected in peripheral blood of acute coronary syndrome (ACS) patients, significantly higher levels of IFN-y, TNF alpha and IL-6, IL-18, IL-12 also exist in ACS. ACS sufferers activated immune system and worse inflammation. There are a lot of immune cells, including neutrophils, macrophages, T lymphocytes and dendritic cells, which constituted the main component of inflammatory cells in AS lesions, especially in unstable plaques. Thus accumulated immune cells leading to enhanced inflammation prompted the unstable plaque formation. As a key for natural immunity linked with acquired immune, DCs are the strongest antigen presenting cells in body. With antigen stimulation, DCs experience from immature to mature, peripheral tissue to subprime lymphopid organs, finally active initial T cells and specific immune response. As the key cell to regulate inflammatory immune response, DCs play an important role in the development AS. On the one hand, peripheral blood DCs are in maturation and activation state in CHD. AS and CHD related risk factors, such as nicotine (main ingredients in tobacco), oxidized low-density lipoprotein (LDL), ox-homocysteine, male hormones, CRP, etc. could induce the maturation of DCs, and strengthen immune response, ultimate promote AS. On the other hand, the distribution of DCs is closely related to AS. DCs are exist in normal artery, pre-AS and AS artery. DCs participated in AS plaque formation and progress, and along with the progress of stable plaques to vulnerable plaques, the quantity and immune response ability of DCs existing in plaques are enhanced. In addition to strong effect of stablizing plaques, statins could reduce the quantity and maturation of DCs. In addition, DCs gathering and mediating immune response are one of the important factors in the formation and development of AS lesions.At present, most viewpoints think that accumulated DCs in plaque are migrated from peripheral with the help of related risk factors. Our and others research showed that the proportion myeloid dendritic cells (mDCs) in peripheral blood is significantly lower in CHD, especially the ACS patients than in normal controls. Because the content of vDCs in CHD are limited, the proportion and quantity of leukocytes subset and the generating function of bone marrow are normal in patients with CHD, so there does not exist factors harming DCs formation or apoptosis. We assume that the increased DCs gathering in AS may partly migrated from the peripheral blood. Clinical and basic research further confirmed that the relevant condition promoting DCs migration exists in patients with CHD. In patients with coronary artery disease, adhesion molecules such as E-selecting, P-selecting, ICAM-1, and chemotactic factors such as fractalkine, CCL19 and CCL21 are increased obviously, the related risk factors for coronary heart disease, such as oxidation low density lipoprotein, glycosyl albumin, nicotine and heat shock protein (HSP) have ability to induce DCs maturity and high expression adhesion molecules and chemotactic factors receptors. In spontaneous AS animal models, silencing CCR7, CX3CR1 gene can obviously limit DCs migration, slow AS process and the content of DCs in AS lesions. Inhibition DCs migration can obviously delay AS progress. Research on the mechanism of migration of DCs may help to early intervened CHD.MicroRNA, with 21 to 25 nucleotides, is a kind of endogenous non-coding RNA, which regulate gene by silencing gene expression in transcription. In gene transcription level, microRNA play a collaborative regulatory function through complementary combining with target mRNA to control multiple mRNA and protein expression. In addition, microRNA regulates targeting genes expression in a dose-response way. MicroRNAs widely participate in cell proliferation, differentiation, apoptosis, and closely related to DCs introducing antigen presenting function and cardiovascular disease. At present, the role of microRNA in the migration of dendritic cells is still unknown.For further discussion DCs migration mechanism related to AS, the following four aspects based on earlier research will be studied:analysis of the relation between peripheral DCs subset and the major risk factors for coronary artery disease; using gene chip technology detecting the microRNA express patterns in ox-LDL induced DCs; Using bioinformatic approaches screening the targeted gene for most significant differentially expressed microRNA, then dual fluorescent protein reporter assay validating the impact of microRNA on this gene; To determined the effect of microRNA on immature DCs phenotype, migration and