Effect And Mechanism Of MiR-19b On Atherogenesis Through Regulating Macrophage ABCA1in ApoE^-/- Mice

Atherosclerotic diseases are the major causes of mortality and morbidity, whichseverely threatens human’s health in the world. Macrophage cholesterol accumulationand foam cell formation in the intima is a major hallmark of early stageatherosclerotic lesion. Uncontrolled uptake of cholesterl infiltrating into arterial walleventually triggers the collapse of foam cell, and subsequently the release ofintracellular cholesterl, the main chemical component within atherosclerotic lesion,promotes the continuing development of atherosclerosis. Facilitation of macrophagecholesterol efflux and reverse cholesterol transport (RCT), subsequently inhibitingfoam cell formation and lipid deposition in arterial wall, has the importantsignificance in the prevention and treatment of atherosclerotic diseases.ATP binding cassette transporter A1(ABCA1) is integral membrane protein,which transports free cholesterol and phospholipid from intracellular compartments tocell membrane by using ATP as a source of energy. Lipidic cargo is removed soon byapoprotein AI (apoAI). ABCA1-mediated cholesterol efflux to apoAI is essential forHDL formation and defined as the first and rate-controlling step in macrophagecholesterol efflux and RCT. The expression and function of macrophage ABCA1areelaborately regulated at multiple levels, including transcriptional andposttranscriptional processes.MicroRNA is endogenous,≈22nucleotides, noncoding RNA which binds topartially complementary sites primarily found in the3’untranslated region (3’UTR) oftarget mRNA and inhibits gene expression via induction of mRNA degradation andtranslational repression. MiR-19b is reported to be implicated in lipid metabolism. MiR-19b is induced in mice fed with high fat diet. MiR-19b is upregulated in humanaortic atherosclerotic lesion and aortic aneurysm. A growing body of evidenceindicates the important role of miR-19b in atherosclerosis progression. However, it isstill largely unknown about the proatherogenic mechanism of miR-19b.Preliminary bioinformatic prediction reveals that miR-19b can coincidentlytarget ABCA1mRNA. Accordingly, we speculate that miR-19b targets macrophageABCA1expression, inhibits intracellular cholesterol efflux and RCT, subsequentlypromotes macrophage cholesterol accumulation and lipid deposition, which causeatherosclerosis progression. To verify the hypothesis of miR-19b’s proatherogenicmechanism, firstly, our study thoroughly predicted miR-19b’s targeting action onABCA1, and certified the targeting action with luciferase reporter gene in the firstpart. Then, in the second part we observed the role of miR-19b in macropahgeABCA1expression, intracellular cholesterol efflux and lipid content. Next, in thethird part we observed the effect of miR-19b on RCT, plasma lipid profile, and aorticatherosclerosis development in vivo. Finally, we examine the influence of diosgenin(Dgn), which had proved to have significant antiatherogenic feature, on miR-19b level,ABCA1expression and lipid metabolism in vitro, and on RCT, plasma lipid profile,and aortic atherosclerosis development in vivo, and explore its possible mechanismsin the fourth part. Our findings provide new insights for revealing the potential roles,mechanisms and therapeutic value of miR-19b in atherosclerosis progression.Part I: The predicted target gene of miR-19bAims: To investigate the target genes of miR-19b, and identifiy the targetingaction of miR-19b on ABCA13’UTR.Methods: Sequences of mature miR-19b and ABCA13’UTR were obtainedfrom online website miRBase and GeneBank respectively. Based on this, we analyzedthe annealing of miR-19b to ABCA13’UTR, the miR-19b binding sites and the minimum free energy of the hybridisation with online databases miRDB, miRanda,miRNA Viewer, TargetScan and RNAhybrid. The full ABCA13’UTR was amplifiedby RT-PCR from human THP-1macrophage. Wild type and mutant cDNA fragmentswere then cloned into luciferase reporter vector psiCHECK-2using restriction sitesXhoI and NotI linker, to construct the fusional expression plasmids including thepsiCHECK-2-ABCA1-WT3’UTR and psiCHECK-2-ABCA1-Mut3’UTR.Relative luciferase activity and the mRNA level of ABCA13’UTR was detected inHEK293T cell cotransfected with miR-19mimic and the fusional plasmids.Results: Bioinformatic analysis revealed multiple miR-19b binding sites existingwithin human/mouse ABCA13’UTR, high context score of miR-19b binding toABCA13’UTR and very low free energy of this hybridisation. Relative luciferaseactivity and the mRNA level of ABCA13’UTR were down-regulated in HEK293Tcell cotransfected with miR-19mimic and wild type recombinant plasmid. Whencotransfected with miR-19mimic and mutant recombinant plasmid, alteration ofrelative luciferase activity and the mRNA level of ABCA13’UTR had nosignificance.Conclusion: miR-19b putatively targets ABCA13’UTR, and miR-19b is provento directly bind to macrophage ABCA13’UTR.Part II: MiR-19b targets macrophage ABCA1gene andimpairs intracellular cholesterol effluxAims: To observe the effect of miR-19b on ABCA1expression, intracellularcholesterol