The Mechanism Of MiR-155 Regulate The Inflammatory Response Of PDCD4 In Atherosclerotic Plaque

Background:Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipids and infiltration of inflammatory cells.The evidence shown that inflammation plays a central role in the cascade of atherosclerotic events that eventually results in plaque erosion.Macrophages,as the principal inflammatory effector cells in atherosclerosis,were differentiated from peripheral blood monocytes those respond to chemotactic factors and cytokines,and then migrate to the arterial intima.Macrophages involve multiple inflammatory signaling pathways during atherosclerosis.For example,inflammatory mediators can activate TLR4 signal pathway to induce inflammatory response and enhance lipid accumulation in macrophages;NF-?B signal pathways is one of crucial importance in the proinflammatory activation of macrophages;Macrophage derived CCL2 can induce additional macrophage accumulation through a positive feedback regulatory mechanism;Depressing BCL6 can induce CCL2 expression in macrophages in atherosclerotic lesions and promote atherosclerosis.Therefore,macrophages play a key role in the inflammatory response during atherosclerosisMiR-155,one of the mircro-RNA,is a non-coding RNA transcribed from the B-cell Integration Cluster(BIC)that is located on chromosome 21.It is expressed in activated immune cells and has been linked to the function of the immune system.MiR-155 is closely associated with cardiovascular diseases,especially in atherosclerosis.More studies shown,despite in vitro or in vivo,there are many contradictions about miR-155 in atherosclerosis:(1).some researches thought miR-155 inhibited inflammatory response and reduced adhesion and chemokine factors expression.However,other researches shown opposite results.(2).in the animal studies,miR-155-/-/LDL-R-/-double knockout mice appeared to be more susceptible to atherosclerosis induced by a high cholesterol diet.However,in miR-155-/-/Apo E-/-double knockout mice,miR-155 deficiency decreased macrophage inflammation and attenuated atherogenesis.Furthermore,recent researches results demonstrated overexpression miR-155 promoted inflammatory response,foam cell formation and then accelerated atherogenesis.(3).In clinical research,Fichtlscherer et al reported the expression of miR-155 in serum decreased significantly in patients with stable coronary artery disease than normal control.But inconsistent results were reported that the miR-155 level of plasma was the most significantly elevated both in atherosclerotic mice and coronary artery disease patients to the normal control.Furthermore,the expression of the miR-155 was found to be slightly enhanced in the AMI group than non-AMI group.The contradictory outcomes from aforementioned in vivo and in vitro studies illustrate the complexity of the role of miR-155 in atherosclerosis.Analysis of the reasons may be: in the aspect of the cell:(1)specific micrornas can be combined with a variety of messenger RNA(mRNA),namely the one-to-many phenomenon;(2)different m RNA can be combined with a specific micrornas competition,namely the many-to-one phenomenon;(3)the expression of micrornas quantity difference under different conditions and the interaction between the miRNA.In the body(1)the different stages of atherosclerosis act the different role;(2)the role of miR-155 act different effect in different animal model.So its specific mechanism still has a lot of unknown,but currently in to clarify the reason and mechanism of related research is still less.Although there are many contradictions,due to the close of miR-155 and cardiovascular disease,it can be as a new biomarkers and therapeutic targets has been one of the important research content,so we can go to further study and clarify.Programmed cell death 4(PDCD4),play an key role in the development of various human cancers.While PDCD4 was seen as a proinflammatory protein involved in LPS-induced Toll-like receptor 4(TLR4)related inflammatory signaling pathways and may be play an important role in the inflammatory diseases.Frederick F Jet al found PDCD4 can promote inflammatory response through activation of the transcription factor NF-?B and suppression of interleukin 10 in macrophages.In addition,other studies shown PDCD4 have a close relation with cardiovascular cells biology.It can inhibit proliferation and induce apoptosis of many cardiovascular cells.Such as endothelial cells,vascular smooth muscle cells,cardiacmyocytes and fibroblasts.Cheng et al found PDCD4-/-/ApoE-/-double knockout mice with fed a high-cholesterol diet reduced atherosclerotic plaque areas than ApoE-/-knockout mice.Based on the above studies,we found that in cardiovascular diseases,especially in atherosclerosis,PDCD4,as an inflammatory factor,play an important role in promoting inflammatory response and developing atherosclerosis.But