Studies On Regulation Machanism Of MTDH Protein Degradation Mediated By Ubiquitin Pathway

Breast cancer,one of the multiple malignant tumors in women,has now leapt to the first malignant tumor and the second cause of tumor related death in women.It is a serious threat to human health with incidence increased year by year.Although it has made great progress in the early detection,early diagnosis and comprehensive treatment of breast cancer in recent years,the 5-year survival rate of breast cancer patients with metastatic increases slowly.Unfortunately,there is not an effective treatment for solving the problem of tumor recurrence and metastasis fundamentally.The occurrence and metastasis in breast cancer,which has not been elucidated,is a multi-factor,multi-link and multi-stage complex evolution process.Therefore,it is largely extent rely on a comprehensive and depth study about its occurrence and transfer mechanism for improving the prognosis and quality of life of breast cancer patients.MTDH protein is overexpressed in many malignant tumors,especially in breast cancer.MTDH protein is closely related to the occurrence and metastasis of breast cancer,but the mechanism of MTDH abnormal accumulation in tumor cells has not been elucidated until now.Our previous study found that MTDH protein polyubiquitination level is significantly lower in breast cancer cells MDA-MB-231 and SKBR3 than in the normal breast epithelial cell line MCF10 A,which suggested that the imbalance between transcription and degradation of MTDH may result in its overexpression,and the abnormal degradation of MTDH may be the one of the main reasons for its promoting metastasis in breast cancer.In our previous study,the results of immunoprecipitation and LC/MS/MS assay showed that MTDH binds to FBXW7,suggesting that FBXW7 may be a specific ubiquitin ligase of MTDH protein.Objective: to explore the interaction mechanism between ubiquitin ligase FBXW7 and MTDH protein.(1)Identify the interaction between FBXW7 and MTDH protein by immunoprecipitation and GST-Pulldown in vitro and in vivo.(2)Investigate the effect of different subtypes of FBXW7 on the stability of MTDH.(3)Study the effect and regulation mechanisim of ubiquitination level of MTDH mediated by FBXW7.(4)Investigate the regulatory effect of FBXW7 on the half-life of MTDH protein and elucidate the mechanism of MTDH abnormal accumulation in breast cancer.(5)Investigate the regulation of cell proliferation,migration,invasion and apoptosis mediated by the FBXW7-MTDH ubiquitin degradation pathway in breast cancer cells.This project will not only elucidate the the role of FBXW7 in the occurrence and metastasis through MTDH ubiquitin degradation of breast cancer cells,but also lay the theoretical foundation for exploring new targets for breast cancer treatment.Methods:(1)Firstly,MDA-MB-231 cells were transfectd with pcDNA-3.1-MTDH eukaryotic expression vector,and then used for screening MTDH interaction protein by immunoprecipitation and LC/MS/MS assay.Secondly,MTDH and FBXW7 were used as the bait protein to verify the interaction between these two proteins by immunoprecipitation assay.Finally,we expressed and purified GST-FBXW7? protein using prokaryotic expression system,and identified the interaction between MTDH and FBXW7 by GST-Pulldown in vitro.(2)Firstly,the Flag-CMV-FBXW7?,pFlag-CMV-FBXW7?,pFlag-CMV-FBXW7? and pSlilencer4.1-shFBXW7 vectors were constructed and transfected into breast cancer MDA-MB-231 cells and human embryonic kidney HEK293 cells respectively,then the effect of FBXW7 on the stability of MTDH protein was detected.Secondly,the effect of MTDH ubiquitination mediated by FBXW7 under the condition of overexpression or knocking-down was detected by immunoprecipitation assay.Last,we observed the endogenous level of MTDH protein under the condition of overexpression and knocking-down of FBXW7 in MDA-MB-231 cells and HEK293 cells respectively followed by treatment with CHX(cycloheximide,protein synthesis inhibitor).(3)We investigated the effects of MTDH degradation mediated by overexpression of FBXW7,on cell biological behavior by MTT,cell scratches,transwell and apoptosis detection methods in breast cancer cells.Results:(1)We screend out eleven MTDH interacted proteins,including ubiquitin ligase protein FBXW7,through immunoprecipitation and LC/MS/MS assay in MDA-MB-231 cells.Then we identified the interaction between MTDH and FBXW7 in positive and negative aspects using immunoprecipitation method with MTDH or FBXW7 as bait,respectively.Finally,we successfully expressed GST-FBXW7 fusion protein by prokaryotic expression system,and confirmed that MTDH could bind to FBXW7 by GST-Pulldown in vitro.(2)We successfully constructed p Flag-CMV-FBXW7?,pFlag-CMV-FBXW7? and pFlag-CMV-FBXW7? vectors,and then transfected these vectors into breast cancer MDA-MB-231 cells.Our result showed that all the three isoforms have similar impact on MTDH.Indeed,overexpression of Flag-CMV-FBXW7?,? and ? could all reduce the MTDH abundance,and the FBXW7? protein has the most significant effect.Then we knocked down the FBXW7 expression in HEK293 cells by pSlilencer4.1-shFBXW7 vetors,the result revealed that depletion of endogenous FBXW7 expression elevated MTDH abundance.Finally,we conducted the immunoprecipitation assay with anti-MTDH antibody,western blotting results showed that the level of poly-ubiquitination MTDH was markedly increased in the presence of FBXW7,but the MTDH ubiquitination level was significantly reduced followed by the deletion of endogenous FBXW7.Lastly,our studies indicated that MTDH half life was decreased in the condition of overexpression FBXW7? compared with control groups.The half life of MTDH was ~12 h in wild type cells,but it decreased to ~6 h in the condition of FBXW7? overexpression.MTDH protein was almosst no degradation in the condition of FBXW7 knocking-down.(3)We found that MTDH was overexpressed in breast cancer MDA-MB-231 and SKBR3 cells,and the level of FBXW7 was negatively correlated with MTDH,which suggesting that the abnormal degradation of MTDH mediated by FBXW7,may be the one of the main causes of MTDH abnormal accumulation in breast cancer cells.Our studies showed that FBXW7 overexpression suppressed breast cancer cell proliferation,migration and invasion,and promoted cell apoptosis.And the changes of cell behavious were reversed by forced expression of MTDH.So it can be concluded that FBXW7 inhibited the malignant phenotype of breast cancer cells through mediating the degradation of MTDH by ubiquitination.Conclusions:(1)MTDH binds to FBXW7 in vivo and in vitro.(2)FBXW7 negatively regulates MTDH protein stability,and the FBXW7? has the most obvious effect.(3)FBWX7 regulates MTDH protein stability through ubiquitination.(4)FBXW7 negatively regulates the half-life of MTDH mediated by MTDH degradation.(5)The level of FBXW7 was negatively correlated with MTDH protein level,which suggesting that the abnormal degradation of MTDH mediated by FBXW7 may be one of the main causes of MTDH abnormal accumulation in breast cancer cells.(6)FBXW7 suppresses breast cancer cell proliferation,migration,invasion and promotes cell apoptosis through regulating MTDH protein degradation...