MiR-590 Inhibits Atherogenesis Via Targeting Macrophage LPL In ApoE^-/- Mice

Atherosclerosis(As) is the pathological basis of cardio-cerebral vascular diseases. Lipid accumulation and inflammation response in macrophages are the two key factors in atherogenesis. Therefore, reducing lipid accumulation and inflammatory response in macrophages has the important significance in the prevention and treatment of atherosclerotic cardio-cerebral vascular diseases.Lipoprotein lipase(LPL) is the key rate-limiting enzyme to hydrolyze triglycerides, which is also closely associated with atherosclerosis. Increasing studies showed that specific knockout of macrophages LPL in apoE-/- mice or rabbit model could obviously improve lipid deposition and atherosclerotic plaque area in aortic wall of the animals, which demostrated macrophages LPL could promote atherosclerosis. Macrophages LPL can cause excessive lipid accumulation by promoting lipid intake and can also stimulate expression of various inflammatory factors. Researchers are trying to find regulatory factors that directly inhibit macrophage LPL expression in vivo, to explore a XII new therapeutic approach for the prevention and treatment of cardiovascular and cerebrovascular disease.MicroRNAs are single strand(about 18-22 nucleotides long) endogenous non-coding RNAs, which match 3’untranslated region of mRNAs(3’UTR) completely or incompletely via the formation of RNA induced silencing complex(RISC), inducing degradation of mRNAs and inhibition of mRNA translation into protein. MiR-590 is located in the proximal long arm of chromosome 7 in human genome. Recent studies have found miR-590 has the effects on blood vessels protection, lipid regulation, prevention of myocardial infarction and so on. MiR-590 expression is down-regulated in cardiovascular diseases, which suggests MiR-590 may be a protective microRNA, but the specific mechanism is not yet clear.Through bioinformatics analysis, we found that miR-590 can coincidently target LPL mRNA. In preliminary experiments, we found that overexpression of miR-590 could silence LPL mRNA and protein expression in THP-1 macrophages. Therefore, we put forward a working hypothesis that miR-590 can silence LPL expression by directly targeting LPL 3’UTR and play an anti-As role by inhibiting lipid accumulation and inflammatory reaction in macrophages.Part I: Effect of miR-590 on Lipid Accumulation and Inflammatory Cytokines Production in Macrophages.Aims: To investigate the effect of over-expression or inhibition of miR-590 on lipid accumulation and inflammatory cytokines production in macrophages.Methods: Human THP-1 monocytes and mouse RAW264.7 monocytes were cultivated and induced to differentiate into macrophages. Both macrophages were divided into 4 groups: miR-590 mimic transfected group, mimic-negnative control group, miR-590 inhibitor transfected group and inhibitor-negnative control group, respectively. The mi R-590 levels were examined by real-time quantitative PCR. The lipid accumulation was assessed by Oil red O staining and the content of total cholesterol(TC), free cholesterol(FC) and cholesterol ester(CE) were observed by HPLC in THP-1 macrophages. The concentrations of interleukin-6(IL-6), interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), IL-6 and monocyte chemoattractant protein-1(MCP-1) in cell culture supernatants were measured by enzyme-linked immunosorbent assay(ELISA).Results: ① miR-590 mimic transfection could significantly increase miR-590 expression, miR-590 inhibitor transfection could inhibit miR-590 expression. ②The data of Oil red O staining and HPLC showed that miR-590 mimic transfection could significantly reduce volume and quantity of lipid droplets, decrease the content of TC, FC and CE in macrophages, however miR-590 inhibitor transfection could significantly XIV add volume and quantity of lipid droplets, increase the content of TC, FC and CE in macrophages. ③ ELISA data showed that miR-590 mimic transfection could significantly reduce secretion of IL-6、IL-1β、TNF-α and MCP-1, however, miR-590 inhibitor transfection could promote the production of inflammatory cytokines.Conclusion: ① miR-590 inhibits lipid accumulation and in macrophages. ② miR-590 inhibits the secretion of inflammatory cytokines in macrophages.Part II: Reduction Effect of miR-590 on Lipid Accumulation and Inflammatory Cytokines Production by targetly Inhibiting LPL in MacrophagesAims: ①To identify whether mi R-590 is combined with gene LPL and inhibit its expression. ②To