Elevated DLL3 Enhanced Cell Proliferation Of Stomach Cancer By Accelerating G1/S Transition And Activating Notch Signaling

The Notch ligand DLL3 possesses diverse roles functionally in different cancers but its effects on stomach cancer and the mechanism are barely understood.Here,we studied the effect of DLL3 on stomach cancer.1.mRNA and exon expression of DLL3 in stomach cancer patients and its connection to overall survival were analysed by TCGA and UCSC datasets.It showed that high DLL3 expression in patients was associated with poor survival.Expression of DLL3 was detected by immunohistochemical staining in stomach cancer tissue samples(n=35)and the corresponding juxtacancerous tissues.It showed that expression of DLL3 was higher in stomach cancer tissue samples.These results suggested DLL3 was an oncogene.2.Proliferation of MKN45 and BGC823 cell lines was detected by MTT assay after DLL3 was overexpressed or inhibited by siRNA.It showed that elevated DLL3 promoted stomach cancer cells proliferation and down-regulated DLL3 inhibited cells proliferation.Cell cycle of MKN45 and BGC823 was detected by PI staining after DLL3 was inhibited by siRNA.It showed that cells in S phase was decreased.Cell scratch assay was performed to detect the migration ability of MKN45 and BGC823 after DLL3 overexpression.It showed that cell migration ability was enhanced.MTT assay was performed to detect the sensitivity of MKN45 and BGC823 towards anti-cancer drug Annonaceous acetogenins and Oxaliplatin.It showed that elevated DLL3 made cells more vulnerable to ACGs and Oxaliplatin.These results suggested that elevated DLL3 could enhance cell proliferation.3.Expression of cell cycle related genes was analysed by TCGA dataset and it showed that alteration of G1/S transition related genes was correlated with stomach cancer;Expression of G1/S transition related proteins including RB,CyclinD1,CyclinE1,CDK2,CDK4,CDK6,p16 and Notchl and Notch2 intracellular domain(NIICD and N2ICD)was detected by western blot after DLL3 was overexpressed or inhibited by siRNA.It showed that N1ICD,N2ICD,RB,CyclinD1,CyclinE1,CDK2,CDK4 and CDK6 were up-regualted and p16 was down-regulated.These results suggested that elevated DLL3 could up-regulate G1/S transition related proteins and activate Notch signaling.4.Co-immunoprecipitation was performed to detect whether DLL3 interacts with N1ICD or N21CD in MKN45 and BGC823.It showed that DLL3 did not interact with N1ICD,N2ICD,RBPJ,CyclinDl,CyclinEl or p16.Nuclear and cytoplasmic extraction and cell immunofluorescence were performed to detect whether DLL3 was expressed in nucleus of MKN45 and BGC823.It showed that DLL3 could translocate into nucleus and accumulate in nucleus.Co-immunoprecipitation and tandem mass spectrometry were performed to detect what proteins interact with DLL3 in MKN45 cell lines.It showed that among 58 proteins detected,26 proteins were reported as tumor enhancers and 2 proteins were reported as tumor suppressors The biological processes mostly involved among 58 proteins were cell cycle,cell growth,cell apoptosis,transcription regulation,protein biosynthesis,lipid metabolism,ion transport and innate immunity,suggesting DLL3 did affect cancer cell proliferation and also regulated cancer progression by many other mechanisms.Furthermore,24 proteins located in nucleus,of which XPO1,DDX5,RPS3,NPM1 and EEF1A1 located in nucleolus.Above all,these results for the first time confirmed that elevated DLL3 enhanced cell proliferation of stomach cancer by accelerating G1/S transition and activating Notch signaling.