Expression of TRF1, TRF2, TIN2, TERT, KU70, and BRCA1 proteins is associated with telomere shortening and may contribute to multistage carcinogenesis of gastric cancer

Abstract

Purpose: Telomere dysfunction is believed to be a significant factor in carcinogenesis. To elucidate the carcinogenesis mechanism in gastric cancer, the expression of telomeric proteins and changes in telomere length were investigated during multistage carcinogenesis of gastric cancer.

Methods: Tissue samples were obtained during surgical operations from the normal gastric mucosa of 10 patients, the precancerous lesions of 15 patients, the gastric cancer tissues (GC) of 20 patients, and of tumors due to gastric cancer with lymph node metastasis (GCLM) from 5 patients. The expression of TRF1, TRF2, and TIN2 proteins was measured by Western blotting, while the expression of TERT, KU70, and BRCA1 proteins was detected using the immunohistochemical method. The mean telomere length was determined by Southern blotting.

Results: Compared with normal gastric mucosa tissues, the expression of TRF1, TRF2, and TIN2 proteins was significantly higher in precancerous lesions, GC, and GCLM (P < 0.01). The expression of TRF1, TRF2, and TIN2 proteins was significantly higher in GC and GCLM than in precancerous lesions (P < 0.01). The expression of TERT and Ku70 proteins in precancerous lesions and GC tissues was significantly higher than that in normal gastric mucosa tissues (P < 0.01). The expression of TERT and Ku70 proteins in GC tissues was significantly higher than in precancerous lesions (P < 0.01). In normal gastric mucosa, the BRCA1 protein was primarily located in the cell nucleus. In precancerous lesions and GC, the expression of the BRCA1 protein was apparent in the cell cytoplasm. The mean telomere length in precancerous lesions, GC, and GCLM was significantly shorter than that in normal gastric mucosa tissues (P < 0.05). The mean telomere length in GC and GCLM was significantly shorter than that in precancerous lesions (P < 0.05). The mean telomere length in all tissue samples was inversely correlated with the level of TRF1, TRF2, TIN2, TERT, and Ku70 proteins.

Conclusions: Our results suggest that the over-expression of telomeric proteins, TRF1, TRF2, TIN2, TERT, and Ku70, and the transposition of the BRCA1 protein may work together to reduce the telomere length in precancerous lesions and gastric cancer, and could contribute to the multistage carcinogenesis of gastric cancer. These findings offer new insight into the mechanism of carcinogenesis in gastric cancer.

Fig. 1

Expression of TRF1, TRF2, TIN2 in normal gastric mucosa, precancerous lesions, and GC a The expression of TRF1, TRF2, and TIN2 protein was evaluated by Western blotting. Compared with normal gastric mucosa tissues, the expression of TRF1, TRF2, and TIN2 proteins was significantly higher in precancerous lesions, GC, and GCLM. The expression of TRF1, TRF2, and TIN2 proteins was significantly higher in GC and GCLM than in precancerous lesions. b–d Data of TRF1, TRF2 and TIN2 protein levels in various lesions were represented, respectively (mean ± SD). ^* P < 0.01 vs N. ^Δ P < 0.01 vs PL. N normal gastric mucosa, PL precancerous lesions, GC gastric cancer, GCLM gastric cancer with lymph node metastasis. The levels of TRF1, TRF2, TIN2 protein = [target protein’s gray density/β-actin’s gray density] × 100

Fig. 2

Expression of TERT, Ku70, and BRCA1 proteins in normal gastric mucosa, precancerous lesions, and GC (the S–P immunohistochemical method. original magnification 200×). a Positive results appeared as brown-stained particles in the cells. The expression of TERT and Ku70 protein was significantly up-regulated in precancerous lesion and in GC tissues. In normal gastric mucosa, the BRCA1 protein was primarily located in the cell nucleus. In precancerous lesions and GC, expression of the BRCA1 protein was apparent in the cell cytoplasm. b Data of TERT and Ku70 protein were represented, respectively (mean ± SD). ^* P < 0.01 vs N. ^Δ P < 0.01 vs PL

Fig. 3

Telomere restriction fragment length (TRF) in normal gastric mucosa, precancerous lesion and gastric cancer was analyzed. a The TRF length was examined by Southern blotting. b Data were represented by mean ± SD. The mean telomere length in precancerous lesions, GC, and GCLM was significantly shorter than that of normal gastric mucosa tissues. The mean telomere length of GC and GCLM was significantly shorter than that of precancerous lesions. c The TRF length in each sample. ^* P < 0.01 versus N. ^Δ P < 0.01 versus PL. N normal gastric mucosa, PL precancerous lesions, GC gastric cancer, GCLM gastric cancer with lymph node metastasis. The size markers and control-DNA are indicated on the side

Fig. 4

TRF1, TRF2, TIN2, TERT, and Ku70 associated with telomere length regulation. The levels of TRF1, TRF2, TIN2, TERT, and Ku70 proteins were analyzed for correlations with telomere length and shown in a–e, respectively