| Part 1.LncRNA MIAT promotes atherosclerotic plaque instability Objective:To collect clinical patients with acute ischemic stroke with unstable carotid atherosclerotic plaques,comparing them with asymptomatic patients with stable carotid atherosclerotic plaques and normal healthy people,analyze the difference in plasma LncRNA MIAT expression.Atherosclerosis model was established in Apo E-/-mice fed with high-fat diet,and the differences in LncRNA MIAT expression in plasma and aortic plaque tissues of Apo E-/-mice fed with high-fat diet at 8 weeks and 16 weeks were analyzed to explore the correlation between LncRNA MIAT and atherosclerosis.Methods:Twenty patients with acute ischemic stroke with unstable carotid atherosclerotic plaques,18 patients with asymptomatic stable carotid atherosclerotic plaques and 20 healthy controls were examined by ultrasound and high-resolution 3.0MR to confirm the nature of carotid atherosclerotic plaques.LncRNA MIAT expression in peripheral blood was analyzed by q RT-PCR.The atherosclerosis model was established in Apo E-/-mice fed a high-fat diet.Thirty Apo E-/-mice aged 7 weeks were randomly divided into the normal diet group(NCD,n=15)and the high-fat diet group(HFD,n=15).QRT-PCR was used to analyze the differences in LncRNA MIAT expression in plasma and aortic plaque tissues of Apo E-/-mice fed with high fat for 8 weeks and 16 weeks.Results: Compared with asymptomatic patients with stable carotid plaque and normal healthy people,LncRNA MIAT was significantly increased in the plasma of acute ischemic stroke patients with unstable carotid plaque,and the difference was statistically significant(P< 0.05).The expressions of LncRNA MIAT in plasma and aortic plaque tissues of Apo E-/-mice after 8 weeks and 16 weeks of high-fat feeding were higher than those in the normal diet group,and the difference was statistically significant(P< 0.05).The plasma and aortic plaque tissue of Apo E-/-mice after 16 weeks of high-fat feeding were significantly higher than that after 8 weeks of high-fat feeding,and the difference was statistically significant(P< 0.05).Conclusion: LncRNA MIAT in peripheral plasma and plaque tissues is significantly correlated with the stability of atherosclerotic plaques,and LncRNA MIAT is a key target for monitoring and intervention of atherosclerotic plaque vulnerability.Part 2.LncRNA MIAT regulates efferocytosis of macrophages in atherosclerotic plaques Objective: To observe the expression and distribution of LncRNA MIAT in atherosclerotic plaques and macrophages.To investigate the effect of down-regulated LncRNA MIAT on plaque load and plaque stability of Apo E-/-mice.To observe the effect of down-regulation of LncRNA MIAT on efferocytosis of macrophages in Apo E-/-mice plaques.Methods:(1)Ox-LDL was used to treat macrophages to construct an in vitro atherosclerosis(AS)model,and q RT-PCR and RNA fluorescence in situ hybridization(FISH)were used to analyze the expression and distribution of LncRNA MIAT in macrophages.(2)Atherosclerosis model was established in Apo E-/-mice fed a high-fat diet.Thirty male Apo E-/-mice aged 7 weeks were randomly divided into normal diet group(NCD,n=15)and high-fat diet group(HFD,n=15).The expression pattern of LncRNA MIAT in macrophages and smooth muscle cells in atherosclerotic plaques was observed by immunofluorescence.(3)Atherosclerosis model was established in Apo E-/-mice fed a high-fat diet.70 male Apo E-/-mice aged 7 weeks were randomly divided into PBS group(n=10),scr-sh RNA group(n=30)and MIAT-sh RNA group(n=30).PBS,Ad-scr sh RNA and Ad-MIAT sh RNA were administered intraperitoneally at 11 weeks of age(4 weeks of high-fat feeding).Intraperitoneal injection of MIAT-sh RNA packaged adenovirus was conducted to down-regulate the expression of LncRNA MIAT in vivo,50?l/g(virus titer: 1.0×1013 viral genomes/m L)intraperitoneally every 4 weeks.At the age of 23 weeks,Apo E-/-mice were sacrificed and the aorta was separated from the whole aorta.Continuous sections(6?m thick)of the aorta root were frozen,and the effects of LncRNA MIAT on plaque load and plaque stability were quantitatively analyzed by oil red O staining,HE staining,MASSON staining,alpha-SMA staining and MOMA-2 staining.Positive caspase-3 and TUNEL staining were used to evaluate the apoptotic cells in the plaque,and whether the apoptotic cells co-located with macrophages was used to evaluate the efferocytosis effect in the plaque.Results:(1)FISH detection revealed that LncRNA was mainly expressed in macrophages in the progressive plaques of Apo E-/-mice,while a small amount of LncRNA was expressed in smooth muscle cells in the plaques.