泛素连接酶Cul7调控人滋养层细胞Notch1蛋白稳定性  

Ubiquitin Ligase Cul7 Regulates the Stability of Notch1 Protein in Human Trophoblast Cells.

作　　者：焦寒 徐阳 范冰凌 林欣欣 付杰军[1] Jiao Han;Xu Yang;Fan Bingling(Key Laboratory of Longevity and Aging-related Diseases of Chinese Ministry of Education,Center for Translational Medicine,Guangxi Medical University,Guangxi 530021,China)

机构地区：[1]广西医科大学长寿与老年相关疾病教育部重点实验室、转化医学研究中心,南宁530021 [2]广西医科大学基础医学院,南宁530021

出　　处：《医学研究杂志》2021年第8期41-46,共6页Journal of Medical Research

基　　金：国家自然科学基金资助项目(82060532,81560483);广西壮族自治区自然科学基金资助项目(2018GXNSFAA281177)。

摘　　要：目的探讨泛素连接酶Cullin 7(Cul7)在人胎盘绒毛外滋养层细胞HTR8/SVneo中对Notch1蛋白稳定性的影响,初步阐明Cul7对Notch1蛋白表达的调控作用。方法Western blot法检测Cul7基因敲除的小鼠胚胎成纤维细胞(MEF)中Notch1蛋白表达的变化。慢病毒稳定转染构建Cul7低表达HTR8/SVneo细胞系,通过real-time PCR检测Notch1 mRNA的表达。Western blot法检测干扰或过表达Cul7后Notch1蛋白的表达。应用蛋白免疫共沉淀(Co-IP)确定Cul7与Notch1蛋白结合情况。结果与野生型MEF细胞比较,Cul7基因敲除MEF细胞Notch1蛋白表达增加。在HTR8/SVneo细胞也观察到干扰Cul7后Notch1蛋白表达显著升高,但对Notch1 mRNA的表达无显著影响。用放线菌酮(CHX)处理细胞后,与对照组比较干扰Cul7使Notch1蛋白降解的半衰期延长。将Flag-Notch1重组质粒和HA-Cul7真核表达质粒导入HTR8/SVneo细胞后,Co-IP结果显示外源性Cul7与Notch1无结合。结论Cul7调控Notch1降解速度,但对Notch1转录表达无显著影响。Cul7可能通过间接方式调控Notch1蛋白稳定性。Objective To investigate the effect of ubiquitin ligase Cullin 7(Cul7)on the stability of Notch1 protein in human placental extravillous trophoblast cells(HTR8/SVneo),and to illustrate the regulatory effect of Cul7 on Notch1 protein expression.Methods The expression of Notch1 protein in embryonic fibroblasts of Cul7 knockout mice was detected by Western blot.After interfering with the expression of Cul7 in HTR8/SVneo cells by lentivirus-mediated shRNA,the expression of Notch1 mRNA was detected by real-time PCR.Western blot was used to detect the expression of Notch1 protein after interference or overexpression of Cul7.Co-immunoprecipitation(Co-IP)was used to detect the combination between Cul7 and Notch1 protein.Results Western blot results showed that compared with wild type,the expression of Notch1 protein in Cul7 knockout mouse embryonic fibroblasts cells was increased.The expression of Notch1 protein was significantly increased after interference with Cul7 in HTR8/SVneo cells.Real-time PCR results showed that interference with Cul7 had no significant effect on the expression of Notch1 mRNA.After treated with Cycloheximide(CHX),the half-life of Notch1 protein degradation was prolonged in Cul7 knockdown HTR8/SVneo cells which interferenced with Cul7.Co-IP results showed that there was no binding between exogenous Cul7 and Notch1 in HTR8/SVneo cells which overexpressed Cul7 and Notch1.Conclusion Cul7 regualtes the degradation rate of Notch1 protein,while has no significant effect on Notch1 transcriptional expression.Cul7 may indirectly regulate Notch1 protein stability.

关 键 词：NOTCH1 CULLIN 7 泛素化 胎盘发育 

分 类 号：R34[医药卫生—基础医学]