Study On The Mechanism Of Hippo-YAP Signaling Pathway Regulate Autophagy By Beclin1 In Phenotypic Transition Of Vascular Smooth Muscle Cells

Objective:To explore interaction and mechanism between Hippo-YAP signaling pathway and autophagy in phenotypic transition of vascular smooth muscle cells（VSMCs）.Methods:（1）To construct phenotypic transition model of mouse VSMCs:Mouse VSMCs were cultured and subcultured.the 4th-8th generations of VSMCs were treated with angiotensinⅡdiluted with DMSO(AngⅡ,1×10^-7mol/L)for 24 hours,and the control group was treated with DMSO of the same concentration.（2）Detection of target gene:Western Blot and q RT-PCR were used to detect the expression of YAP,OPN,α-SMA,autophagy-related markers LC3、P62 and Beclin1 at the protein and m RNA levels.Observe the change of expression level.（3）Interfere the expression of target gene:Small interfering RNA（si RNA）of YAP and Beclin1 were constructed to knock down the two genes,respectively.Also,the plasmid（pc DNA-YAP）was constructed to overexpress YAP.WB and q RT-PCR were used to verify the expression changes of each gene.Results:（1）The cell model was successfully constructed:Compared to the control group,the expression ofα-SMA,VSMCs contractile phenotypic markers treated with AngⅡin the experimental group,was significantly down-regulated,While the expression of synthetic markers OPN was up-regulated.Double validation at protein and m RNA levels showed that The cell model of phenotypic transition was successfully constructed.（2）The expression of target gene changes:Compared to the control group,YAP and autophagy-related markers LC3Ⅱ、Beclin1 were up-regulated,and the expression of P62 was down-regulated in the VSMCs group treated by AngⅡ.The above expression changes were statistical differences at the level of protein and m RNA.（3）Compared to the control group,the expression of YAP in VSMCs were significantly down-regulated after YAP si RNA transfection at the level of protein and m RNA.After the overexpression of YAP plasmid transfection,YAP was significantly up-regulated.Similarly,compared to the control group,the expression of Beclin1 in VSMCs was significantly down-regulated at the protein and m RNA levels after Beclin1 si RNA transfection.（4）Compared to the simple AngⅡstimulation group,the phenotypic transition and the up-regulation of autophagy-related markers were significantly inhibited in the group treated with both AngⅡand YAP si RNA.While in the YAP overexpression group,phenotypic transition and up-regulation of autophagy-related markers were further enhanced.（5）Compared to the simple AngⅡstimulation group,the phenotypic transition and the up-regulation of autophagy-related markers were inhibited in the group treated with both AngⅡand Beclin1 si RNA,while the expression of YAP in the two groups was no statistical difference.Conclusion:The expression of autophagy-related markers and YAP were up-regulated in VSMCs during phenotypic transitioning,and YAP may promote autophagy by upreglute the expression of Beclin1 and ultimately accelerate phenotypic transition.