Expression Of GKN2 In Gastric Cancer And The Inhibition Effect Of Gkn2 On MKN28 Cells

[Objective] GKN2(gastrokines 2), specifically expressed in normal gastric epithelium, its expression is significantly down-regulated or even absent in gastric cancer,and may act as tumor suppressor gene. GKN2 often forms a heterodimer with another protein secretion TFF1(Trefoil factor1), and play the role of protecting gastric epithelial homeostasis in the normal stomach with TFF1. We firstly detect GKN2 and TFF1 protein expression in gastric cancer tissues, adjacent gastric mucosa and distal gastric mucosa tissues by using tissue chip, and analyze the correlation between these two proteins’ expression in gastric cancer. The effect of GKN2 over-expression on growth of human gastric cancer cell line MKN28, in order to illuminate role of GKN2 as tumor suppressor gene, and provide a theoretical basis for clinical and experimental evidence for prevention and treatment of gastric cancer.[Methods]Tissue microarray and immunohistochemistry were used to detect GKN2 and TFF1 expression in gastric cancer tissues, adjacent gastric mucosa and distal gastric mucosa tissues, and determine its clinicopathological significance.Then, the GKN2 gene eukaryotic expression vector was transfected into human gastric cancer MKN28 cells, screened with G418, which establish a stable transfection cell line. After that,GKN2 m RNA and protein expression was verified by q RT-PCR and Western Blot. Cell viability was measured by MTT assay to verify the effect of GKN2 overexpression on proliferation. Migration and invasion ability were explained in GKN2-transfected MKN28 cells by Transwell migration assay and Transwell invasion assay. [Result]Tissue microarray and immunohistochemistry results showed that GKN2 expression in gastric cancer tissues(6.67%,6/90) was lower than that of the adjacent normal gastric tissues(43.75%,21/48) and distal gastric mucosa tissues(83.36%,19/22) significantly. Meanwhile, TFF1 expression in gastric cancer tissues(21.11%,19/90) was lower than that of the adjacent normal gastric tissues(62.50%,30/48) and distal gastric mucosa tissues(81.82%,18/22) significantly. So both GKN2 and TFF1 expression were significantly reduced in gastric cancer tissues(P <0.05). However, the correlation analysis showed no correlativity between GKN2 expression and TFF1 expression in gastric cancer tissue. We constructed stably transfected GKN2/MKN28 cells which overexpressed GKN2 protein successfully. The m RNA and protein expression of GKN2 was examined by RT-PCR and Western blot respectively. Both of them in transfected MKN28 cells expressed significantly increasingly compared with control group.(P<0.05). In the MTT assay, the viability of MKN28 cells with GKN2 transfection was significantly decreased compared to those with vector transfection and non-transfected in 24 h, 48 h and 72 h.(P<0.05), prompting that GKN2 can inhibit the proliferation of MKN28 cells. Transwell test results revealed that the cells’ number passing through the membrane and Matrigel in GKN2 transfected MKN28 cells declined, compared to those in the vector transfected and non-transfected MKN28 cells(P <0.05). But there is no significant difference between vector transfected group and non-transfected group(P> 0.05). The data support that the migration and invasion abilities can be inhibited by GKN2 overexpression. All above suggests that GKN2 overexpression can suppress the growth of MKN28 cells. [Conclusion] 1. GKN2 and TFF1 expression was lowered in gastric cancer tissues, compared to the adjacent gastric mucosa and distal gastric mucosa significantly, which showed that the protein level downregulation of GKN2 and TFF1 may be involved in the development of gastric cancer.2. GKN2 overexpression can inhibit the proliferation, migration and invasion abilities in MKN28 cells.