Unknown,Transcriptomics,Genomics,Proteomics

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Oncogenic roles of SETDB2 histone methyltransferase in gastric cancer

ABSTRACT: Histone H3 lysine 9 tri-methyltransferases (H3K9me3) are related to transcriptional gene silencing. Although SETDB2 has H3K9me3 activity, it is unknown whether SETDB2 is linking to carcinogenesis. Here, we studied alterations and functions of SETDB2 in gastric cancers (GCs). In human clinical samples, overexpression of SETDB2 protein was observed in 30 of 72 (41.7%) primary GC tissues compared with their normal counterparts, and significantly associated with poor prognosis of the patients (P<0.05). SETDB2 protein was significantly detected in late stage of GCs. Moreover, SETDB2 protein was strongly expressed in four (30.8%) of 13 GC cell lines, and knockdown of SETDB2 led to decrease the cell proliferation, migration and invasion. According to the microarray analysis on a GC cell line after knockdown of SETDB2, the expression of WWOX and CADM1 tumor suppressor genes was significantly up-regulated. ChIP analysis showed that the H3K9me3 levels at the promoter regions of WWOX and CADM1 genes were closely regulated by the SETDB2 in GC cells. We also found that SETDB2 bound to the promoter regions after SETDB2 overexpression. Our data suggest that SETDB2 is associated with transcriptional repression of WWOX and CADM1, through H3K9me3, and hence overexpression of SETDB2 may contribute to gastric progression. Transfection of SETDB2 siRNA into MKN74 cells were performed by electroporation. After 48hrs, cells were harvested. Total RNA was used for cDNA microarray.

ORGANISM(S): Homo sapiens

SUBMITTER: Nishikawaji Taketo

PROVIDER: E-GEOD-76135 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:A large-scale characterization of the methylation states of candidate CpG islands (CGIs) throughout the gastric cancer methylome has not previously been conducted. Genome-wide DNA methylation profiles were compared between 4 metastatic and 4 non-metastatic gastric carcinomas (GCs) and their surgical margins (SMs). The GC genome showed significantly higher proportions of hypomethylation in the promoter and exon-1 regions, as well as increased hypermethylation of intragenic fragments when compared to SMs. Differential methylation was observed in CGIs near transcription start sites of 546 genes between GCs and SMs, and 601 genes between metastatic and non-metastatic GCs. From the list of differentially methylated CGIs, 68 candidate genes and 10 known tumor-related genes were selected for further characterization based on their known molecular function using DHPLC. Significant differential methylation was validated in the CGIs of 15 genes between GCs and SMs (Ps<0.05) and confirmed using bisulfite-sequencing. These genes include BMP3, BNIP3, CDKN2A, ECEL1, ELK1, GFRA1, HOXD10, KCNH1, PSMD10, PTPRT, SIGIRR, SRF, TBX5, TFPI2, and ZNF382. Hypomethylation of CGIs correlated with up-regulation of GFRA1 expression in GCs, while hypermethylation of other genes inactivated their transcription. Most importantly, prevalence of GFRA1, SRF, and ZNF382 methylation alterations were inversely and coordinately associated with GC metastasis and the patients’ overall survival throughout discovery and testing cohorts in China as well as independent validation cohorts in Japan and Korea. In conclusion, methylation changes in the CGIs of 15 genes correlated strongly with GC development. GFRA1 hypomethylation and SRF and ZNF382 hypermethylation are potential synergistic biomarkers for the prediction of GC metastasis. To identify differential methylation of CGIs related to GC development and metastasis, genome-wide DNA methylation changes in 8 pairs of GC and SM samples were analysed using the MCAM assay with a 99K custom-designed Agilent oligonucleotide microarray composed of 99,027 probes targeting 6,177 unique protein-coding genes containing at least two methylation-sensitive/insensitive SmaI/ XmaI restriction sites (CCC|GGG/ C|CmCGGG) as described in Shen et al, PLoS Genet 3, 2023-2036 (2007).

Project description:Background & Aims: E-cadherin expression disruption is commonly observed in epithelial cancers and metastasis. Such event is also recognized as a crucial step in gastric cancer (GC) initiation and progression. As aberrant expression of microRNAs often perturb the normal expression and function of pivotal cancer-related genes, we characterized and dissected a pathway initiated by loss of microRNA-101 that causes E-cadherin dysfunction through upregulation of EZH2 expression in GC. Methods: Microarrays were used to profile the expression of microRNAs in human GC. Array-CGH revealed DNA copy number changes that were validated by genomic quantitative PCR and Snapshot. Expression levels of microRNAs, mRNA and protein were determined by quantitative-real time PCR and western-blot/co-immunofluorescence. CDH1 inactivating mechanisms were analyzed. Gain and loss of function experiments were done in KatoIII cells. E-cadherin functionality was assessed by immunofluorescence and flow cytometry. Results: MiR-101 expression was significantly decreased in tumors in comparison with normal gastric mucosas (P<.0001). In 65% of the analyzed GC cases, miR-101 downregulation was caused by deletions and/or microdeletions at miR-101-2 locus. EZH2 overexpression and consequent loss/aberrant E-cadherin expression was found in concomitance with miR-101 downregulation in 41% of the analyzed GC cases. This occurred preferentially in cases retaining allele(s) untargeted by classical CDH1 inactivating mechanisms. MiR-101 gain of function experiments or direct inhibition of EZH2, led to a strong depletion of endogenous EZH2 protein and consequent rescue of functional E-cadherin at the cell membrane, mimicking results obtained with clinical GC samples. Conclusions: Deletions and/or microdeletions at miR-101-2 locus underlying mature miR-101 downregulation and consequent EZH2 overexpression represented a novel cascade of genetic events leading to E-cadherin disruption, preferentially affecting the intestinal-type of GC. In the present study, 37 primary GCs and a pool of 10 normal gastric mucosas were used to acquire the expression of 703 known human mature miRNAs deposited in the Sanger miRBase Sequence Database, Release 10.0 (http://microrna.sanger.ac.uk), as well as 393 novel human miRNAs discovered through deep sequencing and validated by qRT-PCR and array profiling (NCodeM-bM-^DM-" Human miRNA microarray probe set V3 from Invitrogen). The 10 normal gastric mucosa RNA samples were pooled to obtain the normal gastric reference sample, which was labelled and hybridised four times to check arrays' reproducibility.

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Oncogenic roles of SETDB2 histone methyltransferase in gastric cancer

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