efflux and lipid accumulation in THP-1macrophage/MPM.Methods: THP-1macrophage/MPM was cultured with medium1640andtransfected with miR-19b mimic, inhibitor or ABCA1siRNA. The protein and mRNAexpression of ABCA1were examined by western blot and real-time quantitative PCR,respectively. Intracellular3H-cholesterol efflux was detected with liquid scintillator. The levels of cellular total cholesterol (TC), free cholesterol (FC) and cholesterol ester(CE) were assayed with HPLC. Intracellular lipid droplet was stained with oil red O.Results: The mRNA and protein levels of ABCA1were down-regulated inTHP-1macrophage/MPM transfected with miR-19b mimic. ApoAI–mediated(ABCA1dependent) cholesterol efflux was dramatically suppressed by enhancingmiR-19b function with its mimic, resulting in the increase in the content of TC, TCand CE. The numerous droplets densely presented all over the intracellular regions ofmacrophages. The exactly opposite results were observed by anti-miR-19b in THP-1macrophage/MPM.Conclusion:①miR-19b shows the ability to regulates macrophage ABCA1expression.②miR-19b impairs macrophage cholesterol efflux and promotes lipidaccumulation, and that ABCA1plays an important role in this process.Part III: Effect of miR-19b on RCT, aortic lipid deposition andatherosclerotic lesion in apolipoprotein E knockout miceAims: To observe the effect of miR-19b on RCT, plasma lipid profile, aortic lipiddeposition and atherosclerotic lesion in apoE knockout (apoE-/-) mice.Methods: Forty five six-week-old male apoE-/-mice were fed with Western dietand randomly divided into3groups: control (Con) group, miR-19b agomir (Ago)group, miR-19b antagomir (Ant) group. Every4weeks, Ago group and Ant groupwere received tail vein injection of80mg/kg/d miR-19b agomir or antagomirrespectively. The control group was received intravenous injections the same volumeof PBS at the same time. MPM was radiolabeled with3H-cholesterol andintraperitoneally injected into the apoE-/-mice at2day before sacrifice. After8weeks,all animals were euthanized. Blood, feces and hepatic tissue were collected todetected RCT efficiency with liquid scintillation counting. Plasma lipids were determined via commercially enzymatic methods. Atherosclerotic area of aorticintima was stained with Sudan IV. Atherosclerotic lesion in aortic sinus was tested byHE stain. Lipid accumulation in aortic sinus was evaluated by Oil Red O stain.Masson’s staining was for collagen content. Protein expression of ABCA1in aortawas detected by western blot.Results: The excretion of3H-cholesterol originating from cholesterol-ladenMPM into feces and plasma HDL-C level was decreased in apoE-/-mice injected withmiR-19b agomir. MiR-19b agomir increased atherosclerosis area and lipid depositionin aortic intima, decreased the stability of atherosclerosis lesion and aortic ABCA1expression. MiR-19b antagomir obviously increased fecal3H-sterol and plasmaHDL-C level. Lipid deposition and atherosclerosis area were reduced, and ABCA1expression was up-regulated in aortas of apoE-/-mice treated with miR-19b antagomir.Conclusion:①MiR-19b overexpression impairs RCT and exacerbates theatherosclerosis progression in apoE-/-mice.②Down-regulated ABCA1expressionin aortas is likely to involved in the proatherogenic mechanism of miR-19b.Part Ⅳ: Effect and mechanism of Dgn on macrophage ABCA1Expression and aortic atherosclerosis in apoE-/-miceAims: To investigate the effect and molecular mechanism of Dgn on ABCA1expression in THP-1macrophage/MPM, and RCT and aortic atherosclerosis in apoE-/-mice.Methods: THP-1macrophage/MPM was treated with Dgn, alone or incombination with miR-19b mimic/inhibitor. The protein and mRNA expression ofABCA1were examined by western blot and real-time quantitative PCR, respectively.Intracellular3H-cholesterol efflux was detected with liquid scintillator. The content ofintracellular lipid is assayed with HPLC. Sixty six-week-old male apoE-/-mice werefed with Western diet and randomly divided into4groups: Con group, Dgn group, Dgn+Ago group, Dgn+Ant group. In vivo RCT efficiency was detected with liquidscintillation counting. Plasma lipids were determined via commercially enzymaticmethods. Atherosclerotic lesion in aortic sinus was tested by HE stain. Lipidaccumulation in aortic sinus was evaluated by Oil Red O stain. Masson’s staining wasfor collagen content.Results: Dgn up-regulated macropahge ABCA1expression in time-and dose-manner. Dgn significantly decreased macrophage miR-19b level and intracellularcontent of TC, CE, and FC. Dgn dramatically increases fecal3H-sterol and plasmaHDL-C level, and decreases plasma LDL-C level. Dgn up-regulated aortic ABCA1expression, decreased aortic atherosclerotic area, relieved lipid accumulation in aorticsinus and strengthened the stability of atherosclerotic lesion. MiR-19b overexpressionabrogated the antiatherogenic effect of Dgn, while miR-19b inhibition enhancedDgn’s effect.Conclusion:①Facilitation of macrophage cholesterol efflux and in vivo RCTis implicated in the antiatherogenic effect of Dgn.②Partial mechanism of Dgn isthat Dgn up-regulates ABCA1expression by inhibiting miR-19b level.