observe the effect and related mechanism of mi R-590 on on lipid accumulation and inflammatory cytokines production after overexpression or silence of LPL in macrophages.Methods: MiR-590 conservation in each species, LPL3 ’UTR sequence, mi R-590 target genes, binding grade and sites miR-590 and LPL, stem loop structure and free energy were predicted and analysed by using multiple public database and online software. MiR-590 combined targetly with LPL was detected by the method of dual luciferase report gene. Human THP-1 monocytes and mouse RAW264.7 monocytes were cultivated and induced to. differentiate into macrophages. Both macrophages were divided into 4 groups: mi R-590 mimic transfected group, mimic-negnative control group, miR-590 inhibitor transfected group and inhibitor-negnative control group, respectively. The LPL mRNA and protein expression were examined by real-time quantitative PCR and western blot. LPL activity in macrophages was detected by kits. After using inhibition of LPL siRNA or addition of exogenous LPL in THP-1 macrophages, the effect of miR-590 mimic or miR-590 inhibitor on lipid accumulation were observed by Oil red O staining and the content of TC, FC and CE were detected by HPLC. In addition, we detected the effect of miR-590 on SRA1 and CD36 expression, the key scavenger receptor. ELISA was used to detect the effect of miR-590 mimic or miR-590 inhibitor on secretion of IL-6、IL-1β、TNF-α and MCP-1. At the same time, we also tested the effect of miR-590 on phospholipid components in THP-1 macrophages.Results: ① Bioinformatics analysis showed that miR-590 is highly conservative in evolution, play a key role in evolution of species and regulation of target genes and has biological basis to regulate the LPL gene. ②Relative luciferase activity of LPL 3’UTR was down-regulated in HEK 293 T cells cotransfected with miR-590 mimic and wild type recombinant plasmid. When HEK 293 T cells were cotransfected with mi R-590 inhibitor and wild type recombinant plasmid, the relative luciferase activity of LPL 3’UTR was up-regulated. ③Real-time quantitative PCR and XVI western blot results showed that miR-590 mimic can reduce LPL mRNA and protein expression, whereas miR-590 inhibitor can increase LPL mRNA and protein expression in human THP-1 and mouse RAW 264.7 macrophages. ④miR-590 mimic could down-regulated LPL activity, but up-regulated LPL activity in human THP-1 and mouse RAW 264.7 macrophages.⑤Macrophages transfected with miR-590 mimic could reverse the reduction effect of lipid levels by mi R-590 mimic in macrophage after added with bovine LPL. Macrophages transfected with mi R-590 inhibitor could reverse the increase effect of lipid levels by mi R-590 inhibitor in macrophage after treated with LPLsiRNA. ⑥The SRA1 and CD36 expression was decreased obviously in macrophages transfected with mi R-590 mimic. Addition of LPL in miR-590 mimic group could reverse the trend. The SRA1 and CD36 expression were increased obviously in macrophages transfected with miR-590 inhibitor. Silence of LPL in miR-590 inhibitor group could reverse the trend. ⑦Macrophages transfected with miR-590 mimic could reverse the reduction effect of inflammatory cytokines production by miR-590 mimic in macrophage after added with bovine LPL. Macrophages transfected with mi R-590 inhibitor could reverse the increase effect of inflammatory cytokines production by miR-590 inhibitor in macrophage after treated with LPLsiRNA. ⑧ The lyso-PC levels were decreased and phosphatidylcholine levels were increased in miR-590 mimic group. The. lyso-PC levels were increased and phosphatidylcholine levels were decreased in miR-590 inhibitor group. Addition or silence of LPL in miR-590 mimic group and miR-590 inhibitor group could turn lyso-PC levels into the same levels compared with control group.Conclusion: ①miR-590 putatively targets LPL 3’UTR, and miR-590 is proven to directly bind to macrophage LPL 3’UTR. ②miR-590 can inhibit LPL mRNA and its protein expression in macrophages. ③mi R-590 inhibits lipid accumulation and the secretion of inflammatory cytokines in macrophages by suppressing targetly LPL.Part III: Effects of miR-590 on Atherosclerotic Lesions and Inflammatory Cytokines Expression in apoE-/- MiceAims: To observe the effect of mi R-590 on As lesion, lipid accumulation in the aortic wall, plasma lipid level, inflammatory response and the LPL expression in apoE-/- mice.Methods: Eight-week old male apoE-/- mice were fed with western diet and randomly divided into four groups(n=15 