Functional Study On The Interaction Between TMEPAI And Immune-related Gtpase IRGM

Autophagy is a process in which cells degrade their damaged organelles and macromolecules through the autophagy-lysosomal degradation pathway.Autophagy plays an important role in cell metabolism and homeostasis.Transmembrane prostate androgeninduced protein(TMEPAI)mainly locates in lysosomes.It has been reported that TMEPAI is highly expressed in a variety of tumor cells,so it may be closely related to the occurrence and development of tumors.Previous studies in our lab have proved that TMEPAI can promote autophagy.Based on this,this paper explores autophagy related proteins that interact with TMEPAI and conducts in-depth studies on their interaction functions.The research results of this project mainly include :(1)using yeast two-hybrid system to detect autophagy associated proteins(immunity-related GTPase family M,IRGM)that interact with TMEPAI.The interaction between TMEPAI and IRGM in vivo and in vitro was further verified by GST pull-down and co-IP assay.(2)TMEPAI-KD,IRGM-KD,TMEPAI&IRGM-KD cell lines with stable knockdown expression were constructed,and then Western blot,cell transfection and fluorescence microscopy were used to detect the effect of the interaction between TMEPAI and IRGM on autophagy.The results showed that TMEPAI and IRGM synergistically regulated autophagy.(3)MTT assay,monoclonal formation assay and scratch assay were used to detect the interaction between TMEPAI and IRGM on the activity,proliferation and migration of tumor cells.The results showed that the interaction between TMEPAI and IRGM could significantly enhance the activity of tumor cells and promote the proliferation and migration of tumor cells.In summary,this paper found and verified the interaction between TMEPAI and IRGM,and promoted autophagy through synergistic regulation of the interaction.In addition,TMEPAI and IRGM interaction significantly increased the activity,proliferation and migration of tumor cells.Therefore,by exploring the interaction and function of the two,this paper provides new methods and ideas for targeted therapy of tumors.