The Impact Of Mir-497 On The Progression Of Atherosclerosis Vulnerable Plaque And The Effect Of Probucol/rosuvastatin

Aims:MicroRNAs(miRNAs)are a class of small non-coding RNAs that post-transcriptionally regulate gene expression.Recent evidences indicate that special miRNAs may play a critical role in regulating atherosclerosis(AS)plaque vulnerability.ERK activation is essential for promoting AS and increasing plaque vulnerability,and the activation of MEK/ERK signaling has been shown to regulate the expression of MMP-9.Probucol and rosuvastatin are common used to improve serum lipid profiles,other effects including anti-inflammation,anti-oxidation and plaque regression.But it is unknown whether miRNAs play an important role in stabilizing plaque of probucol and rosuvastatin,or whether combination of both can enhance the effect is underreported.In this study,we focused on the effects of miR-497 on stabilizing plaque as well as the protective effects of probucol and rosuvastatin.Methods:we used bioinfonnation method and identified miR-497 as the candidate miRNA of MEK1.After modeling HUVECs with high expression of MMP-9,miR-497 mimics/inhibitor was transiently transfected into HUVECs,miR-497 and the mRNA levels and protein levels of MEK1,other members of MEK/ERK signaling and MMP-9 were detected with real-time RT-PCR and western blotting,in order to confirm the regulating role of miR-497 in target gene expression.Then after modeling HUVECs with high expression of MMP-9,probucol,rosuvastatin and combination of the two drugs were added into the system,with or not miR-497 inhibitor was transiently transfected into HUVECs,24 hours post transfection,miR-497 and the mRNA levels and protein levels of MEK1,other members of MEK/ERK signaling and MMP-9 were detected with real-time RT-PCR and western blotting.In the last part,male apoE deficient mice(8-weeks old)were fed with high-fat diet for 12 weeks to construct AS models(M,n=5).And for probucol group(P,n=6),rosuvastatin group(R,n=6)and combination group(PR,n=6)apoE deficient mice were administered with probucol,rosuvastatin or both separately,simultaneously with high-fat diet for 12 weeks.Other male C57BL/J6 mice(8-weeks old)were raised with high-fat diet as control group(C,n=6).At the end of 12 weeks,miR-497 and protein levels of MEK1,other members of MEK/ERK signaling and MMP-9 in aorta were detected with real-time RT-PCR and western blotting.Results:The expression of miR-497 was lower(P<0.01),the levels of MEK1,ERK1/2,p-ERK1/2 and MMP-9 protein were higher(P<0.01)in HUVECs induced by TNF-? than those in the control group.The level of MEK1 mRNA increased(P<0.01),the levels of MEK1,ERK1/2,p-ERK1/2 and MMP-9 protein decreased(P<0.01)in HUVECs after overexpression of miR-497,and the adverse results(P<0.01)were obtained after suppression of miR-497.The levels of MEK1 mRNA and MMP-9 mRNA descended(P<0.01)and the levels of MEK1,p-MEK1,ERK1/2,p-ERK1/2 and MMP-9 protein descended too after administering with probucol,rosuvastatin or two drugs separately(P<0.01),while the levels were upregulated after suppression of miR-497(P<0.01).For the animal experiment,the levels of TC,LDL and apoB obviously increased(P<0.05),and the level of HDL decreased(P<0.05)in M group.Both probucol and rosuvastatin could bring down the levels of TC and LDL(P<0.05),and the effects were enhanced when the two drugs were used together(P<0.05).The high levels of miR-497,MEK1,p-MEK1,ERK1/2,p-ERK1/2 and MMP-9 protein were detected in aorta of M group(P<0.05),and the expression of miR-497 was upregulated and the levels of MEK1,p-MEK1,ERK1/2,p-ERK1/2 and MMP-9 protein were downregulated after drugs were used(P<0.05).Probucol could bring down the levels of MEK1 and p-ERK1/2 mainly,and rosuvastatin could bring down the levels of p-MEK1 and MMP-9 mainly.The effects were enhanced when the two drugs were used together.Conclusions:Our results indicate that miR-497 functions as a suppressor gene and inhibits the translational process of MEK1 and eventually down-regulates the expression of MMP-9,leading to AS plaque lost stability.Probucol and rosuvastatin can stabilize the AS plaque probably through up-regulating the expression of miR-497.The combination of probucol and rosuvastatin can enhance the effect on stabilizing AS plaque.