NEDDylation-dependent LSD1 Stability And Its Mechanism Research Of Inhibiting Gastric Cancer Cell Stemness

Histone lysine specific demethylase 1(LSD1)removes mono-and di-methylase groups from the fourth lysine residue on histone 3 as well as other non-histone substrates.It has been reported LSD1 is highly expressed and plays an important role in tumorigenesis,metastasis,invasion,growth and differentiation in various cancers such as breast cancer,gastric cancer,liver cancer,lung cancer,prostate cancer and malignant hematological diseases.LSD1 is a potential therapeutic target for many cancers.At present,there are some LSD1 targeted drugs have entered the clinical research stage.Neural precursor cell expressed developmentally down-regulated protein 8(NEDD8)is a ubiquitin-like molecule that participates in the process of protein posttranslational modification.The process,binding to target protein,is called NEDDylation.It has been reported that LSD1 can undergo NEDDylation in the proteomic analysis of NEDD8.NEDDylation has been proven to regulate protein stability,but it is still unclear whether it affects the stability of LSD1 and further regulates the biological function of gastric cancer.This project is dedicated to studying the effects of LSD1 NEDDylation on its stability and the mechanism of its regulation of gastric cancer.Based on this,the research contents and results of this thesis were as follow:(1)LSD1 can be NEDDylated and made it unstableIn order to make sure LSD1 can undergo NEDDylation,the interaction between LSD1 and NEDD8 was first confirmed in gastric cancer cells.Second,the effect of LSD1 NEDDylation on its stability was further explored.Though using MLN4924 and overexpressing NEDD8 to detect the expression of LSD1,the results indicated that LSD1 NEDDylation reduced its stability and shortenen its half-life.In summary,LSD1 can undergo NEDDylation and caused its stability decreasing in gastric cancer cells.(2)LSD1 NEDDylation was at K63 through UBE2MIn order to determine the E2 ligase of LSD1 NEDDylation,it was found knocking down Ubiquitin Conjugating Enzyme E2 M(UBE2M),not Ubiquitin Conjugating Enzyme E2 F(UBE2F),can increase the level of LSD1 and extended half-life of LSD1,proving that UBE2 M was the E2 ligase of LSD1 NEDDylation.In order to explore the sites of LSD1 NEDDylation,using NEDDpreddy to predict,the result showed that sites of LSD1 NEDDylation were K5/6/63/117.Based on this result,the site-directed mutation vectors of LSD1 were constructed and then transferred to 293 T cells.The results showed that LSD1 NEDDylation was at K63,and then it was verified that LSD1-K63R(lysine was mutated to arginine)prolonged the half-life of LSD1.In summary,it was confirmed that LSD1 NEDDylation was at K63 through UBE2 M.(3)NEDDylation of LSD1 inhibited the stemness of gastric cancer cells and enhanced drug sensitivityIn order to determine the biological function of LSD1 NEDDylation,firstly using CRISPR/Cas9 technology gastric cancer cell AGS-KO-LSD1 was constructed.Based on this,cell lines were constructed via overexpressing LSD1-WT and LSD1-K63R(also called AGS-KO-LSD1-WT and AGS-KO-LSD1-K63R).Secondly,the experimental results in cellular and animal level showed that LSD1-K63 R promoted the self-renewal of gastric cancer cells by increasing the expression level of LSD1 and reduced the sensitivity of the gastric cancer cells to 5-Fu and oxaliplatin.In summary,LSD1 NEDDylation inhibited the stemness of gastric cancer cells and enhanced drug sensitivity.To sum up,it was the first time explored that LSD1 can be NEDDylated at the63 rd lysine though UBE2 M.The project also proposed that NEDDylation reduced the stability of LSD1.At the same time,the project put forward that LSD1 NEDDylation inhibited the stemness of gastric cancer and enhanced drug sensitivity.After the research of this subject,the way that LSD1 regulates the stemness of gastric cancer is shown by a new perspective,which not only further consolidated the irreplaceability of LSD1 as a target for the treatment of gastric cancer,but also proposed that it was also a new way to treat gastric cancer by inducing the endogenous degradation of LSD1.