| Atherosclerotic cardiovascular diseases, including coronary artery disease(CAD),peripheral artery disease and carotid artery stenosis, are of the leading causes of morbidity and mortality. The prevention as well as the treatment of atherosclerosis,has been a hot issue of cardiovascular clinical and basic research. The progression of atherosclerosis contributes vigorously to the differentiation of monocytes into macrophages, which accumulate cholesterol to form foam cells(FCs) in the vessel wall. Therefore, as for the prevention of atherosclerosis, it is pivotal to prevent the formation of foam cells.High-density lipoprotein(HDL-C) is positively associated with a decreased risk of CHD. HDL-C plays a key role in protecting against atherosclerosis, mainly through reverse cholesterol transport(RCT), which is a process delivers excessive cholesterol back to the liver from the arterial wall. The RCT pathway is currently considered to be the unique mechanism by which the human body clears the excessive cholesterol away. Besides, RCT can prevent the formation of FCs effectively and play a critical role in the process of antiatherosclerosis. ATP-binding cassette(ABC) A1, which is a member of ATP-binding cassette transporter superfamily, has been proven to mediate the active efflux of cholesterol to lipoprotein acceptors in the promotion of RCT. The active efflux of cholesterol is mediated to the lipid-poor acceptor apolipoprotein(apo)A-I by ABCA1,which is considered to be the rate-limiting step in RCT. In this context, how to regulate the expression of ABCA1 and accelerate the RCT has become the focus of anti-atherosclerotic target.A micro RNA(mi RNA) is a kind of small conserved non-coding RNA molecule(containing about 22 nucleotides), which usually exists in plants, animals, and some viruses. mi RNAs can regulate the gene expression via RNA-induced silencing complexes.They can inhibit the translation of m RNAs or destruct the cleavage directly to achieve thegoal of genetic regulation. Generally speaking, a single mi RNA could target more than 200 m RNAs. Similarly, one gene could be regulated by multiple mi RNAs. As one of the post-transcriptional regulation factors, mi RNA is viewed as an important tool to manipulate the pathophysiological procedure of atherosclerotic cardiovascular diseases. It is also essential to discover the specific regulatory mechanisms of mi RNAs. Recently,mi RNAs have been shown to contribute to the maintenance of cholesterol homeostasis by several studies. It has been reported that mi R-33, mi R-758, mi R-128, mi R-27, mi R-26,mi R-106 b and mi R-19 b, can regulate RCT by decreasing the expression of ABCA1. These results indicate that mi RNAs play critical roles in regulating ABCA1 expression in RCT process.Compared with the normal wall tissues, the expression of mi R-20 a was upregulated in atherosclerotic abdominal aortic aneurysm wall tissues significantly. Meanwhile, the clinical data showed a significant decreased expression of mi R-20 a for the patients with CAD in the comparison to the healthy volunteers. On the other hand, the expression of mi R-20 b dropped in the patients with type 2 diabetes. These results indicate that mi R-20a/b play important roles in formation and development of atherosclerosis and CAD.However, it remains unclear about the specific mechanisms of mi R-20a/b regulation in the atherosclerosis. In our study, we try to predict the potential mi RNA target by using a serial of bioinformatic tools. According to the results, we concluded that mi R-20a/b was conserved and ABCA1 is the target of mi R-20a/b.Based on the bio-information of ABCA1 longer 3’UTR and prediction, we made a reasonable speculation that mi R-20a/b has the ability to recognize the ABCA1 3’UTR and consequently inhibits the ABCA1 translating. By reducing the protein expression of ABCA1, the level of RCT can be declined, which eventually result in the decreased atherosclerosis formation and alleviated progression; To