expression of target gene, and further validating the effect of target gene on DCs migration and function. microRNAs regulating the migration of DCs and its regulation mechanism may be screened through the above research. The results were as follows:1 Analysis of the relation between risk factors with the proportion of DCs subsets in peripheral blood of subjects1.1 General clinical data of objectsThere have no statistically difference in sex ratio, age distribution, the proportion of risk factors for CHD in subjects such as high blood pressure, smoking and diabetes, as well as drugs, total cholesterol, triglyceride, uric acid, leukocyte count, creatinine level among four groups (P> 0.05). There have statistically significant difference in glycated hemoglobin, low density lipoprotein cholesterol (LDL-c), cardiac troponin cardiac troponinⅠ(cTnI) among four groups (P< 0.05).1.2 The proportion of dendritic cells subsets in peripheral blood in all subjects.There have a statistically significant difference in the proportion of mDC among four groups(P<0.01), the proportion of mDC in AMI group and UAP group are obviously lower than in control and SAP group (P<0.01). There have statistically difference in the proportion of pDC among four groups (P=0.043).1.3 The concentration of related factors and Genisi score in all subjectsThere have statistically significant difference in plasma CCL2, ox-LDL, Fractalkine, CCL21, CCL19 and Genisi score among four groups (P<0.01). Among them, there have a statistically significant difference and successive increased in CCL2, ox-LDL, Fractalkine, CCL21, Genisi score between each other in the control group, SAP group, UAP group, AMI group. Plasma CCL19 is elevated in UAP group and AMI group than in control group and SAP group (P<0.05).1.4 Multivariate linear regression analyze the factors affectted the proportion of mDCs in all subjectsUsing multivariate linear regression analyzed known factors in all subjects. The following factors influencing on peripheral mDCs proportion were foud:CCL21, ox-LDL, CCL19, CCL2, Genisi scoreand cTnI. Furthermore, linear multivariate analysis stepwise regression showed that ox-LDL, CCL2, Genisi score, and pDCs could determine the mDCs percentage. 1.5 Correlation analysis of Ox-LDL, CCL21, CCL19, Genisi score, cTnI, and CCL2 with the peripheral mDCs proportionBased on correlation analysis CCL21, ox-LDL, CCL19 and CCL2 with peripheral blood mDCs proportion, ox-LDL is strongest correlated with the proportion mDCs (r =-0.710, P< 0.001), then followed by the CCL2 (r=-0.626, P<0.001), CCL21 (r =-0.449, P< 0.001), CCL19 (r=-0.242, P= 0.002). Correlation analysis of Genisi score with mDCs proportion indicates that mDCs proportion was significantly negative correlation with Genisi score (r=-0.807, P< 0.001). But there have no correlation between mDCs proportion with cTnl (r=-0.125, P=0.111) and pDCs proportion(r=0.238, P=0.092).1.6 Correlation analysis of ox- LDL, CCL21, CCL19 and CCL2 with Genisi scoreBased on correlation analysis between CCL21, ox-LDL, CCL19 and CCL2 with Genisi score which is a gold standard for evaluation total load for the coronary atherosclerosis, ox-LDL is the strongest correlated with Genisi score (r=0.684, P< 0.001), the second is the CCL2 (r=0.608, P<0.001); due to CCR7 is the-common receptors for CCL21 and CCL19, partial correlation analysis showed that CCL21, but not CCL19 correlated with Genisi score (r=0.317, P< 0.001).There has no correlation between pDCs proportion with Genisi score (r=0.011, P=0.891).2 Effect of ox-LDL on the microRNA expression patterns of DCs derived from human monocyte2.1 ox-LDL can promote the maturation of DCs derived from human monocyte2.1.1 Morphological observation the cultured DCsAfter cultured for 5 day, cells adhesion to the culture bottle bottom or suspending growth in medium, with round or oval in shape, around with the burr-like or spinous process. Then stimulated cells with 100μg/ml ox-LDL for 24 h, cells suspended growth in medium, with dendritic-like protrusionsin on cell surface in different quatity and shape, matured cell size is 1-2 times of monocyte. In control group, most cells suspension growth, some of them aggregation in clusters, showed similar round and around with dendritic-like protrusionsin.2.1.2 Effect of ox-LDL on phenotype and migration of DCs and secretion of IL-12Compared with the control group, CD1α, CD83, CD86 molecule expressed on DCs surface in ox-LDL group were significantly upregulated by flow cytometry; and there was significant difference between them (P<0.01); IL-12 was significantly increased in DCs cultured supernatant