the specific regulation mechanism remains unclear.Suppressor of cytokine signaling 1(SOCS1),as one of the SOCS family proteins,is a key negative regulator of inflammation,including LPS-induced inflammation,TLR-4 signaling.SOCS1 can also inhibit inflammatory pathways by a wide range of cytokines,such as TNF-?,IL-6,and INF-?.In addition,studies shown SOCS1 is also one of the miR-155 target genes.MiR-155 can through SOCS1 to regulate inflammatory response negatively in macrophages and macrophage miR-155 can also through SOCS1/signal transducer and activator of transcription 3(STAT3)signaling to promotes cardiac hypertrophy and failure.However the functional role of miR-155 related SOCS1/STAT3 signaling in atherosclerosis remains unclear and some researches think it should be further study.Here,we will explore miR-155 and PDCD4 expression and the relationships between each other in aortic atherosclerotic mice tissues and ox-LDL treatment of RAW264.7 cells.We will clarify whether the miR-155 direct inhibition of expression of SOCS1 and affect PDCD4 expression in macrophages.Further study the mechanism of the miR-155 how to acts on the PDCD4 wll be explored.Subjects will from two parts,animal and cell experiment to verify the relationship between miR-155 and PDCD4.In animal experiments,we will establish animal models to prove the relationship between the miR-155 and PDCD4 and it's downstream of inflammation factors,such as IL-10,IL-6,TNF-?.In cells experiments,to establish the model of macrophage with ox-LDL treatment,application of adenovirus infection overexpression SOCS1,transfection siRNA to silence PDCD4 and STAT3 expression,using antagomiR-155 to suppress miR-155.These test methods will be used by oil red O staining,Luciferase ReporterAssay,Western Blot,Real-time PCR and ELISA to detect protein and gene expression and cell colocalization.Finally,we will verify miR-155 through SOCS1/STAT3/PDCD4 axis promote inflammatory response of the macrophages and affect atherosclerotic plaque stability,resulting in plaque rupture and the occurrence of cardiovascular events.We hope theoretical evidence can provide new ideas and therapeutic targets for clinical atherosclerosis disease treatment.Metehods:1.Animal model to observe the relationship between miR-155 and the inflammatory factorsExperimental animals of C57 wild type mice and Apo E-/-mice are purchase from the Model Animal Research Center of Nanjing University,6 weeks old,weighing 17-20 g.C57 wild type mice and normal diet group Apo E-/-mice use conventional diet.High-fat diet(HFD)Apo E-/-mice were fed HFDs(21% fat,1.25% cholesterol).Animal model establishment: The animal were randomly divided into 5 groups,including C57 wild type mice(n=10 group,conventional diet),ApoE-/-mice(n=10 group,conventional diet),ApoE-/-mice(n=10 group,high fat diet),ApoE-/-mice + Anti-NC(n=10 group,high fat diet),Apo E-/-mice + Anti-miR-155(n=10 group,high fat diet).After feeding one week,Aanti-NC and an Anti-miR-155 were injected through tail vein,control group used Physiological saline.Doses is 25 mg/kg Aanti-NC and an Anti-miR-155 for the first week,spaced 36 days apart,and then weekly injections the same dose thereafter for 9 weeks,at which point injections of Aanti-NC and an Anti-miR-155 were stopped.They were both still fed for 1 week.Arterial tissue of mice were frozen immediately and embedded in OCT medium.Used oil red O staining,HE and Masson staining and immunohistochemical method to detect and evaluate the stability of the plaques;Fluorescence in situ hybridization technique(FISH)locate the position of miR-155 expression in plaque;Western blot(WB)technology to detect the expression of SOCS1,p STAT3 and PDCD4 in the mice aorta atheromatous plaque;Real-time PCR technique to detect the expression of miR-155 in the artery atheromatous plaque;ELISA technique to detect the mice serum TNF-?,IL-6,IL-10 levels.2.Cell experiment to observe how miR–155 regulate PDCD4 and the inflammatory factorCell experiment observed miR-155,PDCD4,TNF-?,IL-6 and IL-10 expression in RAW264.7 macrophages stimulated by ox-LDL.RAW264.7 cells were cultured in vitro and stimulated by ox-LDL.The technologies of Western blot,Real-time PCR were used to detct miR-155 and PDCD4 gene and protein expression at different concentrations of ox-LDL.ELISA technique was used to detect TNF-?,IL-6 and IL-10 levels.Application of miR-155 inhibitor,Anti-miR-155 transfect cells to observe the expression of PDCD4 changes and using PDCD4 siRNA silence PDCD4 expression to obseve TNF-?,IL-6 and IL-10 gene and protein expression.To study how miR-155 regulate PDCD4 and three inflammation factors in RAW264.7 macrophages stimulated by ox-LDL.3.To further study the molecular mechanisms of how miR-155 regulate PDCD4Based on the second part results,we further use Targetscan website forecast of miR-155 targets and found SCOS1 is a target genes for