| In China, gastric carcinoma is one of the most common malignant tumor with high fatality rate. The metastasis of gastric carcinoma is fatal. In order to increase the survival rate of patients with gastric carcinoma, it is very helpful for us to understand the molecular mechanism of the metastasis of gastric carcinoma. Helicobacter pylori (H.pylori) infection, Class I carcinogen defined by the World Health Organization, is a major risk factor for gastric carcinoma. However, the correlation between Helicobacter pylori (H.pylori) infection and metastasis of gastric carcinoma remains unclear. It was reported that the expression and function of ezrin protein( a key protein in tumor metastasis) were changed after gastric cancer cells was infected by H.pylori. In this research, we wanted to confirm whether H.pylori. infection influenced the metastasis and invasion of gastric carcinoma by regulating ezrin expression.Objective: 1. To investigate the correlation between invasion and metastasis of gastric carcinoma and H.pylori infection; 2. To explore whether ezrin plays an important role in mediating invasion and metastasis of H.pylori infection-related gastric carcinoma and its molecular mechanism.Methods: 1. By rapid urease test and serum antibody determination, we identified whether the patients were infected H.pylori. H.pylori and AGS cells of gastric adenocarcinoma were cultivated together in vitro. Then the AGS cell morphology was obversed by scanning electron microscopy, the invasion of AGS cell was detected by transwell chamber, AGS cell migration was obversed by scratch test. MTT assay was used to analyze the impact about loss of nests of AGS cells.2. The expression of ezrin, CD44 and E-cadherin in tissues of gastric carcinoma were detected by immunohistochemistry. The express levels of ezrin protein in AGS, MKN28, BGC823 cells were determined by western blot and the invasion of the three cells was analyzed by transwell chamber. MKN28 cell lines possessing weak metastasis ability in above-mentioned cells were observed. MKN28 cell lines were divided into two groups, HP infected group and PBS control group. The invasion of two groups were determined by transwell chamber. The expressions of ezrin, CD44, E-cadhrin and Sixl in two groups were detected by western blot.3. The VIL-2 of RNA interference plasmid vector and control plasmid vector were constructed by gene recombinant technique. AGS cells were transfected by these plasmid vectors. The stable expression cell lines AGSezrin was obtainted by G418 resistance screening. The expressions of Ezrin mRNA and protein were detected by semi-quantitative RT-PCR and western blot. The morphology of four groups, including AGS+PBS, AGS+H.pylori, AGSezrin+PBS, AGSezrin+H.pylori was detected by scanning electron microscope. Meanwhile,the ability of cell invasion, migration and adhesion was determined respectively. In addition, the expressions of ezrin, CD44 and E-cadherin protein in four groups were detected by western blot.Results: 1.①Among 103 subjects, 71 cases were positive for serum H.pylori antibody, 67 cases were positive for rapid enzymatic urine analysis. 60 cases were both positive and 23 cases were both negative in serum H.pylori antibody and rapid enzymatic urine analysis. Among 83 patients enrolled in this research, 36 cases (60%) of H.pylori-positive showed lymph node metastasis and 5 cases (21.7%) of H.pylori-negative showed lymph node metastasis. The rate of lymph node metastasis was significantly different between H.pylori-positive cases and H.pylori-negative cases(P<0.05). The depths of tumor invasion between H.pylori positive cases and negative cases did not show significant difference(P>0.05). The rate of H.pylori infection in intestinal type gastric carcinoma (75.6%,34/45) was significantly higher than that in diffuse-type gastric carcinoma (42.1% ,16/ 38,P<0.05).②After incubated by H. pylori,AGS cells showed deformation, a "hummingbird phenotype" and vacuolated changes. The cells exhibited a long spindle-shaped, long-stretched thin pseudopodia after 24 hours of incubation by H. pylori.③The count of the transmembrane AGS cells was 130.4±11.5 from each version under microscope at 200 times after AGS cells were incubated by H. pylori, significantly higher than that of AGS cells without being incubated by H. pylori(87.1±9.3, P <0.05).④Migrate distance of AGS cells incubated by H. pylori was significantly higher than that of AGS cells without being incubated by H. pylori.