| Objectives: Gastric cancer is one of the most common malignancies,the main cause of death is the metastasis and recurrence.Lysyl oxidase-like 2(LOXL2) is a member of the lysyl oxidase(LOX) protein family and is implicated in tumor development, Proliferation and metastasis.The purpose of present study is to investigate the expression of LOXL2 in gastric cancer and its relationship with clinicopathological characteristics, prognosis,and to test expression of LOXL2 in gastric cancer cells, and to investigate the effect and mechanism of LOXL2 to proliferation and metastasis of human gastric cancer cell line BGC823.Method: Immunohistochemistry was used to examine the expression of LOXL2,PCNA,Cyclin D1,MMP-2 and MMP-9 proteins in gastric carcinoma and gastric mucosa adjacent to carcinoma;The expression of LOXL2 in gastric cancer cell lines and gastric epithelial cell line GES-1 by RT-PCR, Western blot, Effects of LOXL2 on proliferation, and invasion of gastric cancer cells were detected by RT-PCR, Western blot, flow cytometry, cell adhesion,migration and so on after silencing of endogenous LOXL2 using LOXL2-siRNA.Results:1 Expression of LOXL2,PCNA,Cyclin D1,MMP-2 and MMP-9 in gastric cancer tissues1.1 Expression differences of LOXL2, PCNA, Cyclin D1, MMP-2, MMP-9between gastric cancer tissue and adjacent normal tissues, and its relationship with clinicopathological characteristicsLOXL2, PCNA, Cyclin D1, MMP-2 and MMP-9 protein expression was significantly higher in cancer tissues compared to that in adjacent normal tissues(all P<0.05). The expression of LOXL2, PCNA, Cyclin D1, MMP-2and MMP-9 in gastric carcinoma on protein levels were significantly correlated with tumor depth of invasion, TNM stages and lymphatic metatasis(all P<0.05), but not related with patient age, sex, tumor size and differentiation(all P>0.05).1.2 The expression of LOXL2, PCNA, Cyclin D1, MMP-2 and MMP-9 in gastric cancer and its relationship with the patient surialThe overall survival rate was lower in patients with a positive expression of LOXL2, PCNA, Cyclin D1, MMP-2 and MMP-9 than those with negative expression(all P<0.05).1.3 The correlation between the expression of LOXL2, PCNA, CyclinD1,MMP-2 and MMP-9 in gastric carcinomaThere was a positive correlation between LOXL2 and PCNA, Cyclin D1,MMP-2, MMP-9 in gastric carcinoma protein levels(r=0.279,0.287,0.380,0.310; all P<0.05), There was a positive correlation between PCNA and Cyclin D1 in gastric carcinoma protein levels(r=0.270,P<0.05), There was a positive correlation between MMP-2 and MMP-9 in gastric carcinoma protein levels(r=0.545,P<0.05).2 Effect of LOXL2 on the proliferation of gastric cancer cells BGC8232.1 Expression of LOXL2 between differences cell linesLOXL2 m RNA and protein expression was significantly higher in gastric cancer cells BGC823 compared to that in gastric epithelial cell line GES-1(P<0.05).2.2 Effect of si RNA on LOXL2 expression in BGC823 cellsAfter si RNA transfection, there was a significant decrease in BGC823 m RNA and protein levels. The lowest expression levels of LOXL2 m RNA and protein were detected 24 h after transfection(90.47% and 89.40% relative to blank control, respectively; both P<0.05).2.3 Effect of LOXL2 on the proliferation activity of BGC823 cellsAfter siRNA transfection, MTT assay indicated that cell proliferation was restrained remarkably, LOXL2 expression inhibited time-dependently by LOXL2-siRNA treatments.2.4 Comparison of cell cycle distribution of si RNA on LOXL2 expression in BGC823 cellsAfter siRNA transfection, The rate of G1 phase increased, and S phase decreased in BGC823 after LOXL2-si RNA tansfected(both P<0.05)2.5 Effect of LOXL2 on the cell proliferation-related gene expression in BGC823 cellsAfter siRNA transfection, the expression of PCNA and Cyclin D1 mRNA and protein were significantly down-regulated in BGC823 cells(both P<0.05)3 Effect of LOXL2 on the migration and invasion of BGC823 cells3.1 Effect of LOXL2 on migration of BGC823 cellsThe wounding assay was used to observe the effect of LOXL2 on migration of BGC823 cells, the results showed that the migration inhibition rate of BGC823 cells increased significantly by LOXL2-si RNA transfection;while the control si RNA had no significant effect on cell migration compared with those treated with Lipofectamine alone.3.2 Effect of LOXL2 on invasion of BGC823 cellsThe transwell chamber assay was used to observe the effect of LOXL2 on invasion of BGC823 cells, the results showed that the invasion inhibition rate of BGC823 cells increased significantly by LOXL2-si RNA transfection; while the control si RNA had no significant effect on cell invasion compared with those treated with Lipofectamine alone.3.3 Effect of LOXL2 on the gene expression of MMP-2 and MMP-9 in BGC823 cellsAfter siRNA transfection, the expression of MMP-2 and MMP-9m RNA and protein were significantly down-regulated in BGC823 cells(both P<0.05).Conclusion:1 LOXL2 expression was up-regulated in gastric cancer tissue and the its expression level were significantly correlated with tumor depth of invasion,TNM stages and lymphatic metatasis, the patients with overexpression of ZNF139 had poorer prognosis, and overexpression of ZNF139 was factor for prognosis.2 LOXL2 promoted proliferation of BGC823 cells by down-regulating the expression of PCNA and Cyclin D1.3 LOXL2 promoted migration and invasion of BGC823 cells, which is probably related with its down-regulating MMP-2 and MMP-9 expression. |
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