Expression of pleomorphic adenoma gene like-2 in gastric carcinoma and its possible mechanism on invasion and epithelial-mesenchymal transition of gastric carcinoma

Abstract

Objective To detect the expression of pleomorphic adenoma gene like-2 (PLAGL2) in human gastric cancer and cell lines, and its role in cell invasion and epithelial-mesenchymal transition (EMT) and the molecular mechanism involved. Methods Fifteen cases of pathologically confirmed gastric cancer tissues and their adjacent normal tissues were collected during operation in our hospital from Jan. 10, 2016 to Mar. 20, 2018. Gastric cancer cell lines MKN-28, BGC-823, NCI-N87 and SGC-7901 were cultured. The expression levels of PLAGL2 in gastric cancer tissue samples and MKN-28, BGC-823, NCI-N87 and SGC-7901 cells were determined by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting. After successful transfection with PLAGL2-small interfering RNA (siRNA) in SGC-7901 cells, the expression levels of PLAGL2 mRNA and protein were detected by Real-time PCR and Western blotting respectively. Cell invasion was detected by Transwell assay. The expression levels of N-cadherin, Vimentin and E-cadherin, which were EMT related markers, and Wnt/β-catenin related proteins were detected by Western blotting. After PLAGL2-siRNA SGC-7901 cells were treated with Wnt pathway agonist SKL2001 and inhibitor XAV939, the changes of cell invasion ability were retested by Transwell assay. Results The expression levels of PLAGL2 mRNA (1.11±0.27) and protein (0.98±0.26) in gastric cancer tissues were significantly higher than those in the adjacent tissues (mRNA: 0.37±0.10; protein: 0.28±0.11; t=-20.941, P<0.01; t=-20.186, P<0.01). The expression levels of PLAGL2 mRNA and protein in MKN-28, BGC-823, NCI-N87 and SGC-7901 were significantly higher than GES-1 cells (mRNA: t=-12.327, P<0.01; t=-19.812, P<0.01; t=-13.080, P<0.01; t=-15.218, P<0.01; protein: t=-11.816, P<0.01; t=-21.162, P<0.01; t=-14.517, P<0.01; t=-15.899, P<0.01). The expression levels of PLAGL2 mRNA and protein in SGC-7901 cells were significantly decreased after PLAGL2-siRNA transfection (mRNA: 2.10±0.29; protein: 1.96±0.27) as compared with control siRNA group (mRNA: 6.61±0.73; protein: 6.46±0.71; t=-1.000, P<0.01; t=-1.000, P<0.01). The number of invasive cells at 24, 48, 72 hin PLAGL2-siRNA transfection group [(30.75±5.74), (48.75±6.90) and (64.50±4.80) cells] was significantly less than in control siRNA group [(51.00±4.97), ( 75.75±4.92) and (132.50±5.57) cells; t=-1.000, P<0.05; t=-1.000, P<0.01; t=-1.000, P<0.01]. The expression of N-cadherin, Vimentin, β-catenin, matrix metalloproteinase (MMP)-7 and c-Myc proteins was significantly down-regulated, and that of E-cadherin protein was significantly up-regulated (t=-1.000, P<0.01; t=-1.000, P<0.01; t=-1.000, P<0.01; t=-1.000, P<0.01; t=-1.000, P<0.01; t=-8.205, P<0.01). SKL2001 could reverse the inhibitory effect on EMT and invasion of SGC-7901 cells after PLAGL2-siRNA transfection, while XAV939 enhanced the above effects of PLAGL2-siRNA. Conclusion PLAGL2 is highly-expressed in gastric cancer. Inhibition of PLAGL2 gene by siRNA can inhibit the invasion of gastric cancer cells probably by down-regulating the Wnt/β-catenin signaling pathway. Key words: Gastric cancer; Invasion; Pleomorphic adenoma gene like-2; Epithelial-mesenchymal transition; Wnt/β-cateninn signaling pathway