per group) miR-590 agomir group(AG), miR-590 scrambled agomir group(AG-Con), mi R-590 antagomir group( AN), mi R-590 scrambled antagomir group(AN-Con). Every 4 weeks, AG group and AN group were received tail vein injection of 80 mg/kg/d mi R-590 agomir or antagomir respectively. The control group was received intravenous injections the same volume of PBS XVIII at the same time. After 8 weeks, all animals were sacrificed, blood was obtained, and tissues were collected for further analysis. Lipid deposition in blood vessel wall was observed by stereoscopic microscope. Triglyceride(TG), total cholesterol(TC), HDL-C and LDL-C were determined by commercially enzymatic methods. Lipid accumulation in aorta and aortic sinus were evaluated by Sudan stain and Oil Red O stain. Collagen levels were evaluated by Masson’s staining. Colocalization of LPL and macrophages in plaque were detected by immunofluorescence double labeling. The LPL mRNA and protein expression in aorta tissue and peritoneal macrophage were examined by real-time quantitative PCR and western blot. Lipid levels in abdominal cavity macrophages were detected by HPLC. The mRNA and protein expression of SRA1 and CD36 in peritoneal macrophage were examined by real-time quantitative PCR and western blot. Plasma inflammatory cytokines(IL-6、IL-1β、TNF-α and MCP-1) expression were detected by ELISA.Results: ① Stereoscopic microscope and Sudan Ⅳ staining results showed that aortic plaques were decreased significantly in AG group compared with AG-Con group. However, aortic plaques were increased significantly in AN group compared with AN-Con group. ②Oil red O staining showed that lipid accumulation in aortic sinus in AG group was reduced compared with AG-Con group. Compared with the AN-Con group, lipid accumulation in aortic sinus in AN group was increased significantly.. ③Masson trichromatic staining results showed that collagen fiber content in the aortic sinus was decreased in AG group compared with AG-Con group. Compared with the AN-Con control group, the collagen fiber content was increased significantly in AN group compared with AN-Con control group. ④ Immunofluorescence double labeling results displayed positive area of LPL expression is consistent with rich area of macrophages expression in As lesions in aortic root. Compared with AG-Con group, macrophage LPL expression was reduced significantly in aortic sinus in mi R-590 AG group. The macrophage LPL expression was increased significantly in aortic sinus in mi R-590 AN group compared with the AN-Con group. ⑤ Real-time quantitative PCR and western blot results showed that LPL mRNA and protein expression in aorta tissue and peritoneal macrophage were decreased in miR-590 AG group compared with AG-Con group. LPL mRNA and protein expression in aorta tissue and peritoneal macrophage were increased in miR-590 AN group compared with AN-Con group. ⑥TG levels were increased and TC、LDL-C levels were decreased in miR-590 AG group compared with AG-Con group. TG levels were decreased and TC、LDL-C levels were increased in mi R-590 AN group compared with AN-Con group.⑦HPLC results showed that lipid levels in peritoneal macrophages were significantly reduced in miR-590 AG group compared with AG-Con group. Compared with the AN-Con group, lipid levels in peritoneal macrophages in mice were increased significantly XX compared with mi R-590 AN group. ⑧ Real time quantitative PCR and Western blotting results showed that overexpression of mi R-590 could obviously reduce the mRNA and protein expression of SRA1 and CD36 in peritoneal macrophages of the apoE-/- mice. Inhibition of miR-590 could obviously increase the mRNA and protein expression of SRA1 and CD36 in peritoneal macrophages of the apoE-/- mice. ⑨ELISA results displayed that, compared with AG-Con group, secretion of plasma IL-6、IL-1β、TNF-α and MCP-1 were significantly decreased in miR-590 AG group. Compared with the AN-Con group, inflammatory cytokines in plasma were significantly increased in mi R-590 AN group.Conclusion: ①miR-590 has the inhibition effect on As lesion, lipid accumulation in the aortic wall, plasma lipid level, inflammatory cytokines in apoE-/- mice.