Study On The Stability Of RNA M~6A Methyltransferase Interaction Protein METTL14 Regulated By HSP90

Heat shock protein 90(HSP90)is a highly conserved and ubiquitous chaperone molecule.It has been reported in the literature that the expression of HSP90 in tumor cells increases sharply,and HSP90 can participate in the development and metastasis of tumors through the regulation of substrate proteins.Methyltransferase-like 14(METTL14)is one of the important components of the RNA m~6A methyltransferase complex.Its function is to help METTL3 recognize the m~6A modification site on RNA and bind to RNA.In recent years,with the continuous research on RNA m~6A methyltransferase,researchers have discovered that METTL14 has many biological functions that have not been previously recognized.According to reports,METTL14 is highly expressed in a variety of tumor cells,and plays a vital role in the development and invasion and metastasis of certain tumors.The focus of this project is to investigate whether METTL14 is regulated by its inhibitors as a substrate of HSP90 and which genes may be involved in the development of cancer by regulating the stability of METTL14.Our results show that:1.After heat shock(about 42℃),the level of METTL14 protein in the cells increased significantly.2.Through immunoprecipitation experiments,it was found that METTL14 interacts with the heat shock proteins HSP90,HSP70,and HSC70,and the region where the METTL14 protein interacts with HSP90 is the C-terminal of METTL14(166-465aa).3.The interaction between METTL14 and HSP70’s C terminus of HSP70 interacting-protein STUB1 was discovered by immunoprecipitation experiments.Through stability experiments,it was found that STUB1 can regulate the stability of METTL14protein.4.After treatment with HSP90 inhibitor 17-AAG in a variety of cells,both exogenous and endogenous METTL14 protein levels decreased significantly,fully proving that METTL14 is a customer protein of HSP90.5.After treatment of colorectal cancer cells with HSP90 inhibitor 17-AAG,it was found that the expression of endogenous SOX2 protein decreased,while the expression of exogenous SOX2 protein did not change significantly.The significance of this experiment is that,unlike STUB1 found in previous articles that directly degrade C-Myc and other oncogenes,we found that METTL14 as a client protein of HSP90 can also be regulated by STUB1,and at the same time,it was found that HSP90inhibitors expressed SOX2 protein levels.It may eventually reduce the stemness of colorectal cancer stem cells.Therefore,the discovery that the HSP90 inhibitor 17-AAG regulates the stability of METTL14,which may lead to a decrease in endogenous SOX2protein expression,may provide a basis for more systematic and in-depth functional research in the future,and provide a new target for the treatment and prognosis of colorectal cancer.