Effect And Mechanism Of MiR-467b On Atherosclerotic Lesion By Directly Regulating LPL In Macrophage Of Mice

Atherosclerosis is one of the major underlying factor leading to dysfunctionalcardiovascular events in obesity, type2diabetes, heart attacks and strokes, andatherosclerosis-related cardiovascular disease is one of the leading causes of death inchina, and it is well established that macrophages play a crucial role in atherogenesisthrough the uptake of modified low-density lipoprotein (LDL) and secretion ofinflammatory modulators, cytokines, and matrix-degrading enzymes. Thus, theprevention and treatment of atherosclerosis, which is usually thought to be a complexpathological process, have become an important topic in medical study areas.Withadvances in our understanding of the molecular mechanisms of atherosclerosis, lipidaccumulates and inflammation of macrophage are regarded as the main pathogeneticpathway of both early atherogenesis and advanced plaque rupture.miRNAs are endogenous, noncoding, single-stranded RNAs of22nucleotidesand constitute a class of gene regulators. miRNAs are initially transcribed by RNApolymerase II (Pol II) in the nucleus to form large pri-miRNA transcripts. Thepri-miRNAs are processed by the RNase III enzymes, Drosha and Dicer, to generate18-to24-nucleotide mature miRNAs. More than700miRNAs have been cloned andsequenced in the human and it is believed that miRNAs control thepost-transcriptional regulation of30%of mammalian genes. The mature miRNAsnegatively regulate gene expression depending on the degree of complementaritybetween the miRNAs and its targets; miRNAs that bind to the3’ UTR of mRNA withimperfect complementarity block protein translation, while miRNAs that bind tomRNA with perfect complementarity induce targeted mRNA cleavage.Recent extensive studies have demonstrated that miR-467b may play importantroles in atherosclerosis initiation and progression via regulating key vascular cellular events through their target genes. The expression of miR-467b is upregulated inapolipoprotein E-deficient mice (apoE-/-) fed diets supplemented with Cafeic acidwhich shows altered lipid metabolism in the liver and spontaneously developsatherosclerotic lesions, and miR-467b*expression is downregulated in apoE-/-mice bytreated with polyphenols diets. Interestingly, miR-467b and miR-467b*are bothmature miR-467molecules arising from the same precursors, their nucleotides is thebasic complementary. However, the intracellular molecular mechanisms by miR-467baffecting atherosclerosis development have yet been studied.Here, we examined whether miR-467b regulates LPL expression inoxLDL-induced mouse RAW264.7macrophages and apoE-/-mice, thereby affectinglipid accumulation and proinflammatory cytokine secretion. Using oxLDL-treatmentRAW264.7macrophages transfected with miR-467b mimics or inhibitors, we haveshowed that miR-467b significant decreases lipid accumulation and IL-6, IL-1β,TNF-α and MCP-1secretions. Using of miR-467b agomir or antagomir investigateeffect of miR-467b in atherosclerosis of apoE-/-mice. The results showed thatmiR-467b decreased inflammatory cytokines levels and the accumulation ofmacrophages in plaques of apoE-KO mice. Furthermore, our studies suggest anadditional explanation for the regulatory mechanism of miR-467b regulation to itsfunctional target, LPL in RAW264.7macrophages and apoE-/-mice. Thus, ourfindings suggest that miR-467b may regulate the occurrence and development ofatherosclerosis by targeting LPL gene of macrophages.Part I: The predicter target genes of miR-467bAims: To investigate the target genes of miR-467b, and identification ofmiR-467b target site in the3’UTR of LPL transcript.Methods: The miRNA sequences were obtained from the miRBase Sequencedatabase and the genomic sequence was obtained from EnsEMBL. MicroCosmTargets (http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/) and RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/) were used to identifymiR-467b binding sites. MicroCosm Targets is a web resource developed by theEnright Lab at the EMBL-EBI. It contains computationally predicted targets formicroRNAs across many species. RNAhybrid is a tool for finding the minimum freeenergy hybridisation of a long and a short RNA. The hybridization is performed in akind of domain mode. The tool is