Effect and mechanism of RNAi targeting WWTR1 on biological activity of gastric cancer cells SGC7901

Abstract

Gastric cancer (GC) is one of the most common malignancies in the world. It is essential to develop novel targets and therapeutic approaches for GC, which requires identification of novel functional molecules. WW‑domain containing transcription regulator 1 (WWTR1) may activate many transcriptional factors and exhibit an important role in the development of various tissues in mammals. The results of the present study demonstrated that mRNA and protein levels of WWTR1 are increased in GC tissues and cell lines. The SGC7901 cell line was selected to perform RNA interference (RNAi) targeting WWTR1, and for subsequent study. Compared with control groups (cells without any treatment) and mock groups (cells treated with nonspecific siRNA), cell proliferation of siWWTR1 cells (cells treated with WWTR1 siRNA) was detected using a Cell Counting Kit‑8 assay at 12, 24 and 48 h, and decreased in a time‑dependent manner. Cell cycle and apoptosis status were determined by flow cytometry, and it was demonstrated that G1/S transition was blocked in the cell cycle and apoptosis promoted in siWWTR1 cells, compared with control and mock cells. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to detect the mRNA and protein levels of cell cycle and apoptosis‑associated factors. The expression of Cyclin D1, cancer Myc and B cell lymphoma/leukemia‑2 (Bcl‑2) decreased and Bcl‑2 associated X protein increased significantly in siWWRT1 cells, at the mRNA and protein level, compared with control and mock cells. With the exception of the Hippo pathway, siWWTR1 regulated downstream factors, including mothers against decapentaplegic homolog family member 3 (SMAD3) and inhibitor of DNA binding 1, HLH protein (ID1), HLH protein in the transforming growth factor (TGF)‑β pathway. The expression of asparagine synthetase was decreased whereas ID1, SMAD3 (proteins that participate in intracellular TGF‑β transduction) and betacellulin increased notably in siWWRT1 cells. In conclusion, WWTR1 promotes cell proliferation and inhibits apoptosis of GC cells by regulating cell cycle/apoptosis‑associated factors, and effectors in the TGF‑β pathway.

Figure 1.

WWTR1 expression in gastric cancer tissue and cell lines. (A) WWTR1 mRNA expression was measured by RT-PCR in gastric cancer tissue and the adjacent normal tissue. (B and C) Protein levels of WWTR1 were determined by western blotting in gastric cancer tissue and adjacent normal tissue. Several representative western blotting images were presented. The average protein level was presented in (C). (D) WWTR1 mRNA expression was measured by RT-PCR in normal gastric mucosa epithelia cell GES1 and gastric cancer cell lines including SGC7901, BGC823, HGC-27 MGC-803 and MKN45. (E and F) Protein levels of WWTR1 were determined by western blotting in cell lines (GES1, SGC7901, BGC823, HGC-27 MGC-803 and MKN45). GAPDH was detected as the control of sample loading. Data were presented as mean ± SD, n=6; *P<0.05 and **P<0.01 compared with the control cells. WWTR1, WW-domain containing transcription regulator 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2.

Effect of siRNA-WWTR1 on cell viability of SGC7901 cell. (A) After siRNA-WWTR1 transfection for 48 h, mRNA expression of WWTR1 in SGC7901 cells was quantified by RT-PCR. (B and C) Protein levels of WWTR1 in SGC7901 cells were measured by western blotting after siRNA-WWTR1 transfection for 48 h. (D) After siRNA-WWTR1 transfection for 12, 24 and 48 h, CCK-8 assay was performed to verify the cell viability of SGC7901 cells. Data were presented as mean ± SD, n=6. **P<0.01 compared with the control cells and ^##P<0.01 compared with the mock cells. WWTR1, WW-domain containing transcription regulator 1; CCK-8, Cell Counting Kit-8; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 3.

Effect of siRNA-WWTR1 on cell cycle progression and apoptosis of SGC7901 cell. (A and B) At siRNA-WWTR1 48 h transfection, cell cycle analysis of SGC7901 was determined, checking the proportion of cells in G1, S and G2 phases respectively. (C and D) At siRNA-WWTR1 48 h transfection, cell apoptosis of SGC7901 was detected by Annexin V/PI double-stain analysis. Data were presented as mean ± SD, n=6. *P<0.05 and **P<0.01 compared with the control cells, ^#P<0.05 and ^##P<0.01 compared with the mock cells. WWTR1, WW-domain containing transcription regulator 1; PI, propidium iodide.

Figure 4.

Effect of siRNA-WWTR1 on expression of cell cycle and apoptosis related factors. (A) RT-PCR was conducted for mRNA expression detection of cell cycle factors such as Cyclin A, Cyclin D1, Cyclin E, c-Myc, and apoptosis related factors such as Bcl-2, XIAP and Bax, in SGC7901 cells after siRNA-WWTR1 transfection for 48 h. (B and C) The protein levels of Cyclin D1, c-Myc, Bcl-2 and Bax in SGC7901 cells were analyzed by western blotting after 48 h of siRNA-WWTR1 treatment. GAPDH was detected as the control of sample loading. Data were presented as mean ± SD, n=6, **P<0.01 vs. control; ^##P<0.01 vs. mock. WWTR1, WW-domain containing transcription regulator 1; Bcl-2, B cell lymphoma/leukemia-2; XIAP, X-linked inhibitor of apoptosis; Bax, Bcl-2 associated X protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; c-Myc, cancer Myc.

Figure 5.

Effect of siRNA-WWTR1 on expression of WWTR1 downstream factors. (A) After siRNA-WWTR1 transfection for 48 h, the mRNA expression of ASNS, ID1, SMAD3 and BTC were measured by RT-PCR. (B and C) ASNS, ID1, SMAD3 and BTC protein expression were determined by western blotting after siRNA-WWTR1 transfection for 48 h. GAPDH was also detected as the control of sample loading. Data were presented as mean ± SD, n=6, **P<0.01 vs. control; ^##P<0.01 vs. mock. WWTR1, WW-domain containing transcription regulator 1; ASNS, asparagine synthetase (glutamine-hydrolyzing); ID1, inhibitor of DNA binding 1, HLH protein; SMAD3, mothers against decapentaplegic homolog family member 3; BTC, betacellulin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.