Preliminary Study Of MIR-132 Involved In The Regulation Of The Process Of AS

Atherosclerosis (atherosclerosis, AS) is a common serious hazard to human health, it is the main pathological basis of ischemic cardiovascular and cerebrovascular diseases, such as coronary heart disease, cerebrovascular disease and thromboembolic disease. So far, the pathogenesis of AS is yet not entirely clear, many theories exist, involving a variety of risk factors, as well as the lack of effective prevention and treatment of drugs. A lot of basic and clinical studies have shown that the risk factors caused As, including hyperlipidemia, hypertension, high blood sugar (diabetes), hyperfibrinogenemia, hyperhomocysteinemia, hyperuricemia, obesity, renin-angiotensin-aldosterone system (RAAS) activation, smoking, high blood coagulation (tissue factor, thrombin), microelement metabolic disorders (such as iron, copper, zinc, selenium, chromium, manganese, germanium, etc.), autologous biologically active substances (such as serotonin, NO, endothelin-1, etc.) metabolic disorders, chronic stress-induced cortisol secretion of catecholamines and other stress hormones in the blood flow and changes in blood pressure, leading to endothelial dysfunction and platelet adhesion. In these hypothesis, the inflammatory mechanisms of As are relatively new doctrine, causing great concern of scientists, and a lot of research work and achieved fruitful results, leading the formation of a general consensus. In 1999, Professor ROSS emphasized that As is a chronic inflammatory disease. According to the performance of the inflammatory response, As can be divided into acute inflammation exudative inflammation and chronic proliferative inflammation. Acute exudative inflammation is mainly seen in the early stage of As, as the monocyte-macrophage cell infiltration; and chronic proliferative inflammation exists usually in the progression of As, mainly for VSMCs proliferate excessively and extracellular matrix synthesisAbnormal lipid metabolism, is a major factor in the smooth muscle and endothelial injury occurrence during the development of AS. Oxidized low density lipoprotein (ox-LDL) on monocyte exhibits chemotaxis, which upregulates endothelial cells to produce monocyte colony-stimulating factor (MCSF) and monocyte chemotactic factor (MCP-1). The above factors lead to monocyte-macrophage cell recruitment and proliferate in to foam cells, which involved in the formation and development of AS. During inflammatory stimulation, endothelial cells express inflammatory mediators and lipid peroxidation regulation protease, such protease cut off the connection between endothelial cells and their endometrial stromal, promoting vascular endothelial cells to detach form vascular intima and causing intimal injury. Inflammation can induce low-density lipoprotein cholesterol (LDL-C) oxidative modification, and modified LDL-C may further lead to arterial intimal inflammation, in all, inflammation promotes the effect of lipoprotein to accelerate the formation of AS.The formation of As is due to a variety of risk factors of vascular endothelial cells and smooth muscle cells, leading to a loca excessive proliferation of chronic inflammatory reaction in vascular. Endothelial cells, macrophages and smooth muscle cells composed of the three important elements during the formation of As. Many microRNA express in these cell, which regulate endothelial cell, macrophages and smooth muscle cells to involve in the formation of As. Currently, most results are from the research of vascular diseases and angiogenesis, mainly in endothelial cells integrity and its ability to participate in angiogenesis, vascular smooth muscle cell proliferation and inflammatory response to As lipid. miRNA regulates the ruction of monocytes/macrophages , endothelial cells and vascular smooth muscle cells. Therefore, miRNA, by adjusting these three cells’function and affect the development of atherosclerosis, can become anti-atherosclerosis targets,micro RNA affects the function of vascular endothelial cells, and involves in the migration and proliferation of vascular smooth muscle cells. During the formation of atherosclerosis, the transformation of blood vessel wall cells, such as EC and VSMC played an important role. Endothelial dysfunction increased the endothelial layer permeability and the expression of corresponding cytokines and adhesion molecules, reducing the endothelium regeneration. VSMC migration and proliferation promotes plaque formation, accelerated coronary atherosclerosis process. The dysfunction of endothelial cells and smooth