Homocysteine Improves The Proliferation Of Human Aortic Smooth Muscle Cell By Downregulating MiR-146

Objective:Homocysteine(Hcy)has been proved to be an independent risk factor for atherosclerosis,and it can be involved in the regulation of cell proliferation through the PI3K/Akt signaling pathway.Proliferation of smooth muscle cells is one of the main causes of atherosclerosis.MicroRNA is an important small molecule involved in a variety of cardiovascular diseases including atherosclerosis.However,in homocysteine regulation pathways,the role of micro RNA in the proliferation of smooth muscle cells is rarely reported.In this study,human aortic smooth muscle cell(HASMC)was used as the model,and the effect of Hcy on smooth muscle cells was confirmed by observing the proliferation of Hcy on smooth muscle cells,the expression of mir-146 and PI3K,Akt and EGFR,so as to reveal the mechanism of Hcy in the development of atherosclerosis.Methods:1.HASMC was cultured in vitro with DMEM containing 2%fetal bovine serum.2.The 3rd~4th generation of HASMC were transfered to the 96-well plate.After incubation of 5 x 10 ~3/well overnight,complete culture medium containing Hcy with 4 different concentrations(0.025μM,0.2μM,0.5μM,1.0μM)was used to stimulate HASMC for 48h,and the relative cellular activity of HASMC was detected by MTS cell proliferation test.Then,the morphological changes of HASMC with the stimulation of 0.5μM Hcy were observed through the transmission electron microscope,and the proliferation effect of Hcy on HASMC was determined.3.The mRNA expression levels of miR-146,EGFR,PI3K and Akt in HASMC stimulated by 0.5μM Hcy were detected by Real time PCR.Western Blot was used to determine the protein expression levels of EGFR,PI3K and Akt,and the mechanism of Hcy on HASMC proliferation was determined.4.After transfection of HASMC with a miR-146 mimic(100nM)and stimulation of HASMC with Hcy(0.5μM),the relative cellular activity of the HASMC was detected by MTS.The changes in cell morphology were then observed by transmission electron microscopy to determine the role of miR-146 in Hcy proliferation of HASMC.5.After transfection of HASMC with a miR-146 mimic(100 nM)and stimulation of HASMC with Hcy(0.5μM),the mRNA expression levels of mir-146 and EGFR,PI3K and Akt were detected by Real time PCR,and the protein expression levels of EGFR,PI3K and Akt were determined by Western Blot to determine whether miR-146 was a key node of Hcy activated EGFR/PI3K/Akt pathway.Results:1.After HASMC was stimulated by Hcy at different concentrations,compared with the blank control group,Hcy of 0.025μM presented no obvious stimulation effect on HAMSC,while 0.2-1.0μM showed significant stimulation effect on HAMSC proliferation(all P<0.01),and Hcy of 0.5μM showed the most significant effect.The changes of cell morphology induced by Hcy(0.5μM)were observed by transmission electron microscopy.Compared with the normal control group,intracellular vacuoles increased.The results showed that Hcy could induce cell proliferation in a certain concentration range,and make HAMSC to present the changes in early atherosclerosis.2.It was found by Real time PCR that the expression level of miR-146was significantly reduced after Hcy treatment of 0.5μM in HAMSC.The mRNA and protein expression of EGFR,PI3K and Akt were significantly increased.Hcy can weaken the expression of miR-146 in HASMC and activate the EGFR/PI3K/Akt signaling pathway.3.After transfection by miR-146 mimics,HAMSC proliferation was significantly reduced by Hcy(0.5μM)stimulation,suggesting that the proliferation effect of Hcy on HASMC could be weakened by miR-146mimics.The miR-146 mimic was also observed by the transmission electron microscope to weaken the changes of HASMC in early atherosclerosis,endoplasmic reticulum expansion and mitochondrial consolidation,after stimulation with Hcy.4.After transfection with miR-146 mimics,the expression levels of EGFR,PI3K and Akt mRNA and protein in HASMC were significantly reduced after stimulation with Hcy(0.5μM).Conclusions:1.Homocysteine improved proliferation of human aortic smooth muscle cell and make HAMSC to present the changes in early atherosclerosis.2.Hcy downregulating the expression of miR-146 in HASMC and activated EGFR/PI3K/Akt signaling pathway.3.Overexpression of miR-146 mimic undermined the function of Hcy on EGFR/PI3K/Akt pathway,At the same time,Overexpression of miR-146mimic diminished HASMC proliferation caused by Hcy,and weaken the changes of HASMC in early atherosclerosis.