primarily meant as a means for microRNA targetprediction. The full3’UTR of LPL was amplified by RT-PCR from RAW264.7macrophages. The amplified cDNA fragments were then cloned into p-LPL luciferasereporter vector using restriction sites XhoI and NotI. The integrity of3’UTR of LPLwas verified by DNA sequencing. Approximately,2×104293T cells were seeded into24-well plates24h before transfection and these cells were transiently cotransfectedwith p-LPL UTR miRNA luciferase reporter vector and miR-467b mimic/inhibitorusing lipofectamine2000. MiRNA mimic/inhibitor-neg was used as a control forthese experiments. Luciferase assays were performed4h later using theDual-Luciferase reporter system. Relative reporter activity was obtained bynormalization to the Renilla control. The ratio of the luciferase activity of eachconstruct was calculated using a luminometer.The miR-467b mimic and inhibitors were purchased from Dharmacon RNAiTechnologies. The mimics are double-strand RNA oligonucleotides that arechemically modified with the Dharmacon ON-TARGET modifications and can mimicendogenous miR-467b effects with a high specificity. The inhibitors are microRNAhairpin inhibitors with chemical modifications that can effectively inhibit endogenousmiR-467b function. The negative controls of miRNA mimics and inhibitors were alsopurchased from Dharmacon.Results: To search for a possible involvement of miR-467b in regulation of LPL,we scanned the3’UTR sequence of the mouse LPL transcripts using two databases:Microcosm Targets and RNAhybrid. We found that miR-467b is predicted to bind tomouse LPL3’UTR based on the two databases. Microcosm data give a binding scoreof publicly available algorithms at15.7745, and a free energy score at-11.51kal/mol.RNAhybrid data give an even lower free energy score at-17.9kal/mol, which contains three common sequence of LPL3’UTR (GCAUAU). The data indicate that miR-467blikely interacts with the3’UTR of LPL mRNA and then downregulates its expressionposttranscriptionally. Based on analyses of prediction algorithms, to determine ifregulation of LPL by miR-467b is specifically mediated by the microRNA mechanism,we performed luciferase reporter assays to determine if miR-467b specifically targetsLPL post-transcriptionally by binding to its3’UTR. The3’UTR of the LPL genecontaining miR-467b recognition sites was cloned from RAW264.7cells by RT-PCRand inserted downstream to a luciferase reporter.293T cells were transientlycotransfected with p-LPL UTR miRNA luciferase reporter vector and miR-467bmimic, or inhibitor. Cotransfection of miR-467b mimic dramatically reducedpromoter activity of the LPL3’UTR. On the other hand, cotransfection of miR-467binhibitor significantly increased the promoter activity. Similar result was also foundusing western immunoblotting to determine the expression of LPL proteins. Takentogether, these findings suggest that miR-467b directly targets LPL gene.Conclusion: Conclusion: miR-467b likely interacts with the3’UTR of LPLmRNA, and miR-467b can directly regulating LPL gene of macrophage.Part II: MiR-467b targets LPL gene in RAW264.7macrophagesand attenuates lipid accumulation and proinflammatorycytokine secretionAims: To observe the effect of miR-467b on LPL expression, lipid accumulationand proinflammatory cytokine secretion in RAW264.7macrophages.Methods: RAW264.7macrophages were preincubated with oxidized lowdensity lipoprotein (ox-LDL) to form foam cells. Cells were treated with miR-467bmimic, inhibitor, and (or) intervention LPL or siRNA of LPL. The association ofDil-oxLDL to RAW264.7cells was then determined. The levels of cellular totalcholesterol, free cholesterol and cholesterol ester by HPLC. The secretion ofinflammatory cytokines including IL-6, IL-1β, TNF-α and MCP-1were examed with ELISA. The protein and mRNA expression of LPL were examined by westernimmunoblotting assays and real-time quantitative PCR, respectively.Results: RAW264.7cells were transfected with miR-467b mimic or inhibitor,mRNA and protein levels of LPL were determined by qPCR and Western blot,respectively. When compared to cells transfected with nonspecific miRNA, cellstransfected with miRNA mimic showed a significant reduction in LPL mRNA andprotein levels. On the other hand, inhibition of miR-467b expression with miRNAinhibitor increased both mRNA and protein