| Colorectal cancer(CRC)is the third most common malignancy and a major cause of cancer-related deaths worldwide.The morbidity and mortality of CRC have increased in the past few years,especially in China.In recent years,despite the advances of surgery resection,radiotherapy and chemotherapy to regulate various localized tumors,relapse and development of tumor metastasis are the main causes of failure in the treatment of CRC.Therefore,it is crucial to clarify the molecular mechanisms underlying CRC progression and metastasis.Specificity nucleoprotein AT enrichment combined with sequence 1(special AT-rich sequence binding protein 1,SATB1)gene in human chromosome 3 3 p23 area,its coding protein with 763 amino acids,contains a MAR combining structural domain(MAR binding domain),a homologous structure domain(homeodomain),a dimer function domain(dimerization domain).SATB1 is a tissue specific expression of nuclear matrix sequence specificity binding protein.Studies have shown that in colon cancer and breast cancer high express SATB1 and play an important role.SATB1 in breast cancer can change the expression of thousands of genes at the same time,raised to promote cancer gene,cut tumor-suppressor genes,and promote the growth of cancer cells and metastasis.SATB1 to participate in the negative regulation of gene expression,formed by the combination of composite anchor SATB1 and MAR due to ring base and form a space truss structure,chromosome remodeling and the required enzymes histone to ubiquitin provides' landing platform,and put them to BUR as the breakthrough point into chromosomes,which will be linked to the packing of the chromosome and gene regulation.SATB1 involved in T cell development,which can coordinate the T cells development at various stages of the expression of genes in normal and orderly,the mechanism is likely to SATB1 mobilization enzyme histone to ubiquitin regulating factors such as changing the structure of the certain ring structure domain and influence transcription factors into chromosomes.The ubiquitin-proteasome pathway is an important regulation system of protein degradation in cells.Through the ubiquitination of substrate proteins and proteasomal degradation,a variety of cellular processes can be affected or regulated,ranging from transcription,cell cycle,immune response,receptor function,tumorigenesis,and inflammatory processes.This pathway is also a strictly regulated reversible process in which E3 ubiquitin ligase and deubiquitin enzyme play key regulatory role.Here,we first found the deubiquitinating enzyme USP47 and E3 ubiquitin ligase SMURF2,which are involved in the reversible regulation of SATB1 ubiquitination,and studied their function in the regulation of colon cancer cell growth and metastasis by knock-out or overexpression.The main results of the research and the contents are as follows:1.USP47 regulating the stability of SATB1 promote colon cancer cell growth and metastasisDUB library screening,endogenous protein--protein interaction analysis is found to ubiquitin enzyme USP47 and SATB1 specific interaction exists.In 293 t cells,the overexpression of USP47 specificity of lower level of SATB1 protein of ubiquitin.In HCT116 colorectal cancer cells,knockout USP47 enhance SATB1 protein ubiquitin.In 293 t and HCT116 cells,the overexpression of USP47 significantly extend the half-life of SATB1 protein.In HCT116,however,knockout USP47 greatly shorten the half-life of SATB1 protein,and join in the knockout USP47 HCT116 cells after MG132,extended the half-life of SATB1 protein.In HCT116 and DLD1 cell,knockout USP47 significant inhibition of cell growth and metastasis,and the expression USP47 significantly promote cell growth and metastasis.And USP47 influence through regulating SATB1 colon cancer cell growth,migration,and invasion.In HCT116 colorectal cancer cells,knockout USP47 significantly inhibit the growth of subcutaneous tumor.Proteomic analysis showed that knockout USP47 significantly affect multiple growth of colon cancer cells and tumor metastasis related gene expression profile.2.SMURF2 mediated SATB1 degradation control colon cancer cell growth and metastasisExogenous and endogenous protein-protein interactions analysis revealed that E3 ubiquitin ligase SMURF2 interacts with SATB1.Overexpression of SMURF2 significantly increased the level of ubiquitination of the SATB1 protein in 293 T cells.Overexpression of SMURF2 significantly shortened the half-life of the SATB1 protein in 293 T and HCT116 cells.However,knockdown of SMURF2 significantly prolonged the half-life of the SATB1 protein in HCT116.Knockout of SMURF2 significantly promoted cell growth and metastasis,whereas overexpression of SMURF2 significantly inhibited cell growth and metastasis.And SMURF2 affects the growth,migration and invasion of colon cancer cells by regulating SATB1.In colon cancer cell HCT116,knockout of SMURF2 significantly promoted subcutaneous tumor growth.3.USP47 negative regulation of SMURF2 affects colon cancer cells resistant to 5-FUIn HCT116 cells,SMURF2 expression was significantly increased after knockout of USP47,whereas SMURF2 expression was significantly decreased after overexpression of USP47.In HCT116 cells,USP47 protein expression was not affected after knockout of SMURF2.In clinical colorectal cancer samples,the expression levels of USP47 and SMURF2 were found to be negatively correlated.The anticancer drug 5-fluorouracil inhibited the expression of USP47,and after knocking out USP47 in colon cancer cell HCT116,the cells were more sensitive to apoptosis.At present,Compelling evidence indicates that SATB1 is highly expressed in various cancers such as breast cancer and colon cancer,and plays a very important role in tumor growth,migration and invasion.Given the observation that USP47 and SMURF2 mediate the stability of SATB1 involved in the regulation of colon cancer cell growth,metastasis,invasion and tumor formation,selective inhibitior of USP47/SATB1 will provide new targets and strategies for the treatment of colon cancer. |
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