muscle, as well as migration, proliferation can be regulated by micro RNA. mi R-let-7c by inhibiting the expression of Bcl-xl, promotes apoptosis of endothelial cells and is involved in promoting atherosclerosis, while mi R-126 by enhancing the endothelial cell repairmen, exert anti-artery atherosclerosis effect, mi R-126 may also directly inhibit vascular endothelial growth factor (VEGF) pathway negative regulators, such as Sprouty associated proteins SPRED1 and type Ⅱ phosphoinositide 3-kinase regulatory subunit (PIK3R2/p85-b) to regulate endothelial cell response to VEGF. mi R-221 mi R-222 inhibit their targets p27 and p57 to promote the proliferation of VSMC. Both mi R-143 and mi R-145, are largely expressed in smooth muscle cells, and are highly conservative, they participate in the differentiation and migration of smooth muscle cells. Deletion or reduciton of mi R-143 and mi R-145, can promote VSMC migration, proliferation and differentiation, thus promoting coronary atherosclerosis. More and more evidence indicates mi RNAs involved in many important physiological and pathological processes, such as development, organogenesis, apoptosis, cell proliferation, and even tumors formation. Atherosclerosis is a vascular endothelial injury and lipid metabolic disorders characterized by chronic inflammatory diseases. Some recent studies have shown that mi RNA plays an important role in the regulation of angiogenesis, inflammation and lipid metabolism, which are closely related to atherosclerotic lesion progression.Atherosclerosis (AS) is a clinical events caused by a variety of factors, leading to cholesterol and other lipid deposition in the artery intima, complicated by smooth muscle cells (SMC) accumulation, fibrosis and calcification, plaque hemorrhage and thrombosis, and ultimately cause arterial wall hardening, stenosis or occlusion. AS is a chronic complex disease process, in the situation of abnormal blood lipids, vascular endothelial cells (VEC) is injured, platelet aggregates and releases platelet factor, circulated monocytes enter into endothelial and activates into macrophages, they phagocytic oxidative modificated low density lipoprotein (ox-LDL), middle vascular intimal SMC migrated to injury lesion and proliferation, causing intimal thickening and fibrosis, the formation of plaque and stenosis or occlusion. Eventually lead to cardiovascular and cerebrovascular events. Thus, VEC, SMC, macrophages and some cytokines are all involved in the occurrence and development of ASObjective:To investigate the miR-132 pro-inflammatory role in the atherosclerotic process.Method:1:Using online software DIANA microT v4.0 (http://diana.cslab.ece.ntua.gr/) to predict microRNAs that can be combined with SIRT 1; retrieving SIRT1 3’-UTR sequence according GeneCards (http://www.genecards.org), using Vector NTIAdvance 11.5.1 software to design PCR amplification primers, restriction enzyme digestion sites are EcoR V and BamH I. The primer sequences are:upstream:5’-GGGAAGTACATCAAGAGCTTCGT-3’; downstream:5’-CCCCCTGAACCTG AAACATAAA-3’.2:Cultured human umbilical vein endothelial cells, transiently transfected with miR-132 mimics and inhibitor, cultured cells, extracted endothelial cells total RNA and total protein for western blot and real-time PCR to detect the expression levels of SIRT1 and its downstream regulation genes SREBP, FASN and HMGCR;3:Cultured human umbilical vein endothelial cells with different concentrations of TNF-a for 4 hours, then extract cellular proteins from endothelial cells, using western blot to detect expression levels of inflammation-related protein ICAM1 (intercellular adhesion molecule-1) and MMP9 (matrix metalloproteinase-9). In addition, we also use Elisa to detect ICAM (sICAM1) level in cell culture supernatant solution;4:Cultured human umbilical vein endothelial cells, pretreat endothelial cells with miR-132 or miR-132 inhibitor for 48 hours, then treat these cells with or without TNFa (10 ng/mL) for 24 hours. Then extracted cells total protein for western blot to detect the expression levels of inflammation-related protein ICAM1 (intercellular adhesion molecule-1) and MMP9 (matrix metalloproteinase-9);5:Cultured human umbilical vein endothelial cells, design oligonucleotide to blocking expression of endothelial cells derived miR-132, and with or without the use of TNF-a proinflammatory process, total protein was extracted for western blot to detect SIRT1, SREBP-lc gene expression and its downstream expression;Result:1:First, we use online software DIANA microT v4.0 (http://diana.cslab.ece.ntua.gr/) to predict whether there is one or