Role And Mechanism Of MiR-150 In Atherosclerosis

BackgroundCardiovascular disease is the leading causes of death in worldwide.The pathological basis of most cardiovascular diseases is atherosclerosis(AS)but the specific mechanism remains unclear.Over the past years,AS has been considered as a chronic pathological process involving multiple risk factors,such as heredity,environment,cellular immunity,inflammation,lipid metabolism disorder,and apoptosis.Currently,inflammation has been recognized as an important factor in AS pathogenesis since manipulation of inflammatory factors could change the development and ending of AS.Therefore,discovering AS-related genes that could influent AS pathogenesis is of great meaning for the prevention and treatment of AS.MiR-150 is a 22 nucleotide non-coding small molecule RNA which located at the 19q13.33 locus in human and widely observed in various mammals.Physiologically,miR-150 is highly expressed in mature lymphocyte,regulatory T cells,B cells,lymph gland and spleen.miR-150 has high conservation during evolution and functions through targeting 3 '-UTR and negatively controlling mRNA in the process of material metabolism,cell development,tumor occurrence and immune inflammation.Recent researches showed the important roles of miR-150 in AS-related macrophage foaming and endothelial cell differentiation,and activation/migration of monocyte colonies from human bone marrow.Besides,miR-150 has also been proved to regulate monocyte migration and pro-inflammatory cell products after myocardial infarction.The above researches proved the close relationship between miR-150 and AS at cellular level.However,whether miR-150 contribute to the occurrence of AS in vivo is still unknown.ObecjectsTo further discuss the role and potential molecular mechanism of miR-150 in AS by establishing high fat feeding AS mice models in ApoE-/-mice and miR-150-/-ApoE-/-mice.MethodsPart I:Littermate male ApoE-/-mice(8 weeks old,21-23 g)were divided into the high fat diet(HFD)group and normal chow(NC)group and fed with HFD for 16/28 weeks or NC for 16 weeks.Human coronary atherosclerosis specimens and normal coronary samples were collected and evaluated with H&E staining and miR-150 levels were tested with real-time PCR.At the same time,bone marrow-derived macrophages(BMDMs)and vascular smooth muscle cells(VSMCs)were harvested and cultured.After stimulation of Ox-LDL,the miR-150 levels were tested with RT-PCR.Part II:Male ApoE-/-or miR-150-/-ApoE-/-mice(8 weeks old,21-23 g)were divided into the HFD group and NC group,respectively(n=20 in each).After 28 weeks feeding,H&E staining and oil red O staining were applied for morphological evaluation;immunofluorescent staining,PSR and oil red O staining were applied to evaluate plaque stability and inflammatory infiltration;RT-PCR was applied to test Inflammatory cytokines and chemokines;western blot was applied to examine the activation of NF-?B;bone marrow transplant experiment was used to testified the functions of miR-150 in BMDMs;BMDMs from ApoE-/-or miR-15150-/-ApoE-/-mice were harvested and cultured and Ox-LDL stimulation was used to evaluate macrophage migration.Part ?:Targetscan was applied to predict and screen potential target genes with miR-150.RT-PCR was adopted to test the expression of all target genes in BMDMs.Luciferase assay was used to examine the interaction between 3'UTR and miR-150 in all trending target genes.The target genes were then knockdown and examined for pro-inflammatory cytokine levels and macrophage migration.At last,western blot was performed to test P65 phosphorylation in BMDMs after knockdown of target genes.ResultsPart ?:H&E staining showed obvious plaques lipid-rich core in patient with coronary heart disease.PCR results showed miR-150 level in AS patients was significantly elevated(P<0.05 vs Normal).For mice after 16 weeks feeding,HFD group had a larger plaque area than the NC group with a corresponding increase of miR-150 levels and a more significant trending was observed after 28-week high fat feeding(P<0.05).After Ox-LDL stimulation,the miR-150 was significantly upregulated in BMDMs(P<0.05)while there was no changes in VSMCs(P>0.05).Part II:After feeding with HFD/NC for 28 weeks,oil red O staining of aorta tree showed,the areas of the atherosclerotic lesions in miR-150-/-ApoE-/-mice was significantly lower than ApoE-/-mice(21.31%vs.38.21%,P<0.05)in HFD group.Similar results were observed in NC group(5.18%vs.5.95%,P<0.05).H&E staining showed plaque area of the aortic sinus and arch in miR-150-/-ApoE-/-mice were less than ApoE-/-mice(P<0.05)while no significant changes of lipids metabolism were observed(P>0.05).Immunofluorescence,PSR and oil red O staining showed the collagen of AS plaque and a-SMA were increased but CD36 and lipid level were decreased after miR-150 knockout.PCR showed the mRNA level of serum pro-inflammatory cytokines was reduced in miR-150-/-ApoE-/-mice.Bone marrow transplant experiment showed miR-150 promoted AS in BMDMs.Western blot showed after the activation of NF-?B was inhibited after miR-150 knockout.The deficiency of miR-150 in BMDMs results in the reducing of macrophage adhesion/migration and proinflammatory cytokines.Part ?:Targetscan predicted and screened several target genes for miR-150,including PDLIM1,CXCL3,SP1,CD276,IL2RA and CXCL2.PDLIM1 was elevated in BMDMs from miR-150-/-ApoE-/-mice and the interaction zone could be located at 3'-UTR of PDLIM1.Luciferase reporter test showed after transfecting the inhibitor of miR-150,the activity of PDLIM1 was increased and this impact disappeared after the mutation of 3'-UTR.The knockdown of PDLIMI could reverse the reducing of pro-inflammatory factors(TNF-??IL-6?IL-1? and CXCL3)and macrophage infiltration after miR-150 deficiency.Conclusion1.Expression of MiR-150 is upregulated in AS plaque of human and mice;after stimulation of Ox-LDL,miR-150 is upregulated in BMDMs while no significant change was observed in VSMCs.2.After miR-150 knockout,AS plaque can be reduced and stabilized;pro-inflammatory cytokines and chemokine released by macrophage are reduced;miR-150 plays its role in BMDMs.3.miR-150 inhibited the transcriptional activity of PDLIM1 via targeting 3'-UTR of PDLIM1;the knockdown of PDLIM1 could reverse the reducing of pro-inflammatory factors(TNF-??IL-6?IL-1? and CXCL3)and macrophage infiltration after miR-150 deficiency.The deficiency of miR-150 may prevent AS through upregulating PDLIM1 as its target.