Circadian Clock Protein CRY1 Inhibits Ang Ⅱ-induced Phenotypic Transformation Of Mouse Aorta Smooth Muscular Cells

Objective:To investigate the effect of circadian clock protein cryptochrome 1（CRY1）on angiotensin II（Ang Ⅱ）-induced transitions of mouse aortic vascular smooth muscle cells（VSMCs）from a contractile phenotype（VSMC-C）into a synthetic phenotype（VSMC-S）.Methods:For a start,the phenotype transformation model phenotype transformation cell model of C57 mouse aorta VSMCs was induced in vitro by Ang Ⅱ with different concentrations(10^-8 mol/L,10^-7mol/L,10^-6 mol/L,10^-5 mol/L)at different times（12 h,24 h,48 h,72 h）.α-smooth muscle actin（α-SMA）,osteopontin（OPN）and CRY1expression in models was detected via RT-q PCR and Western blot to determine the optimal concentration and time for constructing phenotype transformation model.Then VSMCs were treated with overexpression of CRY1,CRY1 silencing and Yes associated protein 1（YAP1）silencing,respectively.VSMCs were transfected with knockdown vector si CRY1and si YAP1 or overexpression vector pc DNA-CRY1,and the corresponding gene expression was detected with RT-q PCR and Western blot.Lastly,VSMCs were divided into 4 groups:Control group,AngⅡgroup,AngⅡ+si CRY1 group,AngⅡ+pc DNA-CRY1 group.Si CRY1,si YAP1,pc DNA-CRY1 were transfected into VSMCs induced by AngⅡfor observation onα-SMA,CRY1,OPN and YAP1 at m RNA and protein levels detect-ed by RT-q PCR and Western blot.CCK-8 method were used to measure proliferation capacity.Results:VSMCs were treated with different concentrations of Ang Ⅱ for various time,the expression of contractile markers in VSMCs decreased at mRNA and protein levels,and the expression of synthetic markers in VSMCs increased at m RNA and protein levels.At the same time,it was found that the expression of CRY1 decreased at m RNA and protein levels after stimulating VSMCs with Ang II at different concentrations（P<0.01）.After VSMCs were transfected with si CRY1,si YAP1 and pc DNA-CRY1,the expression of CRY1 decreased in si CRY1 group and increased in pc DNA-CRY1 group,while the expression of YAP1 decreased in si YAP1 group and increased in si CRY1group.Knockdown of CRY1 could stimulate the transformation of VSMCs from contractile phenotype into synthetic phenotypes by further activating expression of synthetic markers and CRY1（P<0.05）,inhibiting expression of contrac-tile markers and YAP1（P<0.05）,the proliferation of VSMCs increased significantly（P<0.05）.While overexpressed CRY1 could block the phenotypic transformation,the expression of syn-thetic markers and CRY1 was reduced and contractile markers and YAP1 was increased both at m RNA and protein levels（P<0.05）,the proliferation of VSMCs inhibited（P<0.05）.Conclusion:CRY1 can inhibit switch of mouse VSMCs phenotypes induced by AngⅡand inhibit VSMCs proliferation,which may be related to the Hippo-YAP signaling pathway.