miR-155.We design a SCOS1 3 'UTR extents and carries on the purification,enzyme digestion,transfection,sequencing and fluorescence detection.Which verified SCOS1 is target genes for miR-155.Then we apply adenovirus vector to build a overexpress SOCS1 gene vector and tranfect RAW264.7 macrophages to observe the expression of SCOS1/ STAT3 and PDCD4.cells were transfected with Anti-NC,Anti-miR-155 and STAT3 siRNA to observation SCOS1/STAT3 signaling pathway changes and PDCD4 expression.Western blot and Real-time PCR techniques were applied to detect SCOS1/STAT3 and PDCD4 gene and protein change,in order to further study the molecular mechanisms of how miR-155 regulate PDCD4.Result:1.In animal models,the PDCD4 and proinflammatory factors inhibited while the expression of miR-155 were inhibited.Animal experiments results shown miR-155 expression was highest in Apo E-/-HFD among three groups of C57 wild type mice,ApoE-/-ND and ApoE-/-HFD.The trends of TNF-??IL-6 gene and protein expression was the same in those three groups,but IL-10 was the opposite.Compared to the ApoE-/-HFD to ApoE-/-HFD+Anti-NC,both of them the expression of inflammation factor had no obvious change.Comparison of Apo E-/-HFD+Anti-NC and Apo E-/-HFD+Anti-miR-155,oil red staining found Atheromatous plaque was obviously less in ApoE-/-HFD+Anti-miR-155 group.Genetic testing found that miR-155 and CD68 positive macrophages expression is restrained also.Further study shown while the expression of SOCS1 was increased,the expression of pSTAT3 and PDCD4 were reduced.TNF-? and IL-6 expression were inhibited in the Apo E-/-HFD+Anti-miR-155 groups.Above research suggest the decreasement of miR-155 expression can lead to decrease the levels of inflammatory cells,such as p-STAT3,PDCD4,TNF-? and IL-6.Protective factor expression is strengthened,such as SOCS1.Those implied the miR-155 was closely associated with the occurrence and development of plaque and it may be related to SOCS1/p-STAT3/PDCD4 signaling pathways and regulated the expression of inflammatory factor.2.MiR-155 regulated the expression of PDCD4 and inflammatory factors associated with PDCD4 in RAW264.7 macrophages stimlulated ox-LDL.In the cells experiment,we builed RAW264.7 macrophages model which stimulate by ox-LDL.We inspected the miR-155 expression in the modle that we found miR-155 expression depended on ox–LDL stimulating concentration and time.This result was consistent with animals fed HFD.The similar results of PDCD4,TNF-?,IL-6 and IL-10 also obtained.To prove the relevance among miR-155,PDCD4 and the three kinds of inflammatory factors,we transfected Anti-miR–155 into macrophages which stimulated by ox-LDL and found while miR-155 level was inhibited,PDCD4 gene and express was resisted.Those suggested miR-155 can regulate the PDCD4 secretion.In the model we used PDCD4 siRNA to silence PDCD4,and then we dected TNF-?,IL-6 and IL-10 expression.We found the effect which caused transfected Anti-miR-155 was overtuned.Those results implied miR-155 can through PDCD4 to control the three inflammatory factors changes in macrophages.3.MiR-155 via SOCS1/p-STAT3 to regulate changes of PDCD4 expression in macrophages under ox-LDL stimuli.In this study,we used the same experimental model to study how miR-155 control PDCD4 expression.First,we verified the SCOS1 is one of target genes for miR-155.And then we transfected miR-155 in to macrophages,the results shown while the miR-155 expression increased,the expression of SOCS1 was suppressed and p-STAT3 enhanced.Those explained that enhanced miR-155 can amplify action of pro-inflammatory signaling.The opposite result was got when transfected Anti-miR-155 into macrophages.In order to elucidate the relationship between miR-155,SOCS1/p-STAT3 and PDCD4,in further experiments,we used adenovirus to overexpress SOCS1 gene and STAT3 si RNA to silence the expression of STAT3,monitoring the change of expression of SOCS1/p-STAT3 and PDCD4.Compared between ox-LDL+Adv.SOCS1 group and ox-LDL group,the experimental results show the high expression of p-STAT3 and PDCD4 induced by ox-LDL was significantly inhibited after transfection with SOCS1.The same results were obtained when transfected with siRNA STAT3 into macrophages stimulated by ox-LDL.Therefore,we believe that miR-155 can stimulate the expression of PDCD4 through SOCS1/STAT3 signaling pathway in macrophages stimulated by ox-LDL.Conclusion:1.miR-155 played an important role to promote atherosclerosis and plaque formation in animal model.Inhibited miR-155 can supressed inflammation response in vivo.2.Ox-LDL increased the expression of miR-155,and promoted inflammation mediators relase in macrophage RAW264.7 cells.Inhibition of miR-155 expression reversed ox-LDL mediated inflammation factors relassion though SOCS1-STAT3-PDCD4 aix in vtro.MiR-155 was as protenal target to prevent and slow atherosclerosis and plaque formation.