⑤The survival rate of nest-losing AGS cells incubated by H. pylori was significantly higher than that of cells incubated without being incubated by H. pylori (P <0.05) after 24 hours of incubation.2.①The expression of ezrin protein and E-cadherin in cases of H.pylori-positive gastric carcinoma were higher than those in cases of H.pylori-negative gastric carcinoma (P <0.05). The expressions of Ezrin in different invasion depths in tissues of gastric carcinoma did not show significant difference (P> 0.05).The immunological activity of ezrin protein in gastric carcinoma patients with lymph node metastasis was significantly stronger than that in patients without lymph node metastasis (P <0.05). The expressions of CD44 in H.pylori positive and H.pylori negative group did not show significant difference(P> 0.05).②Among three cell lines, AGS cell showed the strongest invasion ability, followed by BGC823 and MKN28. The expression of ezrin protein in AGS cells was highest in three cell lines, followed by BGC823 and MKN28 cells.③The count of transmembrane MKN28 cells incubated by H.pylori for 24 hours were 51.5±5.5, higher than that without being incubated H.pylori(32.7±6.2, P<0.05).④The promotion to MKN28 cells invasion caused by H.pylori infection was higher than that to AGS cells. (P<0.05). H.pylori infection can significantly promote the expression of ezrin,CD44 and six1 protein in MKN28 cells (P <0.05). H.pylori infection can also lower the expression of E-cadherin in MKN28(P <0.05).3.①The three kinds of specific interference plasmids of ezrin gene transcription (VIL2) were successfully constructed. The stable expression of interference plasmids of ezrin gene cell lines AGSezrin was obtainted by G418 resistance screening. The expression of ezrin mRNA and protein were detected by semi-quantitative RT-PCR and western blot.②The morphology of cell surface was observed under scanning electron microscopy. AGS cells were oval-shaped or long-spindle. AGS cells were rough in cell surface and possessed stumpy lobed pseudopodia. After being added 100:1 (bacteria / cell) H.pylori, the attachment of H.pylori can be observed. AGS cells exhibited a"hummingbird"phenotype with two slender dendritic processes along their long axis, and the length of the processes is more than 2 times of the cellular diameter. AGSezrin cells appeared polygonal or round with a lot of slender filipodium extending around cell surface. Moreover, cell-cell junction and cell apoptosis were also observed. After AGS cells were added H. pylori at the ratio of 100:1(H. pylori/cells), cell surface was almost covered by the attachment of H. pylori. Filipodium was radially arranged from the cell surface. Cells remained polygonal in shape.Under scanning electron microscope, the adhesion numbers of H.pylori were 24.2±11.5 in AGS cells, 51.4±13.1 in AGSezrin cells. The adhesion number of H.pylori in AGSezrin cells was significantly higher than that in AGS cells (P <0.05).③The adhesion rate was ( 76.2±11.7)% in AGS cells, which was significantly lower than that in AGSezrin cells[ (90.5±7.6)%]. When AGS cells were added 10:1 ratio of H.pylori in AGS and AGSezrin cells, the adhesion rate was (61.8±8.5 )% and (92.4±7.3)% respectively. The adhesion rate of H.pylori infection in AGS and AGSezrin cells showed significant difference(P <0.05).④The average number of transmembrane of AGSezrin cells was 27.67±4.50 , which was signicantly lower than that of AGS cells(125.50±8.33, P <0.05). The number of transmembrane of AGSezrin cells infected with H.pylori were 31.25±6.10. H.pylori infection did not affect the number of transmembrane AGSezrin cells.The migration distance of AGSezrin cells were significantly less than that of AGS cells. H.pylori did not affect migration distance of AGSezrin cells.⑤The percentages of anoikis in AGSezrin cells infected with H.pylori at 6-, 12-, 18- and 24-hour was slightly lower than those of cells infected without H.pylori (P>0.05).⑥The expressions of ezrin and CD44 were promoted in AGS cells infected with H.pylori but no significant change was observed in AGSezrin cells.Conclusion: 1. H.pylori can promote the invasion and lymph node metastasis of gastric carcinoma. 2. Ezrin protein plays an important role in invasion and metastasis of gastric carcinoma and also participates the process of invasion of gastric carcinoma mediated by H.pylori. 3. H.pylori can regulate the expression of Sixl to promote the expression of ezrin in gastric carcinoma cells, resuting in the gastric cancer invasion. |
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