more miRNAs that act on SIRT1 in the retrieved miRNA, we found that miR-132 acts on SIRT1 mRNA 3 ’non-coding region. To further validate miR-132 can bind directly to the 3’untranslated region of SIRT1, we detected 3’UTR luciferase reporter gene and found that compared with the control group, after transfection miR-132, endothelial cells SIRT1 3’luciferase activity in the non-coding region (0.338 ± 0.036) was significantly lower (P=0.000). The results confirmed that, SIRT1 of mRNA is a direct target of miR-132.2:In order to verify miRNAs can regulate endothelial cells SREBP- adipogenesis-cholesterol-producing pathway, and SREBP1c, FASN and HMGCR are important enzymes in the de novo synthesis of fatty acids and cholesterol. We transfected miR-132 mimics and inhibitor into human umbilical vein endothelial cells and cultured for 24 hours, extracted total RNA for RT-PCR, the result showed that miR-132 inhibited the expression of SIRT1 mRNA, and also restricted the expression levels of their downstream regulated genes SREBP (0.45 ± 0.07), FASN (0.55 ± 0.09) and HMGCR mRNA (0.62 ± 0.08).After 48 hours of incubation, total cellular protein was extracted for western blot to detect expression levels of SIRT1. The results showed that:miR-132 inhibited the expression of SIRT1 (P<0.01) at the protein levels, and also inhibited the protein expression of their downstream regulation genes SREBP, FASN and HMGCR.3:Studies have shown that, TNF-α can participate in the inflammatory response. To confirm TNF-α is capable of inducing endothelial cell inflammatory response, we use different concentrations of TNF-α to treat human umbilical vein endothelial cells for 4 hours and then extracted cellular proteins of endothelial cells, using western blot to detect to express levels of inflammation-related protein. The results showed that, the expression levels of ICAM1 (intercellular adhesion molecule-1), MMP9 (matrix metalloproteinase-9) and other protein increased significantly (Figure 2-1). In addition, we also use Elisa to detect ICAM (sICAM1) levels in cell culture supernatant and found that compared with the control group, the supernatant soluble ICAM (sICAM1) increased significantly (Figure 2-2:control group:0.38 ± 0.04; 5 ng/mL 0.62 ± 0.11; 10 ng/mL 0.87 ± 0.09; 50 ng/mL 0.93 ± 0.14), these results indicate that:TNF-a induced human umbilical vein endothelial cells in the inflammatory response.4:To determine whether endothelial cells miR-132 can promote inflammation, we first pretreated endothelial cell with miR-132 or miR-132 inhibitor for 48 hours, and then treated with or without TNFα (10 ng/mL) cells for 24 hours. Then we extracted cells total protein for western blot for detection of inflammation-related protein; the results showed that miR-132 is capable of inducing sICAM1 and increased expression levels of ICAM 1 and MMP9 in endothelial cells. Moreover, miR-132 and/or TNFa induced expression of ICAM1, and such expression can be inhibited by miR-132 inhibitor (c vs. e, P<0.01; d vs. f, P<0.01). (Fig 3b:c vs. e, P <0.01; d vs. f, P<0.01) (a,0.38 ± 0.04; b,0.87 ± 0.09; c,1.33 ±0.18; d,1.54 ± 0.14; e,0.18 ± 0.03; f,0.56 ± 0.04). These data indicate that, miR-132 promotes human umbilical vein endothelial cells inflammation.5:In order to study the correlation of miR-132 inducing human umbilical vein endothelial cells inflammation and SREBP-adipogenesis-cholesterol generated, we designed oligonucleotides to block the expression of endogenous miR-132 in endothelial cells, using or not using TNF-a to promote inflammatory process, total protein was extracted for western blot to detect the expression of SIRT1, SREBP-1c and their downstream genes, the results found that after blocking endogenous miR-132, using TNF-α to induce endothelial cell inflammatory response, the gene expression of SIRT1, SREBP-1c and their downstream were increased significantly, but no significant changes when not using TNF-a-induced; and miR-132 can inhibit SIRT1, SREBP-1c and its downstream gene expression. These results indicate that, miR-132 can induce human umbilical vein endothelial cells inflammation that is relevant to SREBP-adipogenesis-cholesterol generation.Conclusion:1, bioinformatics analysis showed, miR-132 can be directly bonded to the 3 ’untranslated region of SIRT1;2, miR-132 inhibits the expression of SREBP 1-c and its downstream SIRT1 gene in human umbilical vein endothelial cells;3, miR-132 shows pro-inflammatory effect in artery atherosclerosis development;4, miR-132 can induce SREBP-adipogenesis-cholesterol generation relevant human umbilical vein endothelial cells inflammation;...