| Objective:β1, 3-N-acetylglucosaminyltransfera 8 (β3Gn-T8), also as known as beta 1, 3-galactosyltransferase 7(β3Gal-T7), which has been cloned and characterized by our group, is a new member of glycosyltransferases.It has been reported thatβ3Gn- T8 may be associated with tumor invasion and metastasis. This thesis aims to analysis the possible mechanism of how the glycolsyltransferase correlates with the invasive and metastatic potential of human gastric cancer.The purpose of this study also was to investigate the effect ofβ3Gn-T8 siRNA and positive-sense expression vector in gastric cancer cells AGS and to find a novel therapeutic target for human gastric treatment.Methods: 1. To follow the principle of selecting RNA interference target sequences and to utilize the web resources, three pairs of small interfering RNA (siRNA) forβ3Gn-T8 gene was designed and transfected into AGS cells. Forty- eight hours after transfection, we collected cells and detected theβ3Gn-T8 mRNA by RT- PCR and real-time-PCR. Protein expression was tested by western blotting .Cell proliferation and apoptosis were examined by MTT, flow cytometry, and Hoechst33258 staining, respectively. The change of Morphological after transfection with siRNA was detected using H-E staining.2. Screened the genes which had high homology withβ3GnT-8 from theβ3GnT family and theβ3GalT family. Screened out the genes which correlated with the invasive and metastasis potential from the homologous gene through bioinformatics and predicted the relationship betweenβ3Gn-T8 and these genes.3. After transfected with effectiveβ3GnT8 siRNA and pEGFP-C1-β3GnT8-sense, detected the expression of genes which was screened out by bioinformatics approach using RT- PCR, western blot .Gelatin zymography was used to measure the enzymatic activity. The proliferation of AGS cells was evaluated with MTT and flow cytometry. The invasion ability was assayed by Transwell. Took different subtype AGS to subcutaneously inoculate to make the nude mice tumor models. Then observe the rate and time of tumor genesis .Measure the weight and volume of tumors.Results: 1. Chemically synthesized three siRNA molecules and transfected into AGS cells.Compared the transfection efficiency of transfection system,the final concen- tration of transfected siRNA experiments was 100 nmol /L per 1×10~5 cells, and filter out the most efficient siRNA sequence.β3Gn-T8 siRNA specifically reducedβ3Gn-T8 expression on the level of mRNA and protein in AGS cells and induced cell apoptosis.2. Through homology comparison, we found thatβ3Gn-T2 andβ3Gn-T8 had greater homology. We found thatβ3Gn-T2 and MMP-2, TIMP-2 had a very close interaction from the database. In addition, PPI network analysis also revealed that Ets-1 and Jun were closely related to the regulation of MMP-2 and TIMP-2.β3Gn-T8 contained the binding sites of transcriptional factors Ets-1 and Jun within several hundred base pairs upstream the TSS.3. We found out that siRNA-mediated suppression of theβ3Gn -T8 could directly reduce the MMP-2 expression and activity as observed in RT- PCR, western blot and gelatin zymography analysis. The cell invasion ability decreased. Meanwhile, TIMP-2 expression had been increased. Furthermore, cells overexpressingβ3Gn-T8 gene (when transfected with pEGFP-C1 plasmid) also expressed MMP-2 gene. TIMP-2 expression had been inhibited.In siRNA goup, the weight and volume of were significantly lower than those in control group. But in pEGFP-C1-β3GnT8-sense group, the results were contrary.Conclusion: We adopted bioinformatic technique to design effective target siRNA; Expression ofβ3Gn-T8 gene could be effectively inhibited by siRNA in AGS cells.β3Gn-T8 had influence on cell growth, proliferation, and invasive potential of AGS. Nude mice experimental evidence suggested thatβGn-T8 might play a very useful role in regulating tumor invasion and metastasis.
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