confirm the hypothesis that mi R-20a/b is a new mi RNA that can regulate ABCA1 expression, we perform the experiments as follows: 1, The effect of mi R-20a/b on the 3′ UTR of ABCA1 was detected by using the luciferase reporter assay; 2, The role of mi R-20a/b in the expression of ABCA1, lipid content, and cholesterol efflux were observed by overexpressing or silencing mi R-20a/b inTHP-1, RAW264.7 FCs; 3, The effect of mi R-20a/b on ABCA1 expression of liver, the efficiency of RCT, plasma lipid profile, and plaque area were examined by overexpressing or silencing apo E-/- mice of mi R-20a/b; 4, The mi RNA-20a/b expression in patients with CAD was investigated and the clinical sense of mi RNA-20a/b was explored. Our objectives were trying to reveal the possible molecular mechanisms of mi R-20a/b-related atherosclerosis and searching for a new promising mi RNA which can target at the regulation of ABCA1.Part 1 Bioinformatic prediction and experiment confirm that mi R-20a/b can recongnize the ABCA1 3’UTR Aims:To predict the target genes of mi R-20a/b, and to confirm the target zone of mi R-20a/b on ABCA1 3’UTR.Methods:mi R-20a/b sequences were from online website mi RBase and ABCA1 3’UTR were obtained from Gene Bank respectively. Based on these bio-information, we analyzed the conservatism of mi R-20a/b, the binding sites of mi R-20a/b with ABCA1 3’UTR and the binding minimum free energy. According to the result of prediction, we amplified the human by RT-PCR from THP-1 macrophage. Then we cloned the wild type and mutant ABCA1 3’UTR into luciferase reporter vector psi CHECKTM-2. After cotransfecting with mi R-20a/b mimic and the fusional plasmids, we detected the relative luciferase activity in HEK 293 T cells.Results:We obtained the sequences of mature mi R-20a/b and ABCA1 3’UTR successfully.Bioinformatic prediction revealed mi R-20a/b sequences are conservative, mi R-20a/b can target human/mouse ABCA1 3’UTR, featuring the very low free energy of binding, and high context score of mi R-20a/b binding to ABCA1 3’UTR. When we cotransfected with mi R-20a/b and wild type ABCA1 3’UTR into HEK 293 T cell, relative luciferase activity were down-regulated. No significant difference were shown, cotransfected with mi R-20a/b mimic and mutant ABCA1 3’UTR.Conclusion:Based on the bioinformatic prediction and luciferase reporter array confirmation, the ABCA1 3’UTR can be recognized by mi R-20a/b.Part 2 mi R-20a/b target ABCA1 gene and reduce cholesterol efflux in foam cells Aims:To investigate the effect of mi R-20a/b on the expression of ABCA1, cholesterol efflux and cholesterol content in THP-1 macrophage-derived foam and RAW 264.7 macrophagederived FCs.Methods:The differentiation from the cultured THP-1 cells to the macrophages was induced with PMA. THP-1 macrophages and RAW 264.7 macrophages were transformed into FCs by incubating with ox-LDL. The FCs were transfected with mimic control, mi R-20a/b,inhibitor control or mi R-20a/b inhibitor. The m RNA and protein expression of ABCA1 were examined by quantitative real-time PCR(q RT-PCR) and western blot respectively.The cellular cholesterol efflux was detected with liquid scintillator. The cholesterol content was detected by high performance liquid chromatography assays.Results:Compared to the cells transfected with nonspecific mi RNA, the mi R-20a/b mimic transfected ones showed a significant reduction in ABCA1 m RNA and protein levels in a dose and time-dependent manner. Compared to the control, mi R-20a/b inhibitor elevated the m RNA and protein expressions of ABCA1. Meanwhile, the mimic control or inhibitor control showed insignificant difference in ABCA1 expression. Besides, mi R-20a/b suppressed the cholesterol efflux and made the cholesterol content proliferated in THP-1macrophage-derived as well as RAW 264.7 macrophage-derived FCs. In contrast to this,the inhibitor of mi R-20a/b showed a totally opposite effects featuring the increased cholesterol efflux and the reduced cholesterol