PGC1?/miR-378a/IGF1&TLR8 Axis Inhibits Proliferation,Migration And Inflammatory Response Of Human Aortic Smooth Muscle Cell

Atherosclerosis and its complications are the most common cause of death in developed countries.In recent years,the number of patients with coronary heart disease in our country has also increased by years.Therefore,how to control the progression of atherosclerosis plaque is an urgent problem to be solved.PGC1? is a transcriptional coactivator that is the core of many cell and organ homeostasis mechanisms.In recent years,PGC1? has attracted extensive attention in vascular regulation.PGC1? a protective role in the vascular system,but the molecular mechanism still needs further elucidation.miRNAs are a class of endogenous,short-stranded,non-coding RNAs,approximately 19-22 nucleotides in length.They complementarily joined their seed sequences with the identified target m RNA and thereby transcriptionally inhibit or degrade to regulate post-transcriptional gene expression.Recent studies have also shown that miRNA plays an important role in maintaining the homeostasis of the cardiovascular system.Studies have shown that miR-378 A is highly expressed in the cardiovascular system and plays a role in the regulation of myocardial development.However,the role of miR-378 A in atherosclerosis remains unclear.1.Materials and Methods 1.1 Clinical Samples and cell culture All atherosclerosis samples and non-atherosclerosis sampled were collected during Coronary Artery Bypass Grafting and Aortic Repair surgery.All samples were collected and stored immediately in liquid nitrogen and transferred to a-80°C freezer.Human aortic smooth muscle cells HASMC were purchased from Sciencell.1.2 miRNA microarray analysis and quantitative real-time PCR(q RT-PCR)miRNA microarray analysis was evaluated to obtain expression profile of miRNAs in rabbit aorta with vascular smooth muscle specific overexpressed PGC1? WT rabbit aorta.Mi RNA with significantly profile changed were analyzed by q RT-PCR.Analysis the relationship between miR-378 a and PGC1?.1.3 The role of miR-378 a in HASMC proliferation,migration and invasion HASMC were transfected with miR-378 a mimic,miR-378 a inhibitor or the corresponding negative controls.Proliferation of HASMC was evaluated by Ed U assay.Wound Healing assay and Transwell assay were evaluated to analyze migration of HASMC.QRT-PCR and ELISA were evaluated to analyze the inflammatory effect of HASMC.1.4 Prediction and analysis of miR-378 a downstream target By using the miRBase and Target Scan,IGF1 and TLR8 were considered to be a direct target of miR-378 a.Luciferase activity assay and western blot were performed to evaluate the relationship between miR-378 a and IGF1 as well as TLR8.1.5 The mechanism of IGF1 and TLR8,the direct downstream target of miR-378 a,on HASMC Function The effect of IGF1 and TLR8 on HASMC proliferation,migration and inflammation were evaluated by performing Ed U assay,Transwell assay,q RT-PCR and ELISA.Overexpressed miR-378 a HASMC were transfected with IGF1 and TLR8 overexpression plasmid to explore that the inhibitory effect of miR-378 a can be reversed.2.Results 2.1 miR-378 a is significantly upregulated in overexpressed PGC1? HASMC.miR-378 a expression was significantly down-regulated in atherosclerosis tissues compared with non-atherosclerotic tissues.Statistical results showed that the expression level of miR-378 a was significantly correlated with PGC1.PGC1? can regulate the expression of miR-378 a through the transcription factor NRF1.2.2 Overexpression miR-378 a significantly decreased proliferation,migration,and inflammation of HASMC.Opposite outcome were observed in downregulation miR-378 a in HASMC.2.3 IGF1 and TLR8 were the direct downstream targets of miR-378 a.The expression of IGF1 and TLR8 in atherosclerosis tissues were significantly higher compared with non-atherosclerotic tissues.Overexpression of miR-378 a significantly reduced the expression of IGF1 and TLR8 in HASMC stimulated with FFA.The expression levels of Mi R-378 a and IGF1 as well as TLR8 in atherosclerosis tissues were negatively correlated.2.4 Over expression of IGF1 and TLR8 expression levels increased the proliferation,migration and inflammation of HASMC,which similar to pattern observed in downregulation of miR-378 a.Furthermore,upregulation of IGF1 and TLR8 reversed the effect of miR-378 a in decreasing proliferation,migration,and inflammation of HASMC in overexpressed miR-378 a cells.3.Conclusion 3.1 miR-378 a expression was significantly down-regulated in atherosclerosis tissues and significantly correlated with PGC1?.PGC1? can regulate the expression of miR-378 a through the transcription factor NRF1,suggesting that PGC1? can regulate miR-378 a and function as an atherosclerotic suppressor in the development of atherosclerosis.3.2 Overexpression of miR-378 a significantly reduced the proliferation,migration and inflammation of HASMC.IGF1 and TLR8 overexpressed HASMC got a totally different pattern.The effect of miR-378 a on reducing proliferation,migration and inflammation of HASMC was reversed by IGF1 and TLR8.These finding suggested that miR-378 a may be involved in the development of atherosclerosis by directly targeting the expression of IGF1 and TLR8 in HASMC.3.3 The expression level of miR-378 a can significantly differentiate atherosclerosis tissue and non-atherosclerotic tissue.Meanwhile,miR-378 a expression was inversely correlated with IGF1 and TLR8 expression level in atherosclerosis.Therefore,miR-378 a may be a potential treatment target for patients with atherosclerosis.