The Role Of MiR-145in The Anti-proliferation And Anti-inflammation Effects Of Aspirin On VSMCs

Background:The abnormal proliferation of vascular smooth muscle cells (VSMCs) caused by the phenotypic change is an important pathologic basis of atherosclerosis, cerebral infarction, and other cardiovascular diseases, but the exact mechanisms of the phenotypic change of VSMCs are still unknown. miR-145is a kind of microRNAs which is closely associated with cardiovascular diseases, mainly expressed in VSMCs, and plays an important role in phenotypic regulation of VSMCs. Both CD40and CD40L are expressed in VSMCs, and activated CD40-CD40L system stimulates the inflammation and proliferation of VSMCs, so it is vital in the occurrence and development of atherosclerosis. By using the bioinformatics software, we found CD40may be an important target gene of miR-145, miR-145may be involved in proliferation and inflammatory cytokines secretion of VSMCs by regulating CD40. Aspirin is a widely used drugs in clinic for cerebral infarction. It is reported that aspirin has antithrombotic and anti-inflammatory effects, aspirin can also inhibit the proliferation of smooth muscle cells, but its exact mechanism of anti-proliferation effect in VSMCs is still unclear. The antithrombotic and anti-inflammatory effect of aspirin is associated with the activity of CD40-CD40L system, but there is no research reported whether aspirin can affect the CD40level in VSMCs and its relation with miR-145.Objectives:1. To investigate whether miR-145can mediate proliferation and inflammatory cytokine secretion of VSMCs by regulating CD40, providing a reference for the study of miR-145in cardiovascular disease.2. To investigate whether miR-145is involved in the regulation of VSMCs proliferation and anti-inflammatory effects of aspirin by regulating CD40, providing a theoretical basis for the new mechanisms of aspirin.Methods:1. TNF-α induced proliferation model of VSMCs were divided into following groups:(1) Control group;(2) TNF-a;(3) miR-145mimic control+TNF-α,(4) miR-145mimic+TNF-α. Cell proliferation was detected by using EdU; Real-time PCR was used to detect the mRNA expression of miR-145, CD40, VSMC differentiation marker genes Calponin, Western-blot method was used to detect the protein expression of CD40; ELISA was used to detect concentrations of inflammatory cytokines IL-6in cell supernatants.2. TNF-α induced proliferation model of VSMCs were divided into following groups:(1) Vehicle group;(2) TNF-α;(3) miR-145inhibitor control+TNF-α;(4) ASA+TNF-α;(5) miR-145inhibitor+ASA+TNF-α. Cell proliferation was detected by using EdU; Real-time PCR was used to detect the mRNA expression of miR-145, CD40, VSMC differentiation marker genes Calponin, Western-blot method was used to detect the protein expression of CD40; ELISA was used to detect concentrations of inflammatory cytokines IL-6in cell supernatants.Results:1. Overexpression of miR-145could inhibit the TNF-α induced VSMCs proliferation, block the decreased expression of Calponin and increased level of CD40induced by TNF-α, overexpression of miR-145could also reduce the level of IL-6released by VSMCs.2. The proliferation of VSMCs was stimulated by TNF-α accompanied by a decreased level of Calponin, TNF-α also caused a decreased level of miR-145, an increased level of CD40, and an increased level of IL-6; Pretreatment with20μg/mL aspirin in VSMCs, could partly block the above effects induced by TNF-a.Conclusions:1. miR-145has a negative regulatory effect on CD40in VSMCs, miR-145also decrease the level of proliferation inflammatory cytokines in VSMCs by inhibiting the expression of CD40.2. miR-145is involved in anti-proliferation and anti-inflammation effects of aspirin in VSMCs by inhibiting the expression of CD40.