Decreased Dp71 expression is associated with gastric adenocarcinoma prognosis

Abstract

For the first time, dramatically decreased Dp71 protein and mRNA was found in 34 pairs of resected primary gastric adenocarcinoma. Immunohistochemistry identified Dp71 expression suppressed in 72.2% of 104 gastric cancer patients. The decreased Dp71 expression was significantly correlated with cancer differentiation (P=0.001) and lymph vascular invasion (p=0.041). Decreased Dp71 expression was associated with a poor gastric adenocarcinoma prognosis (P=0.001). Significantly less Dp71 mRNA and protein were found in BGC823, SGC7901, AGS compared with GES-1. Via increasing lamin B1 mRNA and protein, enforced Dp71d and Dp71f expression resulted in SGC7901 proliferation inhibition. Co-IP proved interaction of Dp71 with lamin B1 in GES-1 cells. Further expression characterization showed reduced lamin B1 in gastric cancer tissue and cancer cells. Increasing lamin B1 expression results in the growth inhibition of SGC7901, which suggests that Dp71-lamin B1 protein complex plays an important role for the newly identified tumor suppressive function of Dp71.

Keywords: Dp71; gastric cancer; gastric cancer cells; lamin B1; prognosis.

Figure 1. Dp71 expression decreased significantly in gastric cancer

A. Dp71 protein expression in 34 paired gastric tissues assessed by western blotting. B. Statistical analysis of Dp71 protein expression (P = 0.024). C. Dp71 mRNA expression in 34 paired gastric tissues assessed by qRT-PCR (P=0.003). Real time RT-PCR was performed to detect Dp71 RNA expression in gastric cancer and adjacent non-cancer tissues, statistically less Dp71 mRNA were expressed in gastric cancer tissues.

Figure 2. Immunohistochemistry of Dp71 expressions in gastric cancer and its prognostic implications

A. Normal gastric tissues, scored as Dp71 (+++); B. Well-differentiated gastric cancer, scored as Dp71 (++) ; C. Moderately differentiated gastric cancer, scored as Dp71 (+); D. Poorly differentiated gastric cancer, scored as Dp71 (-). Original magnification: A–D ×400. E. Kaplan–Meier survival curves of gastric cancer patients (n = 104) after surgical resection. Decreased Dp71 expression correlated with poor patient survival. Patients in high Dp71 expression group exhibited significantly better survival than the low Dp71 expression group (log-rank test: P<0.001).

Figure 3. Decreased Dp71 mRNA and protein expression in gastric cancer cell lines

A. RT-qPCR analysis revealed that the Dp71 mRNA expression was down-regulated in the AGS, BGC823, and SGC7901 cells compared with the normal gastric epithelial cell line GES1. *P <0.05, GES-1 versus AGS, ▲ P < 0.05, GES-1 versus BGC-823, #P < 0.05, GES-1 versus SGC-7901. B. Western blotting analysis of Dp71 expression in GES-1 and and AGS, BGC823, and SGC7901 cells. C. Statistical analysis of Dp71 protein expression of GES-1 and AGS, BGC823, and SGC7901 cells. *P < 0.05, GES-1 versus AGS, ▲ P < 0.05, GES-1 versus BGC823, # P < 0.05, GES-1 versus SGC-7901. There are no statistical differences between AGS, BGC823, and SGC7901 cells.

Figure 4. Increasing Dp71 expressions in SGC7901 cell inhibited its proliferation

A. SGC7901-Dp71d cells displayed inhibited proliferation, *P<0.05, versus SGC7901-Control Dp71d cells; #P<0.05, versus blank SGC7901 cells. B. SGC7901-Dp71f cells displayed inhibited proliferation; *P < 0.05, versus SGC7901-Control Dp71f cells; #P < 0.05, versus blank SGC7901 cells,. C. SGC7901-Dp71d and SGC7901-Dp71f cells increased their Dp71 mRNA and protein D. SGC7901-Dp71d and SGC7901-Dp71f cells increased their laminB1 mRNA expression. SGC7901-Dp71d and SGC7901-Dp71f cells increased their laminB1 mRNA expression compared with SGC7901-Control Dp71d, SGC7901-Control Dp71f and parental cells. *P < 0.05, versus blank SGC7901 cells; Δ P <0.05, versus SGC7901-Control Dp71d cells; #P < 0.05, versus SGC7901-Control Dp71f cells. E. Increased lamin B1 and Dp71 protein in SGC7901-Dp71d and SGC7901-Dp71f cells. After transfection of Dp71d and Dp71f plasmids in SGC7901 cells, western blot were performed to detect Dp71 and lamin B1 expression in SGC7901, SGC7901-Dp71d, SGC7901-Dp71f, SGC7901-Control Dp71d, SGC7901-Control Dp71f cells, lamin B1 and Dp71 protein expression were significantly increased in SGC7901-Dp71d and SGC7901-Dp71f cells.

Figure 5. Dp71 interacts with lamin B1 in GES-1 cells

Whole cell extracts from GES-1 cells were immunoprecipitated with a rabbit anti-lamin B1 antibody A. rabbit anti-Dp71 antibody B. an irrelevant antibody (IgG0) was used as control. Immunoprecipitated proteins were analyzed by western blotting with mouse anti-lamin B1 and anti-Dp71 antibodies. Association between Dp71 and lamin B1 was observed in GES-1 cells.

Figure 6. Decreased lamin B1 expressions in gastric cancer tissue and cancer cells

Normal gastric tissues, scored as lamin B1 (+++); B. Well-differentiated gastric cancer, scored as lamin B1 (++) ; C. Moderately differentiated gastric cancer, scored as lamin B1 (+); D. Poorly differentiated gastric cancer, scored as lamin B1 (-). Original magnification: A–D ×400. E. Western blot analysis identified reduced lamin B1 protein in gastric cancer tissues. F. Lamin B1 protein decreased in gastric cancer cell lines, western blot analysis identified reduced lamin B1 protein expression in AGS, BGC823, and SGC7901 cells compared with GES-1 cells. G. Statistical analysis of lamin B1 protein expression in GES-1, AGS, BGC823, and SGC7901 cells. Statistically less lamin B1 protein was expressed in gastric cancer cells compared with GES-1 cell. *P < 0.05, GES-1 versus AGS, ΔP < 0.05, GES-1 versus BGC823, # P < 0.05, GES-1 versus SGC-7901.

Figure 7. Increased lamin B1 expression inhibited the proliferation of SGC7901 cells

A. Increased lamin B1 protein was detected in SGC7901 cells after transfection of lamin B1-pcDNA3.1 plasmids B. Increasing lamin B1 in SGC7901 displayed inhibited proliferation; *P < 0.05, SGC7901-lamin B1 cells versus SGC7901-Control, # P < 0.05, SGC7901-lamin B1 cells versus blank SGC7901 cells.