A Study Of The Effect Of β1,3-N-Acetylglucosaminyl-Transferase-8(β3Gn-T8) On Tumor Proliferation And Nvasion In Human Astroglioma

Objective:(1) To investigate the difference of β3Gn-T8protein expression in astrogliomatissue and normal brain tissues.(2) To investigate proliferating and invasion regulation of glioma by β3Gn-T8inhuman astroglioma cell lines U251and subcutaneous glioma of nude mice.(3) To investigate the relationship between β3Gn-T8and MMP-2.(4) To investigate the effect of β3Gn-T8on Biosynthesis of polylactosamines.Methods:(1) Detected the expression of β3Gn-T8on human astroglioma and normal braintissues by immunohistochemistry. Collected clinical and pathological parameters of42cases of astroglioma and analyzed the correlation of β3Gn-T8expression withclinicopathological parameters.(2) β3Gn-T8sense vector and its empty vector, β3Gn-T8RNA interference vectorand its control vector were transfected stably into U251cells by Liprofectamine2000.And the stably transfected cell lines were sieved by G418. The optimal cell lines ofβ3Gn-T8upregulation and knockdown were chosed by RT-PCR. There were fivegroups: U251transfected with sense vector and empty vector groups, U251transfectedwith interference vector and control vector groups, U251non-tranfected group. AndRT-PCR, Western blot and Laser Confocal Microscope were used to compare theexpression of β3Gn-T8in the five group cell lines. U251cells transfected withpEGFP-C1-T8or pSilenCircle-T8Si were examined for changes in the cell surfaceexpression of carbohydrate chains by flow cytometric analysis.(3) MTT was used to detect the cell proliferation, and colony formation assay wasused to evaluate the ability of colony formation from single cell in different groups. Wound healing and Boyden chamber were used to compare the ability of cell migrationand invasion in five groups. RT-PCR and Western blot were used to evaluate thevariational characteristics of MMP-2and TIMP-2expression at mRNA and proteinlevel.(4) Constructed model of glioma via subcutaneous inoculation transfected U251cells of pSilenCircle-T8Si and pEGFP-C1-T8to nude mouse. By the time of tumorformation, growth curves and tumor size were detected to research the effect ofβ3Gn-T8on proliferation of glioma. Detected the effect of β3Gn-T8on the invasiveability of glioma of nude mice by HE staining.Results:(1) β3Gn-T8expression in astroglioma tissues was significantly higher than innormal brain tissues (P﹤0.01), and with increasing depth of higher clinical stage,β3Gn-T8expression was increased (P﹤0.05);(2) Vectors were transfected into cells successfully, which was observed throughRT-PCR, Western blot and Laser Confocal Microscope. And we got the stablytransfected cell lines whose β3Gn-T8expressions were upregulated and knockdown.The biosynthesis of polylactosamine chains was decreased in pSilenCircle-T8Si cells，while increased in pEGFP-C1-T8cells.(3) MTT assay showed cell proliferation was higher in over-expressing β3Gn-T8cells than control. And cells with β3Gn-T8knockdown had a poor ability ofproliferation (P﹤0.05). The over-expression β3Gn-T8cells formed more colonies thanthe cells thansfected with empty vetors. And the knockdown of β3GnT8cells formedletter colonies than the control cells (P﹤0.05).Compared to control, over-expression of β3Gn-T8promoted the invasion of U251cells while knock-down of β3Gn-T8inhibited U251cell invasion. Furthermore, woundhealing was performed to investigate the effect of β3Gn-T8on U251cell migration.Consistent with the results of the Boyden chamber assay, over-expression of β3Gn-T8enhanced cell migration (P﹤0.05) in vitro, while knockdown of β3Gn-T8inhibited themigration (P﹤0.05) of U251cells.Our results showed that the expression of MMP-2increased in β3Gn-T8over-expression cells, and decreased in β3Gn-T8knockdown cells both in mRNA and protein levels. But the expression of TIMP-2had no statistical significance in mRNAand protein levels.(4) Glioma model of nude mice was constructed successfully, the tumor form ratewas100%. The forming rate and volume of tumor of nude mice in pSilenCircle-T8Sigroup were significantly smaller than the control group (P﹤0.05); on the contrary side,those except the forming time in pEGFP-C1-T8group were significantly higher than thecontrol group (P﹤0.05). And pEGFP-C1-T8group showed tumor invasion of thesurrounding muscle.Conclusion:(1) β3Gn-T8expression in human astroglioma was increased, and β3Gn-T8expression in human glioma was positive correlated with clinical stage of glioma.(2) The stable upregulation and downregulation of β3Gn-T8U251cells wereestablished(3) The result indicated that β3Gn-T8is involved in the biosynthesis ofpolylactosamine chains(4) β3Gn-T8can promoted the invasion and proliferation of U251glioma cells.Up-regulation of β3Gn-T8increased the mRNA and protein expression of MMP-2.Down-regulation cells showed opposite results. But the expression of TIMP-2had nostatistical significance in mRNA and protein levels. Therefore, these results suggestedthat β3Gn-T8might affect the balance of MMP-2and TIMP-2, and then promote cellmigration and invasion.(5) β3Gn-T8can promote the proliferation and invasion of glioma xenografts innude mice.Therefore, our findings indicated that β3Gn-T8might play a role in cellproliferation and invasion in glioma, indicating a new adjuvant molecular therapystrategy to effective therapy of human malignant glioma.