Transcription factor SOX2 up-regulates stomach-specific pepsinogen A gene expression

Abstract

Purpose: Transcription factor SOX2 is expressed in normal gastric mucosae but not in the normal colon. We aimed to clarify the role of SOX2 with reference to pepsinogen expression in the gastrointestinal epithelium.

Methods: We analyzed expression of SOX2 and pepsinogens, differentiation markers of the stomach, in ten gastric cancer (GC) and ten colorectal cancer (CRC) cell lines. The effects of over-expression and down-regulation of SOX2 on pepsinogen expression were also examined.

Results: Six GC and five CRC cell lines showed SOX2 expression on RT-PCR. Expression of pepsinogen A was detectable in eight GC and seven CRC cell lines, whereas the majority of the cell lines expressed pepsinogen C. Over-expression of SOX2 up-regulated expression of pepsinogen A but not that of pepsinogen C in 293T human embryonic kidney cells, and some GC and CRC cell lines. Moreover, pepsinogen A expression was significantly reduced by SOX2 RNA interference in two GC cell lines.

Conclusion: These data suggest that SOX2 plays an important role in regulation of pepsinogen A, and ectopic expression of SOX2 may be associated with abnormal differentiation of colorectal cancer cells.

Fig. 1

RT-RCR analyses of SOX2, PGA and PGC expression levels in GC and CRC cell lines, and normal mucosae.The SOX2, PGA and PGC expression levels were examined by RT-PCR in GC and CRC cell lines, and normal mucosae. GAPDH expression was used as an internal loading control for RT-PCR, and H₂O indicates no RNA added. We performed semi-quantitative RT-PCR with different cycle numbers for each gene and representative data are shown

Fig. 2

Over-expression of SOX2 in 293T and CaCo2 CRC cells. a Western blot analysis of SOX2 protein expression in 293T and CaCo2 cells. The levels of SOX2 expression were determined with an antibody against human SOX2 (Li et al. 2004). The expression levels of α-tubulin are shown at the bottom as a control. b RT-PCR analyses of PGA and PGC expression in 293T and CaCo2 cells with the pcDNA3 vector or pcDNA3-SOX2 transfection. Cells without transfection were used as a negative control, as denoted as Parental. GAPDH expression was used as an internal loading control for RT-PCR. c Western blot analysis of PGI and PGII expression in 293T and CaCo2 cells after transfection of the pcDNA3 vector or pcDNA3-SOX2. Media from the cultured cells were concentrated and then electrophoresed in 10% SDS-polyacrylamide gels. Cells without transfection were used as a negative control, as denoted as Parental. Secreted PGI and PGII proteins were detected in TGBC11TKB, MKN74 and CaRI cells, which showed corresponding PGA and PGC mRNA levels. Human normal stomach mucosa was used as a positive control

Fig. 3

Effect of SOX2 RNA interference on PGA mRNA expression in a GC cell line. The SOX2, PGA and PGC mRNA levels in TGBC11TKB were measured by semi-quantitative RT-PCR after SOX2 siRNA transfection. Decreased SOX2 expression due to siRNA and resultant PGA reduction were found in TGBC11TKB cells, while no change in PGC expression was observed. GAPDH expression was used as an internal loading control for RT-PCR, and HiPerFect indicates that only the transfection reagent was added

Fig. 4

Effects of the SOX2 deletion mutant on PGA and PGC expression in 293T cells. a Structures of the wild type (Wt) and mutant SOX2 (ΔHMG) cDNAs. 293T cells were transfected with a pTracer expression vector encoding either the wild type SOX2, or SOX2ΔHMG. b The SOX2 constructs were abundantly expressed in 293T cells. Western blot analysis of cell extracts was performed with an antibody against human SOX2. The expression levels of α-tubulin are shown at the bottom as a control. c RT-PCR analysis of PGA and PGC mRNA expression in 293T cells transfected with a plasmid encoding either the wild type or mutant type SOX2. GAPDH expression was used as an internal loading control for RT-PCR