The Molecular Mechanism Of USP13 Promoting Breast Cancer Cell Proliferation By Deubiquitinating TIMELESS

Deubiquitinating enzymes(DUBs)are a family of proteases that remove ubiquitin tags from proteins of interest undergoing proteasomal degradation.In recent years,several studies have found that DUBs family members regulate the post-translational modification of different cancer-related proteins in a variety of cancers.The USP family is the largest group of DUBs and the sub-family which most closely related to breast cancer.USP13 has been found to be significantly up-regulated in many cancer tissues in recent research reports,and it is a potential cancer drug design target.Timeless is one of the core circadian clock genes in mammals,and it plays an important role in regulating cell cycle,apoptosis,metabolism,DNA replication and DNA damage repair.Past studies have found that Timeless is closely related to the occurrence and development of a variety of cancers,including breast cancer.However,the post-translational modification of Timeless is rarely involved in related research.This thesis mainly studies the effect of USP13 on Timeless and its molecular mechanism,and then reveals its effect on breast cancer cell proliferation.This research is mainly carried out around the following contents:1.The effect of USP13 on breast cancer-related protein levels was studied by Western blot experiments,and it was found that USP13 can significantly up-regulate the protein level of Timeless,and the endogenous expression of the two in a variety of cells is positively synergistic;2.Through immunoprecipitation experiment,it is found that there is an exogenous and endogenous interaction between the USP13 and Timeless;Through GST-Pulldown experiment,it is found that there is a direct interaction between the the USP13 and Timeless.The immunofluorescence co-localization experiment proved that the two co-localized in the nucleus,which further proved the possibility of interaction between the USP13 and Timeless.3.In order to determine the effect of overexpression and knockdown of USP13 on the stability of Timeless,we tested the half-life of Timeless with or without USP13.It was found that USP13 can significantly extend the half-life of Timeless.By inhibiting the proteasome pathway,it was found that USP13 can improve the stability of Timeless through the ubiquitin proteasome pathway;Through exogenous ubiquitination experiments,it was found that USP13 can deubiquitinate Timeless,thereby improving its stability;4.Through the MTT and Colony formation experiment,it that when Timeless was overexpressed and USP13 was knocked down at the same time,the proliferation of breast cancer cells was significantly down-regulated,and when the expression of USP13 was supplemented,cell proliferation was also significantly restored.In summary,USP13 increases its protein level by deubiquitinating Timeless,thereby further promoting the proliferation of breast cancer cells.It shows that USP13 can be used as a potential breast cancer molecular drug target,and it will provide new ideas for solving the problem of breast cancer treatment.