levels of LPL. Given that LPL promoteslipid accumulation in cells by anchoring lipoproteins for cellular uptake. We foundthat miR-467b mimic attenuated the Dil-oxLDL binding to the RAW264.7cells,conversely, miR-467b inhibitor enhanced the ability of RAW264.7cells to uptakeDil-oxLDL. We also measured the levels of cellular total cholesterol, free cholesteroland cholesterol ester by HPLC. Enhancing miR-467b function by its mimicsignificantly reduced the levels of total cholesterol (TC) and cholesterol ester (CE) butincreased free cholesterol (FC) levels. On the other hand, miR-467b inhibitorincreased the levels of TC and CE and reduced FC levels. Both mimic-neg andinhibitor-neg had no effect. In addition, we found that mimic-neg and inhibitor-neghad no effect on the secretion of the four proinflammatory cytokines tested. miR-467bmimic significantly inhibited the secretion of all four cytokines. Addition of LPLessentially blocked miR-467b mimic-induced inhibitory effects. In contrast,miR-467b inhibitor treatment significantly increased the secretion of IL-6, IL-1β,TNF-α and MCP-1. Knockdown of LPL expression by siRNA dramatically impairedthe effects of miR-467b inhibitor on cytokine secretion, restoring the cytokinesecretion levels to the normal levels when compared with control cells.Conclusion:①miR-467b regulates the expression of LPL by targeting the LPL3’UTR to facilitate mRNA degradation or translational repression in RAW264.7macrophages.②miR-467b regulates cellular association of Dil-oxLDL and cellularlipid composition in RAW264.7cells.③miR-467b influences proinflammatorycytokine secretion in RAW264.7cells and that LPL plays an important role in thisprocess. Part III: Effects of miR-467b on Atherosclerotic lesion and LPLExpression in Apolipoprotein E Knockout MiceAims: To observe the effect of miR-467b on LPL expression, lipid accumulationand proinflammatory cytokine secretion in apoE-KO mice.Methods: Male16-week-old apoE-KO mice were randomly divided into fourgroups: miR-467b agomir Negative Control（AG-NC） group, miR-467b agomir（AG）group, miR-467b antagomir Negative contro（lAN-NC）group and miR-467bantagomir （AN） group. All of the mice were fed a high-fat/high-cholesterol diet(15%fat wt/wt,0.25%cholesterol wt/wt). After4weeks, animals were killed. Bloodwas collected for the plasma lipid, and inflammatory cytokines expression viacommercially enzymatic methods and ELISA, respectively. Quantification of lesionsin aortic sinus was tested by HE stain; Lipid accumulation in aorta and aortic sinuswere evaluated by Oil Red O stain. The inflammatory cytokines expression in aortawere determted by ELISA, and the mRNA and protein expression of LPL in aorta andabdominal cavity macrophage were detected by RT-PCR and western-blot,respectively. The levels of cellular total cholesterol, free cholesterol and cholesterolester in peritoneal macrophages were used by HPLC.Results: Treatment of apoE-KO mice intraperitoneal injected with miR-467bagomir led to decrease in plasma TC, TG, LDL-C, HDL-C, TNF-α, IL-1β and MCP-1level, while increased in mice treated with miR-467b antagomir, compared withnegative control group. The lipid content and the levels of TNF-α, IL-1β and MCP-1in the aortic sinus plaques was less in mice treated with miR-467b agomir, and wasmore in the miR-467b antagomir group mice compared with negative control groupmice. ApoE-KO mice at miR-467b antagomir-treatment group developed advancedlesions in the aortic sinus, compared with antagomir negative control group. ThemiR-467b agomir-treatment groups were shown with a reduction of aorticatherosclerotic lesion area compared with agomir negative control group. In addition,the lesions in the aortic were decreased in mice treated with AOPPs and AG-490 compared to the AOPPs group. LPL expression of aortic and abdominal cavitymacrophage was significantly downregulated in miR-467b agomir group, while theexpressions were up-regulated in the miR-467b antagomir group mice in compared tothe negative control group.Conclusion:①Overexpression of miR-467b exacerbate the progrees ofatherosclerosis in apoE-KO mice.②miR-467b decreased the plasma inflammatorycytokines levels and the accumulation of macrophages in plaques of apoE-KO mice.③LPL expression of aortic and abdominal cavity macrophage was significantlydownregulated by miR-467b.