in ox-LDL group and there was significant difference between them (P<0.01). Compared with the control group, the number of DCs migrating to the lower chamber significantly increased in ox-LDL group and there was significant differences between them (P<0.01).2.2 Effect of ox-LDL on the expression patterns of microRNAs in DCsWith microarray assay, compared with control group, there are three gene upregulated expression more than 2 times in ox-LDL induced DCs, which in turn is miR-34a, miR-125a-5p, miR-155; and four genes downregulated expression more than 2 times, which in turn is miR-31, miR-1301, miR-212, miR- 423-5p. At the same time, positive control signal detection, such as Spike control, Hybridization controls showed that chip probe mark and hybrid efficiency are normal.2.3 Real time polymerase chain reaction (PCR) verified the results of microarrayThe samples from control group and ox-LDL group were determined by qRT-PCR, respectively. According to the amplication and dissociation curve, and electrophoresis, the specificity of miRNA realTime PCR is very good. Compared with control group, miR-155 was elevated 1.72 times and miR-31 was lowered 5.75 times. 2.4 miR-34a was elevated in ox-LDL induced DCs, and correlated with the migration of DCsCompared with the control group,ΔCt for miR-34a is significantly lower in ox-LDL group, and there is statistically significant difference(P< 0.05). The expression of miR-34a within DCs was obviously up-regulated;ΔCt for miR-34a was negatively correlated with DCs migration (r=-0.851, P< 0.001), suggests that upregulation expression of miR-34a was related to the migrating ability of DCs.3 Dual fluorescent protein reporter system verified regulation relationship between miR-34a and SRC gene3.1 Using targeting genes prediction software analysis showed that SRC may be the potential targeting genes for miR-34a. Forecasting results are as follows:all of miRanda, Targetscan and circuitsDB successfully predicted it.3.2 Rellina luciferase/Luciferase (RLUC/LUC) changed in each groupThere have significant difference in RLUC/LUC among four groups (F=8.726, P =0.007); compared with other three groups, RLUC/LUC in pcDNA3.1 (+)-miR-C2 group is the highest (P< 0.01). There have no difference in RLUC/LUC among pcDNA3.1 (+)-hsa-miR-34 psicheck2-SRC-UTR, psicheck2-3’MUT-3’SRC-UTR (P > 0.05). But the average of RLUC/LUC in pcDNA3.1 (+)-hsa-miR-34 only lowered 10.18% than in pcDNA3.1 (+)-miR-C2.3.3 Effect of miR-34a on SRC expression in DCsThe expression of SRC is up-regulated in miR-34a overexpressed group and ox-LDL group using western blotting.4 miR-34a promotes DCs migration via upregulation SRC expressionThere have significant difference in the number of migration DCs among four goups. Compared with the other three groups, the number of migration DCs was the most in miR-34a group (P< 0.05); the number of migration DCs was higher in miR-34a+PP2 inhibitors group than in control group (P=0.001). The above results suggest that SRC inhibitors PP2 may reduce the migration of DCs, but not complete inhibition of DCs migration induced by miR-34a.4.2 Effect of miR-34a and PP2 on protein expression of SRC and E-cadherin in DCs Weatern blotting showed that SRC inhibition leads to upregulation of E-cadherin, miR-34a can upregulate the SRC expression and inhibit the E-cadherin expression.4.3 Effect of miR-34a and PP2 on the expression of CD86 and secretion of IL-12 in DCsThere have significant difference in the expression of CD86 and secretion of IL-12 between each group (P< 0.001). In which, the expression of CD86 in DCs was lower in the control group and the PP2 inhibitors group than in miR-34a+PP2 inhibitor group and miR-34a group(P< 0.001); the concentration of IL-12 was the highest in miR-34a group(P< 0.001), and there have no obvious difference among other three groups (P> 0.05).According to the above four parts of experiments, we can draw the following conclusions:(1) The proportion of mDCs is downregulated in CHD, and The proportion of pDCs is downregulated in AMI; The proportion of mDCs is depend on ox-LDL, Genisi score, CCL2 and pDCs. Ox-LDL is the most obviously correlated with the proportion of mDCs and Genisi score.(2) Ox-LDL can upregulate three gene expression in DCs:miR-34a, followed by miR-125a-5p and miR-155; and downregulate four gene expression:miR-31, followed by miR-1301, miR-212 and miR-423-5p. The most obviously upregulated gene is miR-34a, which is positively correlated with the migration of DCs induced by ox-LDL.(3) miR-34a promotes DCs migration via upregulation of SRC. |
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