Yin Yang 1 contributes to gastric carcinogenesis and its nuclear expression correlates with shorter survival in patients with early stage gastric adenocarcinoma

Abstract

Background: Yin Yang 1 (YY1) is a transcription factor that regulates diverse biological processes and increasing recognized to have important roles in carcinogenesis. The function and clinical significance of YY1 in gastric adenocarcinoma (GAC) have not been elucidated.

Methods: In this study, the functional role of YY1 in gastric cancer was investigated by MTT proliferation assays, monolayer colony formation, cell cycle analysis, signaling pathway analysis, Western blot analysis and in vivo study through YY1 knockdown or overexpression. Immunohistochemical study with YY1 antibody was performed on tissue microarray consisting of 247 clinical GAC samples. The clinical correlation and prognosis significance were evaluated.

Results: YY1 expression was up-regulated in gastric cancer cell lines and primary gastric cancers. Knocking down YY1 by siYY1 inhibited cell growth, inducing G1 phase accumulation and apoptosis. Ectopic YY1 expression enhanced cell proliferation in vitro and in vivo. Knocking down YY1 in gastric cancer cells suppressed proliferation by inhibiting Wnt/β-catenin pathway, whereas its overexpression exerted oncogenic property by activating Wnt/β-catenin pathway. In primary GAC samples, YY1 nuclear expression correlated with shorter survival and predicted poor prognosis in early stage GACs.

Conclusion: Our data demonstrated that YY1 contributes to gastric carcinogenesis in gastric cancer. In early stage GACs YY1 might serve as a poor prognostic marker and possibly as a potential therapeutic target.

Figure 1

YY1 expression in gastric cancer cell lines and clinical GAC samples. (A) YY1 protein expression in 9 gastric cancer cell lines and 3 normal gastric tissues. (B) Western blot of YY1 in 10 pairs of gastric tumors (T) and the corresponding non-tumorous mucosa (N). (C) The expression of YY1 mRNA in 9 gastric cancer cell lines and 5 normal gastric tissues. (D) YY1 mRNA expression in 28 paired primary GACs (N, adjacent non-tumorous tissue; T, tumor tissue; H, water as negative control).

Figure 2

YY1 knockdown inhibited proliferation in gastric cancer cell lines. (A) Transfection with YY1 siRNA decreased YY1 protein expression in AGS, MKN28 and NCI-N87 cells. (B) 5-day MTT assays revealed YY1 siRNA suppressed gastric cancer cell proliferation (**, p < 0.001). (C) Monolayer colony formation assays suggested transfection with siYY1 reduced monolayer colony formation to 41.1%, 27.8% and 49.7% in AGS, MKN28 and NCI-N87 cells, respectively (**, p < 0.001). The experiments were done in triplicates. (D) Flow cytometry analysis revealed the accumulation of cells in G1 phase 24 hours after siYY1 treatment. Representative data from two independent experiments was shown. (E) Western blot of cleaved-PARP after siYY1 treatment in AGS, MKN28 and NCI-N87 cells.

Figure 3

YY1 knockdown suppressed Wnt/β-catenin signaling pathway and inhibits tumor growth in vivo. (A) Relative luciferase activity of Wnt/β-catenin signaling pathway in 4 gastric cancer cell lines after siYY1 treatment. (B) Relative luciferase activity of TOPflash in AGS, MKN28, NCI-N87 and MGC-803 cells with siYY1 transfection (*, p < 0.05; **, p < 0.001). (C) Western blot of active-β-catenin, total β-catenin, CCND1 and c-Myc after siYY1 transfection in AGS, MKN28 and NCI-N87 cells. (D1) Western blot of YY1 expression in stable clones of shYY1-MKN45 and the vector control. (D2) shYY1-MKN45 formed smaller xenograft tumors than pBABE-MKN45 30 days after subcutaneous injection (**, p < 0.001).

Figure 4

YY1 ectopic expression enhanced tumor growth both in vitro and in vivo. (A) Western blot of YY1, active-β-catenin and total β-catenin after YY1 exogenous overexpression in AGS, MKN28 and NCI-N87 cell lines. (B) 5-day MTT assays revealed YY1 enhanced gastric cancer cell proliferation (*, p < 0.05). (C) Upper: Western blot of active-β-catenin and total β-catenin under siCTNNB1 treatment. Lower: siCTNNB1 inhibits GAC cell proliferation (**, p < 0.001). (D) YY1 enhanced monolayer colony formation to 2.4, 2.3 and 1.9 folds in AGS, MKN28 and NCI-N87 cells, respectively, compared with vector controls (**, p < 0.001). The experiments were done in triplicates. (E1) YY1 ectopic expression activated Wnt/β-catenin signaling pathway in MKN45 cells by consecutively activating active-β-catenin, CCND1 and c-Myc. (E2) YY1-MKN45 formed bigger xenograft tumors than pcDNA3.1-MKN45 20 days after injection (**, p < 0.001).

Figure 5

YY1 nuclear expression correlated with poor survival in early stage gastric cancers. (A) Representative figures of YY1 immunohistochemistry in primary GACs. Left, negative/low expression case; right, positive expression case (original magnification × 100, insertion × 400). (B) YY1 nuclear expression predicted poor prognosis in early stage GAC patients (stage I and II, p = 0.045). (C) YY1 nuclear expression did not associate with disease specific survival in advanced stage GACs (stage III and IV, p = 0.958).