②miR-590 may alleviate As formation in blood vessel walls of apoE-/- mice by inhibiting macrophage LPL.Part Ⅳ : Effect of Nobiletin on Macrophage miR-590/LPL Expression and Atherosclerosis in apoE-/- miceAims: To investigate the effect of Nobiletin on miR-590 levels, LPL expression in THP-1 macrophage, and As lesion, lipid accumulation in the aortic vessel wall, plasma lipid level, inflammatory cytokines in apo E-/- mice..Methods: The miR-590 levels were examined by real-time quantitative PCR after THP-1 macrophages were treated with Nobiletin. When the THP-1 macrophages treated with Nobiletin were transfected with miR-590 mimic/inhibitor, the protein and mRNA expression of LPL were examined by western blot and real-time quantitative PCR, respectively. The effect of Nobiletin on lipid accumulation was observed by HPLC and Oil red O staining. ELISA was used to detect the effect of Nobiletin on secretion of IL-6 、 IL-1β 、 TNF-α and MCP-1 in THP-1 macrophages. The effect of Nobiletin on the As lesions in artery in apoE-/- mice were evaluated by Sudan stain. The effect of Nobiletin on lipid accumulation in aortic sinus in apoE-/- mice was evaluated by Oil Red O stain. The effect of Nobiletin on collagen levels in aortic sinus in apoE-/- mice were evaluated by Masson’s staining. TG, TC, HDL-C and LDL-C were determined by commercially enzymatic methods after treated with Nobiletin in apoE-/- mice. The effect of Nobiletin on plasma inflammatory cytokines(IL-6、IL-1β、TNF-α and MCP-1) expression were detected by ELISA. The LPL protein expression in aorta tissue was examined by western blot.Results: ①Nobiletin down-regulated macropahge LPL expression in time- and dose- manner. ②Nobiletin significantly increased macrophage mi R-590 level. ③ miR-590 mimic transfection obviously enhanced the inhibition effect of Nobiletin on LPL expression. MiR-590 inhibitor transfection reduced the inhibition effect of Nobiletin on LPL expression. ④XXII HPLC and Oil red O staining results showed that Nobiletin decrease lipid levels in macrophages. Nobiletin combined with transfection with mi R-590 mimic could further decrease significantly lipid levels. However Nobiletin combined with transfection with miR-590 inhibitor could increase lipid levels, which has on difference with those in control group. ⑤ELISA results showed that Nobiletin decrease secretion of IL-6、IL-1β 、 TNF-α and MCP-1 in macrophages. Nobiletin combined with transfection with miR-590 mimic could further decrease significantly inflammatory cytokines levels. However, Nobiletin combined with transfection with miR-590 inhibitor could increase inflammatory cytokines levels, which has on difference with those in control group. ⑥Sudan Ⅳ staining results showed that aortic plaques were decreased significantly in Nob group compared with control group. The aortic plaques were further decreased significantly in Nob+AG group. The aortic plaques were increased in Nob+AN group compared with Nob group. ⑦Oil red O staining results showed that lipid accumulation in aortic sinus in Nob group was reduced compared with control group. The lipid accumulation in aortic sinus was further decreased significantly in Nob+AG group. The lipid accumulation in aortic sinus was increased in Nob+AN group compared with Nob group. ⑧ Masson trichromatic staining results showed that collagen fiber content in the aortic sinus was decreased in Nob group compared with control group. The collagen fiber content in the aortic. sinus was further decreased significantly in Nob+AG group. The collagen fiber content in the aortic sinus was increased in Nob+AN group compared with Nob group. ⑨ Compared with control group, HDL-C levels were increased and TG、TC and LDL-C levels were decreased in Nob group. TC and LDL-C levels were decreased more obviously, HDL-C level was increased, TG has on difference in Nob+AG group. TC、LDL-C levels were increased, TG level was decreased in Nob+AN group and HDL-C level has on difference compared with Nob group. ⑩ELISA results displayed that, compared with control group, secretion of plasma IL-6、IL-1β、TNF-α and MCP-1 were decreased in Nob group. The inflammatory cytokines levels were decreased significantly in Nob+AG group. The inflammatory cytokines levels were increased in Nob+AN group compared with control group. 11 Western blot results showed that LPL protein expression in aorta tissue was decreased in Nob group compared with control group. LPL protein expression in aorta tissue was decreased significantly in Nob+AG group. LPL protein expression in aorta tissue was increased in Nob+AN group.Conclusion: ① Anti-inflammation and inhibition of plasma lipids are involved in the mechanisms of Nobiletin treatment alleviates atherosclerosis regression in apoE-/- mice. ②The inhibition effect of miR-590 on LPL was strengthened by Nobiletin involved in the reduction of atherosclerosis.