SM22-CRE Specific Knockout Of SCAP Gene Leads To Proliferation Disorder Of Embryonic Vascular Smooth Muscle Cells In Mice

Objective:Sterol regulatory element binding protein?SREBP?cleaved activated protein?SCAP?,known as cholesterol sensor,can sense the intracellular cholesterol concentration and maintain the homeostasis of cholesterol in the internal environment through the negative feedback mechanism to regulate cholesterol uptake and the expression level of endogenous synthetic genes.SCAP is mainly associated with lipid metabolism and related diseases,such as non-alcoholic fatty liver disease and aortic atherosclerosis.With the continuous expansion and in-depth study of SCAP,it has been found that the expression of SCAP also plays an important role in cell proliferation,differentiation,embryonic development and other fields.In this study,cre-loxp system was used to construct mice with conditioned knockout of SCAP gene of vascular smooth muscle,in an attempt to explore the effect of SCAP gene deletion on embryonic development of mice and further explore its possible molecular mechanism.Methods:1)In vivo experiments:the model mice used in this study were mice with smooth muscle-specific Cre expression?SM22-Cre?and SCAP^flox/flox transgenic tools as the hybridization objects.Female SCAPflox/flox mice were mated with male SM22-Cre^+/-mice in a cage to obtain vsmc-specific knockout SCAP gene mice(SM22-Cre/SCAP^flox/+),mouse tail was cut about 1cm,and tail DNA was extracted for common PCR to identify mouse genotype,and Western blot and other methods were used to identify the degree and specificity of SCAP gene knockdown.The male SM22-Cre/SCAP^flox/+mice were further mated with the female SCAPflox/flox mice,and four genotype progeny mice were obtained,namely SCAP^flox/+,SM22-Cre/SCAPf^lox/+,SM22-Cre/SCAP^flox/flox,and SCAP^flox/flox.Mouse embryos were collected at E12.5,E14.5,E16.5 and E18.5 days of embryonic stage,and the embryonic hind limbs and tail were cut for general PCR for genotype identification,and the proportion of each genotype was calculated.The gross morphology and development of the embryo were observed and recorded by stereomicroscope.Mouse aorta sections were taken for IHC to detect the expression of SCAP.The thickness of vessel wall was observed by H.E.staining.The expressions of PCNA,PI3K and Akt were detected by immunohistochemistry,and the vascular proliferation was observed.2)in vitro experiments:VSMCs cells were transfected with different concentrations of SCAP SiRNA to detect the degree of interference with the SCAP gene of VSMCs;The proliferation rate was detected on VSMCs that interfered with SCAP gene expression.Western Blot was performed to detect the expression of SCAP,PCNA,PI3K,Akt after vascular smooth muscle cells interfered with SCAP gene.Results:1.ObtainSM22-crethroughLoxp/Crereconfiguration system;SCAPflox/+mouse model,SCAP gene knockdown was proved to be low by quantitative PCR detection of SCAP mRNA expression,and the model was stable and available.Meanwhile,SCAP protein and mRNA expression levels of aorta,liver and kidney of mice were detected by Wstern Blot and it was confirmed that SCAP gene was specifically knocked out by vascular smooth muscle in model mice.2.Model mice were mated and bred to obtain offspring embryo mice,and it was found that the birth rate of SCAP homozygous mice was zero,only heterozygous mice with SM22-Cre/SCAP^flox/+and wild type mice with SM22-Cre/SCAP^+/+were born.Through the anatomy of mice at different embryonic stages,it was found that the homozygous knockout of SCAP gradually decreased over time until the embryonic E18.5 days,and the homozygous knockout decreased to 0.3.The embryonic E12.5 and E14.5 day mouse embryos were observed by stereoscopic microscope,and it was found that SCAP knockout homozygous mouse embryos had obvious vascular bleeding and delayed development compared with the wild-type mice.4.Continuous slices of mouse embryos were taken from the vascular layer for H.E staining.Compared with wild-type mice,homozygous knockout by SCAP reduced the thickness of the vascular wall by about50%.The expression levels of SCAP,PCNA,PI3K and AKT in homozygous mice with IHC and SCAP knockout were significantly lower than those in wild-type mice.5.SCAP SiRNA of different concentrations interfered with vascular smooth muscle cells.The expression of SCAP mRNA was detected by rt-pcr.The cell proliferation rate also decreased with the increase of the concentration of transfected SCAP SiRNA.6.The expression levels of SCAP,PCNA,PI3K,Akt,were detected by Wstern Blot after the vascular smooth muscle cells were loaded with SCAP SiRNA.Compared with the wild type,the expression levels of SCAP interference group were significantly decreased.Conlusions:SM22-Cre mouse specific knockout SCAP gene,inhibited the dependence on SCAP/SREBP regulating lipid rafts in cholesterol and fatty acid synthesis,thereby inhibition of lipid rafts in the activation of PI3K/Akt signal pathway,leading to lack specificity SCAP gene of vascular smooth muscle cell proliferation,inducing embryonic vessel wall thinning,embryo hemorrhage may be lead to abnormal embryonic development,and eventually cause death.