(2)Ox-LDL can lead to up-regulation of LncRNA MIAT expression in macrophages in a time-dependent and concentration-dependent manner.FISH suggested that LncRNA MIAT was expressed in both cytoplasm and nucleus of macrophages,mainly in cytoplasm.(3)Down-regulation of LncRNA MIAT can reduce the vascular plaque load of high-fat fed Apo E-/-mice,independent of plasma lipid distribution and body weight.(4)Down-regulation of LncRNA MIAT can increase the stability of atherosclerotic plaques.(5)Down-regulation of LncRNA MIAT can improve the stability of atherosclerotic plaques by promoting efferocytosis of macrophages.Conclusion:LncRNA MIAT promotes the progression and instability of plaques by regulating the efferocytosis of macrophages in plaques.Part 3.LncRNA MIAT sponges mi R-149-5p to inhibit efferocytosis through CD47 upregulation Objective: To induce apoptosis of macrophage RAW246.7 by ox-LDL,and construct AS modelin vitro.To investigate the specific mechanism of LncRNA MIAT onefferocytosis of macrophage in vitro.Methods :(1)Ox-LDL(150?g /ml)was used to stimulate macrophages to induce apoptosis,and apoptotic macrophages in atherosclerotic plaques were simulated in vitro.WB was used to detect the expressions of apoptosis-related proteins Bax,caspase-3,and Cleaved caspase-3.(2)Three si-MIAT was designed to down-regulate the expression of macrophage LncRNA MIAT,and its effectiveness was confirmed by q RT-PCR,and its effect on macrophage apoptosis was confirmed by flow analyzer.(3)Raw264.7 cells were transfected with si-MIAT or si-NC,and ox-LDL(150?g /ml)was used to induce apoptosis.CFSE green fluorescent labeling(green)was used to co-culture with BMDMs,and the effects of si-MIAT on the phagocytosis index of apoptotic macrophages were evaluated by fluorescence microscope and flow analyzer.(4)Raw264.7 cells were transfected with Ad-MIAT or Ad-NC,and ox-LDL(150?g/ml)was used to induce apoptosis.CFSE green fluorescent labeling(green)was used to co-culture with BMDMs,and the effects of Ad-MIAT on phagocytosis index of apoptotic macrophages were evaluated by fluorescence microscope and flow analyzer.(5)Ox-LDL was used to stimulate macrophages,and WB was used to detect the influence of phagocytosis key molecule CD47 protein level.(6)Up-regulated and down-regulated LncRNA MIAT,and WB was used to detect the effect of ox-LDL on the level of CD47 protein,a key molecule ofphagocytosis.(7)Ox-LDL alone was used to stimulate macrophages,or LncRNA MIAT was up-regulated and down-regulated,and q RT-PCR was used to analyze the effect on CD47 m RNA expression.(8)Through bioinformatics database,LncRNA MIAT and CD47 were analyzed,and RIP,luciferase reporter system and western blot were used to verify the correlation between them.Results :(1)Ox-LDL can stimulate the CD47 protein level of macrophages to be significantly up-regulated in a dose-dependent and time-dependent manner.(2)Si-MIAT can down-regulate the increased expression of CD47 caused by ox-LDL.On the contrary,ad-MIAT can further up-regulate the expression of CD47 protein caused by ox-LDL.(3)Ox-LDL alone stimulated macrophages,or LncRNA MIAT alone was regulated,but the expression of CD47 m RNA in macrophages was not affected.(4)Data analysis showed that there were 8 binding sites between human LncRNA MIAT and mi R-149-5p,while there was 1 binding site between rat LncRNA MIAT and mi R-149-5p.Mir-149-5p has binding sites with CD47.(5)RIP detection and results of the dual-fluorescence enzyme reporting system showed that LncRNA MIAT could specifically bind to mir-149-5p.CD47 is a direct target of mi R-149-5p.Conclusion: LncRNA MIAT sponges mi R-149-5p to inhibit efferocytosis of macrophages in advanced atherosclerosis through CD47 upregulation,promoting the progression and instability of plaques. |
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