content.Conclusion:mi R-20a/b can reduce the ABCA1 expression, resist the cholesterol efflux and proliferate the cholesterol content; mi R-20a/b inhibitor can upregulate the ABCA1 expression, enhance the cholesterol efflux and diminish the cholesterol content.Part 3 The effect of mi R-20a/b on ABCA1 expression and atherosclerotic lesion in apolipoprotein E knockout mice Aim:To explore the effect of mi R-20a/b on ABCA1 expression, and atherosclerotic lesions in apo E knockout(apo E-/-) mice.Methods:Male apo E-/- mice were fed with western diet for 10 weeks and established the atherosclerosis model. Mice received subcutaneous injection 2′F/MOE mi R-20a/b agomir or 2′F/MOE anti-mi R-20a/b. The m RNA and protein expression of ABCA1 were examined by q RT-PCR and western blot respectively. RAW 264.7 macrophages were radiolabeled with3H-cholesterol and intraperitoneally injected into the apo E-/-mice at the time point of2 day before the euthanization. After 8 weeks, all animals were sacrificed. Blood, feces and hepatic tissue were collected to detect the RCT efficiency with liquid scintillation counting.Plasma lipids were determined via commercially enzymatic methods, including triglyceride(TG), high density lipoprotein cholesterol(HDL-C) and low density lipoprotein cholesterol(LDL-C). Atherosclerotic lesions in aortic sinus were tested and observed by HE staining.Results:mi R-20a/b inhibited the m RNA and protein expressions of liver ABCA1, resulted in the reduction of the3H-cholesterol in serum, liver, and feces. Moreover, it led to the decreased HLD-C, increased LDL-C, and the exacerbated atherosclerotic lesions in apo E-/-mice. While anti-mi R-20a/b upregulated the m RNA and protein expressions of liver ABCA1, elevated the serum, liver, and feces3H-cholesterol as well as the HLD-C level,made the LDL-C level slipped down, and extenuated atherosclerotic lesions in apo E-/-mice.Conclusion:mi R-20a/b can suppress the ABCA1 expression, impair plasma lipids expression, and exacerbate atherosclerotic lesion. Anti-mi R-20a/b can upregulate the ABCA1 expression,improve plasma lipids expression, and relieve atherosclerotic lesions.Part 4 The expression of mi R-20a/b in patients with coronary artery disease Aim:To investigate the expression of mi R-33 and mi R-20a/b in patients with CAD and explore the clinical significance of mi R-20a/b.Methods:Forty patients with CAD from the department of cardiology in the second hospital of shanxi medical university enrolled, and forty healthy controls were simultaneously recruited from the community examination group to avoid potential bias of participant selection between groups. The LDL-C level of all people was under 3.12 mmol/L. CAD was diagnosed by coronary angiography. In control group, patients with hypertension and diabetes were excluded. The biochemical targets were detected by using biochemical detection kit. The expression of mi R-33 and mi R-20a/b was examined by real-time PCR.We performed the correlation analysis with mi R-33 or mi R-20a/b and HDL or LDL.Then analyze whether mi R-33 and mi R-20a/b is an independent risk factor for CHD by multivariate logistic regression analysis.Results:Compared to the control group, the ratio of hypertension, diabete, smoking and drinking is higher in CAD group. The expression of mi R-33 and mi R-20a/b was lower in CAD group. In control group, mi R-33 and mi R-20a/b were negative correlation with HDLC, while mi R-33 showed insignificance statistically. These mi RNAs indicated no obvious correlation with LDL. In CAD group, mi R-33 and mi R-20a/b were positive correlated with HDL-C, but mi R-20 b showed no statistical significance. Declined mi R-33 and mi R-20 a might be the protective factors for the development of CAD.Conclusion:The expression of mi R-33 and mi R-20a/b in patients with CHD is lower. mi R-33 and mi R-20a/b are correlated with HDL, which might be a potential biomarker and a risk factor of CAD. |
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