去泛素化酶OTUD3调控PTEN肿瘤相关突变体蛋白稳定性的研究

Deubiquitinase OTUD3 regulates protein stability of PTEN tumor-associated mutants

目的 探索去泛素化酶含OTU结构域蛋白3(OTUD3)在乳腺癌中对抑癌蛋白PTEN肿瘤相关突变体蛋白稳定性的调控。方法 收集50例乳腺癌患者样本,提取癌与癌旁组织的RNA后进行逆转录,对PTEN C2结构域进行PCR扩增后测序分析,鉴定乳腺癌PTEN C2结构域的肿瘤相关突变。结合现有数据库中PTEN基因的突变位点信息,将PTEN突变体的编码序列扩增后插入哺乳动物表达载体pCMV-Myc中,构建含有PTEN C2结构域肿瘤相关突变体基因的质粒载体。将上述含PTEN点突变体或截短体基因的质粒载体分别与含OTUD3基因的质粒载体共转染MCF-7细胞,通过免疫共沉淀实验、半衰期实验、泛素化实验及Western印迹实验分别检测PTEN所有突变体与OTUD3结合能力、OTUD3对部分PTEN突变体蛋白稳定性的维持能力及去泛素化修饰能力。结果 从50例乳腺癌病例中鉴定出PTEN C2结构域有3种突变,即I224M、E307K与F341V,其中F341V是本研究在乳腺癌病例中首次鉴定出的PTEN突变体。△M199、F341V以及1-319丧失了与OTUD3结合的能力,从而失去了OTUD3对其的稳定作用。结论 在乳腺癌中,PTEN的一些突变体丧失了与OTUD3结合的能力,不能对其去泛素化修饰,使得蛋白稳定性降低。

Objective To explore the ability of deubiquitinase OTUD3(OTU domain-containing protein 3)to regulate the stability of PTEN tumor-associated mutants in breast cancer.Methods Fifty samples of breast cancer were collected and RNA was extracted from cancer and adjacent tissues before reverse transcription and PCR amplification of PTEN C2domain was performed.Sequencing analysis was performed to identify tumor-associated mutations in the PTEN C2 domain of breast cancer.In combination with the mutation information about the PTEN genes in the database,the coding sequence of the PTEN mutants was amplified and inserted into the mammalian expression vector pCMV-Myc to construct a plasmid vector containing the tumor-related mutant genes of the PTEN C2 domain.MCF-7 cells were co-transfected with the plasmid vectors containing the PTEN point mutantortruncated gene and plasmid vectors containing the OTUD3 gene,respectively.The ability of PTEN mutants to bind to OTUD3,the ability of OTUD3 to maintain the protein stability of some PTEN mutants,and the ability of OTUD3 to deubiquitinate some PTEN mutants were detected by Co-Immunoprecipitation,half life,ubiquitination and Western blotting,respectively.Results Three mutations in the PTEN C2 domain were identified from 50 cases of breast cancer,namely I224M,E307K and F341V,among which F341V was the first PTEN mutant identified in this study in cases of breast cancer.△M199,F341V and 1-319 lost the ability to bind to OTUD3,thus losing the stabilizing effect of OTUD3 on it.Conclusion In breast cancer,some mutants of PTEN lose the ability to bind to OTUD3 and cannot be deubiquitinated,resulting in reduced protein stability.