Effects And Mechanism Of MicroRNA-155 Regulate Lipid Uptake And Inflammatory Response Of Macrophages

Atherosclerosis is the main cause of coronary heart disease,which is also a serious disease that endangers human health.The formation of AS is a process of chronic lipid deposition and chronic inflammatory reaction.In the early stage,the peripheral blood monocytes migrate to the arterial intima and differentiate into macrophages,then devour the oxidized low density lipoprotein to form foam cells.Losing control of ox-LDL absorption,the excess cholesterol accumulation is thought to be the trigger of macrophage foam cell formation.As the main receptor for the absorption of ox-LDL by macrophages,CD36 play a key role in the pathogenesis.Besides,lipid metabolism interacts with the inflammatory response of macrophages.For example,inflammatory cytokines IL-1β and TNF-α destroy the feedback mechanism of LDL receptors to increase the expression of LDLR,and increase the level of intracellular cholesterol,which can aggravate the imbalance of lipid metabolism.And the lipid disorder can promote the transformation of macrophages from the subtype M2(anti-inflammatory)to M1(pro-inflammatory).Therefore,to study the regulation effect and mechanisms of macrophage on inflammatory reaction and lipid uptake is significant in understanding the development mechanisms of atherosclerosis.Small RNA are short non-coding small RNA molecules that target m RNAs at the post-transcriptional level to regulate gene expression,or to make the target m RNAs degrade at the transcriptional level.Recent studies have shown that various mi RNAs play an important role in the uptake of cholesterol and induced inflammation in macrophages,and the mi RNA-155 is the focus of attention.Studies have shown that mi RNA-155 expression was highly expressed in the mouse AS plaques and was involved in promoting the formation of plaques.In macrophages,mi RNA-155 could regulate the uptake of ox-LDL and activate the inflammatory signaling pathways such as STAT3,NF-κB,and promote the expression of downstream inflammatory factors IL-6,TNF-α,etc.Peroxisome proliferator activated-receptors are a kind of nuclear transcription factors activated by ligand.Three subtypes PPARα,PPARβ,PPARγ have been found,and the PPARγ is mainly expressed in adipose tissue and macrophages.A large number of studies have shown that PPARγ play a key role in regulating the uptake of cholesterol and inflammatory in macrophages.In the alcohol treated macrophages,mi RNA-155 could regulate the expression of PPARγ to influence the expression of inflammatory factors.In order to explore the role of mi RNA-155 in macrophage inflammatory response and lipid uptake,we induced macrophages by ox-LDL.Three parts are studied: part 1: detect the regulation of mi RNA-155 and PPARγ on the macrophage lipid uptake;part 2: detect the regulation of mi RNA-155 and PPARγ on the macrophage inflammatory response;part 3: detect the regulation of mi RNA-155 on PPARγ expressiom.Part I The effects of mi RNA-155 and PPARγ on lipid uptake of macrophagesMethod:1.Macrophage RAW264.7 cells were treated with 0,25,50 and 100 μg/m L ox-LDL for 24 h or 50 μg/m L ox-LDL for 0,6,12,24 h,respectively.Evaluated the level of mi R-155 in all above samples through Realtime PCR.2.In our research,RAW264.7 cells were divided into six group: control group;ox-LDL group;ox-LDL/negative control group;ox-LDL/anti-mi R-155 group;ox-LDL/sh RNA negative as control group and ox-LDL/ PPARγ sh RNA group.Oil red O staining,Filipin staining,cholesterol testing kit and western blot were used for above 6 group to evaluated the cellular uptake of ox-LDL,the level of TC and FC and the expression of CD36 protein.Results:1.The relative expression of mi R-155 was increased after ox-LDL stimulation.2.Through Oil red O staining,Fillipin staining,cholesterol testing,we found the relative absorbance,TC and FC,fluorescence intensity of filipin was significantly lower in ox-LDL/anti-mi R-155 group than in ox-LDL and ox-LDL/negative control group.Similarly,the level of relative absorbance,TC and FC,fluorescence intensity of filipin was significantly lower in ox-LDL/ PPARγ sh RNA group than in ox-LDL and ox-LDL/ sh RNA negative control group.3.The expression of CD36 protein was suppressed in ox-LDL/anti-mi R-155,compared to ox-LDL and ox-LDL/negative control group.Similarly,CD36 protein level was decreased in ox-LDL/PPARγ sh RNA,compared to ox-LDL/Vector group.Conclusion:Ox-LDL stimulation raised the expression of mi RNA-155 and CD36 receptor.Inhibition of mi RNA-155 and PPARγ reduced the expression of CD36 and lipid uptake,so the accumulation of lipid and cholesterol was decreased.Part II The effects of mi RNA-155 and PPARγ on inflammatory response of macrophagesMethod:In our research,RAW264.7 cells were divided into six group: control group;ox-LDL group;ox-LDL/negative control group;ox-LDL/anti-mi R-155 group;ox-LDL/sh RNA negative as control group and ox-LDL/ PPARγ sh RNA group.Western blot was used for above 6 group to evaluated the the expression of p-STAT3 and NF-κB p65 protein.Realtime PCR was used to evaluate the expression of TNF-α,IL-1β and IL-6 m RNA.Results:1.The expression of p-STAT3 and NF-κB p65 protein was suppressed in ox-LDL/anti-mi R-155,compared to ox-LDL and ox-LDL/negative control group.Similarly,p-STAT3 and NF-κB p65 protein level was decreased in ox-LDL/PPARγ sh RNA,compared to ox-LDL/Vector group.2.The expression of TNF-α IL-6 and IL-1β m RNA was suppressed in ox-LDL/anti-mi R-155,compared to ox-LDL and ox-LDL/negative control group.Similarly,TNF-α IL-6 and IL-1β m RNA level was decreased in ox-LDL/PPARγ sh RNA,compared to ox-LDL/Vector group.Conclusion:Ox-LDL stimulation can activate the macrophage inflammatory signaling pathway and promote the release of inflammatory factors.Inhibition of mi RNA-155 and PPARγ reversed the activation of macrophage inflammatory signaling pathway and the release of inflammatory factors by ox-LDL.Part III The effects of mi RNA-155 on PPARγ expression of macrophagesMethod:RAW264.7 cells were divided into six group: control group;ox-LDL group;ox-LDL/negative control group;ox-LDL/anti-mi R-155 group;ox-LDL/sh RNA negative as control group and ox-LDL/ PPARγ sh RNA group.Western blot was used for above 6 group to evaluated the the expression of PPARγ protein.Results:The expression of PPARγ protein was suppressed in ox-LDL/anti-mi R-155,compared to ox-LDL and ox-LDL/negative control group.Conclusion:Mi R-155 mediated PPARγ,induced lipid uptake and inflammation response of macrophages via CD36 and STAT3/NF-κB signal pathway.