TGBC11TKB

Analysis of SET7/9 Mutation in TGBC11TKB Cells

SET7/9, a histone methyltransferase, has two distinct functions for lysine methylation. To clarify whether or not SET7/9 is related to carcinogenesis, alterations of SET7/9 in gastric cancers (GCs) were studied. Genetic alterations of SET7/9 were examined in 12 GC cell lines and 25 primary GC tissues. By long RT-PCR from exons 1-8 and PCR-SSCP, TGBC11TKB cells exhibited a deletion of exon 3 and a C to T transition at the first nucleotide of exon 3 in SET7/9 mRNA and genomic DNA, respectively (Fig. 1), which predicts the encoding of a truncated SET7/9 protein. Except for TGBC11TKB cells, no mutation was detected in any cases examined.

Fig. 1 Longer RT-PCR of SET7/9 exons 1-8 in TGBC11TKB and KATO-III cells (left). Sequencing analysis of the PCR product of SET7/9 in TGBC11TKB cells. After the subcloning of the RT-PCR product showing an abnormal SET7/9 transcript in TGBC11TKB cells, sequencing was performed (right). Deletion of exon 3 predicts encoding a truncated SET7/9 protein lacking the SET domain. (Keller S, et al., 2017)

TP53TG1 Shows Cancer-Specific DNA Methylation-Associated Transcriptional Silencing

The DNA methylation-associated silencing of TP53TG, a lncRNA, produces aggressive tumors that are resistant to cellular death when DNA-damaging agents and small targeted molecules are used. To further demonstrate the silencing of TP53TG1 in cancer cells in association with the presence of promoter CpG island hypermethylation, its transcription start site (TSS) and DNA methylation patterns proceeded to characterize. Bisulfite genomic sequencing of multiple clones confirmed the dense methylation of the CpG island in HCT-116 cells and its unmethylated status in normal colorectal mucosa (Fig. 2A). DNA methylation analyses were extended to another 12 gastrointestinal cancer cell lines (Fig. 2A). In addition to HCT-116, TP53TG1 5'-end CpG island hypermethylation was also found in the colorectal cancer cell line KM12 and the gastric cancer cell lines KATO-III and TGBC11TKB. All of the other cell lines were unmethylated at this locus (Fig. 2A). Importantly, the use of the demethylating agent 5-aza-2'-deoxycytidine restored TP53TG1 expression in the hypermethylated HCT-116, KM12, KATO-III, and TGBC11TKB cell lines (Fig. 2B). Subcellular fractioning showed that the TP53TG1 transcript in DKO cells, in addition to being present in both the cytoplasm and the nucleus, was particularly enriched in the cytosolic compartment (Fig. 2C). RNA fluorescence in situ hybridization (RNA-FISH) corroborated this intracellular localization (Fig. 2C).

Fig. 2 Epigenetic silencing of the lncRNA TP53TG1 in cancer cells. (A) Bisulfite genomic sequencing analysis of TP53TG1 promoter CpG island in cancer cell lines and normal tissues. (B) DNA methylation-associated transcriptional silencing of TP53TG1 in cancer cells. (C) TP53TG1 RNA-FISH and intracellular localization. (Diaz-Lagares A, et al., 2016)