UBE2M Interacts With LSD1 And Its Effect On Gastric Cancer Cells Proliferation And Migration

LSD1(Histone lysine specific demethylase 1)is the first histone demethylase found in 2004.LSD1 can remove the monomethyl and bismethyl groups of histones H3K4 and H3K9 in the manner of FAD(Flavin adenine dinucleotide)-dependent enzymatic oxidation.As a kind of ubiquitin-like modification,neddylation modification is a regulatory method for the body to perform normal functions.However,abnormal activation occurs in tumors and neurodegenerative diseases.The inhibitor of E1 activating enzyme NAE for neddylation pathway MLN4924 has entered preclinical research.It is found that it has a good inhibitory activity on a variety of tumors.However,recent studies have found that MLN4924 is resistant during tumor treatment,and the E2 conjugating enzyme of neddylation pathway UBE2M has been shown to be a good therapeutic target in lung cancer.Our research team earlier found that LSD1 can enhance migration and invasion of gastric cancer cell MGC-803.This article aims to investigate(1)whether LSD1 has neddylation modification and whether UBE2M will affect it;(2)whether UBE2M has an effect on proliferation and migration of gastric cancer cells and whether LSD1 plays a role in it.(1)LSD1 is positively correlated with UBE2M/UBE2F expression in gastric cancer tissuesOur research group conducted the immunohistochemical experiment on the collected gastric cancer tissues to detect the protein expression of LSD1 and two neddylation E2 conjugating enzymes UBE2M and UBE2F in the cytoplasm and nucleus.The analysis found that there was a positive correlation between the protein expressions of LSD 1 and UBE2M5 LSD1 and UBE2F,but the correlation between the protein expression of LSD1 and UBE2M(r=0.2843,p<0.0001)was significantly higher than the correlation between LSD1 and UBE2F(r=0.1593,p=0.0002).At the same time,the correlation between the protein expression of LSD 1 and UBE2M in the nucleus(r=0.3254,p<0.0001)was significantly higher than the correlation between LSD1 and UBE2M in the cytoplasm(r=0.1323,p=0.0130).Moreover,the pathological characteristics of gender,lymph node metastasis and degree of differentiation have a certain degree of influence on the correlation between the protein expression of LSD 1 and UBE2M.It can be known from the above results that in gastric cancer tissues,there is a positive correlation between the protein expressions of the two E2 conjugating enzymes UBE2M/UBE2F and LSD1 in the neddylation pathway,but the correlation between the protein expressions of LSD1 and UBE2M is significantly higher.It shows that there is a higher possibility of interaction between LSD1 and UBE2M,and the correlation between the protein expression of LSD 1 and UBE2M in the nucleus is significantly higher than that in the cytoplasm,indicating that the site of interaction between the two proteins may be in the nucleus.(2)Determination of the interaction between LSD 1 and UBE2M and neddylation modification of LSD 1 in gastric cancer cellsThe data of mass spectrometry for the UBE2M protein conjugate in gastric cancer cell MGC-803 revealed the presence of polyubiquitin chains,and the ubiquitination modification site of LSD1 was K280(due to the limitations of the current technology,ubiquitin-like proteins cannot be distinguish from ubiquitin).Through immunoprecipitation experiments,cellular immunofluorescence experiments and western-blot experiments,it was shown that LSD1 and UBE2M were co-localized in the nucleus,and there was protein binding between the two proteins,and LSD1 could be neddylation modified.(3)The interacted proteins of UBE2M promote the neddylation modification of LSD1 and UBE2M negatively regulates LSD1 in gastric cancer cellsBy carrying out the in vitro neddylation reaction of the protein complex of UBE2M in gastric cancer cell MGC-803 with the recombined LSD1 by our group,it could be found that the neddylation modification of LSD 1 was significantly enhanced.These results indicate that the protein interacting with UBE2M as related enzymes in the neddylation pathway completed the neddylation modification of LSD1.Studies in gastric cancer cell lines AGS,BGC-823,HGC-27,MGC-803,MKN45,NCI-N87 have found that after knocking down UBE2M,the expression of LSD1 generally had an increasing trend,and after overexpression of UBE2M,the amount of expression had a downward trend.The above results indicate that UBE2M has a down-regulated function on LSD 1 expression in gastric cancer cells as a whole.(4)UBE2M promotes proliferation and migration of gastric cancer cells with LSD1According to the MTT test,UBE2M showed different results for the proliferation of gastric cancer cells.Reducing the expression of UBE2M could inhibit the proliferation of all the tested gastric cancer cells;overexpression of UBE2M could inhibit the proliferation of BGC-823 cells and promoted the proliferation of AGS and HGC-27 cells,but it had no obvious effect on the proliferation of other gastric cancer cells.The wound healing results indicated that UBE2M could cooperate with LSD1 to promote migration of gastric cancer cell MGC-803 to a certain extent.In summary,the neddylation E2 conjugating enzyme UBE2M interacts with LSD1 in the nucleus and promotes the neddylation modification of LSD 1.UBE2M has a certain effect on the proliferation of gastric cancer cells,but it does not show obvious regularity.UBE2M can down-regulate the expression of LSD1 in gastric cancer cells,and LSD1 can cooperate with UBE2M to promote the migration of gastric cancer cells MGC-803 to a certain extent.This sthdy explores the effects of UBE2M on LSD1 neddylation modification,the expression of LSD 1 in gastric cancer cells,the proliferation and migration of gastric cancer cells,and it presents a new idea for the study of LSD1 inhibitors for gastric cancer.