The Relationship Between The Glycosyltransferases β3GnT8 And The Proliferation, Invasion Of Human Gastric Cancer

Objective:(1) To investigate the difference ofβ3Gn-T8 protein expression in gastric carcinoma tissue and adjacent non-tumor gastric tissues.(2) To investigate proliferating and invasion regulation of gastric cancer byβ3Gn-T8 in gastric cancer tissue, gastric cancer cell lines AGS, subcutaneous gastric carcinoma of nude mice.(3) To investigate the mechanism ofβ3Gn-T8 on invasion of gastric cancer.Methods:(1) Detected the expression ofβ3Gn-T8 on tissue microarray of 97 cases of human gastric cancer and 89 adjacent non-tumor gastric tissues by immunohistochemistry. Collected clinical and pathological parameters of 97 cases of gastric cancer and analyzed the correlation ofβ3Gn-T8 expression with clinicopathological parameters. Detected the expression of tumor metastasis-related genes MMP-2, TIMP-2 and tumor proliferation related genes PCNA in 97 cases of gastric cancer tissues by immunohistochemical, and analyzed the correlation ofβ3Gn-T8 expression with MMP-2, TIMP-2 and PCNA expression.(2) Designed and synthesized the siRNA ofβ3Gn-T8 and transfected it into AGS gastric cancer cells by Lipofectamine, then screened and identificated the effective siRNA by RT-PCR, realtimePCR, western bloting methods and so on. Transfected the negative controlβ3Gn-T8siRNA (β3Gn-T8NCsiRNA), effectiveβ3Gn-T8siRNA (β3Gn-T8siRNA-2) and theβ3Gn-T8 eukaryotic expression vector (pEGFP-C1-β3Gn-T8), empty vector (pEGFP-C1) into gastric cancer AGS cells, the untreated AGS cells was control, detected howβ3Gn-T8 to influence AGS gastric cancer cell proliferation ability by HE staining, MTT, flow cytometry, Hoechst33258 staining; detected MMP-2, TIMP-2 expression in gastric cancer AGS cell by RT-PCR and western bloting after raising and loweringβ3Gn-T8 expression; detected the MMP-2 enzyme activity by gelatin zymography after loweringβ3Gn-T8 expression; detected the invasive ability of AGS by Transwell after raising and loweringβ3Gn-T8 expression.(3) Constructed model of gastric cancer via subcutaneous inoculation transfected AGS cells ofβ3Gn-T8siRNA-2 and pEGFP-C1-β3Gn-T8 to BALB / c. By the time of tumor formation, growth curves, tumor size, tumor weight were detected to research the effect ofβ3Gn-T8 on proliferation of gastric cancer. Detected the effect ofβ3Gn-T8 on the invasive ability and apoptosis levels of gastric cancer of nude mice by HE staining and transmission electron microscopy. Detectedβ3Gn-T8, MMP-2, TIMP-2 and PCNA expression intensity in gastric cancer of nude mice by immunohistochemistry to further determine theβ3Gn-T8 effect on the proliferation and invasion of gastric cancer of nude mice.Results:(1)β3Gn-T8 expression in gastric cancer was significantly higher than in adjacent non-tumor gastric tissues (P<0.001); and with increasing depth of invasion of gastric cancer, occuring of lymph node metastasis,β3Gn-T8 expression was increased (P<0.05); and in higher clinical stage,β3Gn-T8 expression was increased than that in lower clinical stage(P<0.01);β3Gn-T8 expression in gastric cancer had positive significant correlation with MMP-2(P < 0.01), PCNA(P < 0.05), but negative significant correlation with TIMP-2 (P <0.05).(2)β3Gn-T8siRNA significantly inhibited mRNA and protein expression ofβ3Gn-T8 (P<0.05).AGS cells transfected withβ3Gn-T8siRNA showed increased apoptosis by flow cytometry and Hoechst33258 staining (P<0.05); showed decreased cell proliferation by MTT assay (P<0.05); displayed decreased expression of MMP-2, and increased TIMP-2 expression by RT-PCR and western bloting (P<0.05); showed the decreased activity of MMP-2 by gelatin zymography (P <0.05); showed decreased invasion by Transwell assay (P<0.05).AGS cells transfected with pEGFP-C1-β3Gn-T8 showed decreased apoptosis by flow cytometry and Hoechst33258 staining,but there was no obvious difference(P>0.05); showed increased cell proliferation by MTT assay (P<0.05); showed increased expression of MMP-2, and decreased TIMP-2 by RT-PCR and western bloting (P<0.05); showed increased invasion by Transwell assay (P<0.05).(3) Gastric cancer of nude mice was constructed successfully, the tumor form rate was 100%.The forming time, forming rate, weight, volume of tumor of nude mice inβ3Gn-T8siRNA group were significantly smaller than the control group (P<0.05); on the contrary side ,those except the forming time in pEGFP-C1-β3Gn-T8 group were significantly higher than the control group ( P<0.05).There were not metastasis cancer in lymph node, liver and lung by tumor histology observing; pEGFP-C1-β3Gn-T8 group showed tumor invasion of the surrounding muscle;β3Gn-T8siRNA group showed significantly increased apoptosis by transmission electron microscopy;β3Gn-T8, MMP-2 expression was significantly increased, while TIMP-2 expression was significantly decreased in pEGFP-C1-β3Gn-T8 group detected by immunohistochemistry. On the contraryβ3Gn-T8, MMP-2 expression was significantly decreased, while TIMP-2 expression was significantly increased inβ3Gn-T8siRNA group (P<0.05), while the expression of PCNA had no significant difference.Conclusion:(1)β3Gn-T8 expression in human gastric cancer was increased and is associated with invasion and metastasis of gastric cancer positively;β3Gn-T8 expression in human gastric cancer was correlated with MMP-2, PCNA expression positively, but with TIMP-2 negatively.(2)β3Gn-T8 can enhance the proliferation and invasion of AGS gastric cancer cells; reducedβ3Gn-T8 expression in AGS cell , the expression and activity of MMP-2 decreased ,and expression of TIMP-2 increased; on the contrary ,raisingβ3Gn-T8 expression in AGS cells, expression of MMP-2 increased and TIMP-2 expression decreased.(3)β3Gn-T8 can promote the proliferation and invasion of gastric cancer in nude mice; reducing expression ofβ3Gn-T8 induced decreasing of MMP-2 expression and increasing of TIMP-2 expression; raising expression ofβ3Gn-T8, the expression of MMP-2 increased and the expression of TIMP-2 decreased.