The Effect Of GaINAc-T14and IGFBP-3on Glioma Cell Lines U87MG And Its Molecular Regulation Mechanism

Part I: The regulation effects of IGFBP-3on the proliferation of brainglioma cell line U87MG through ERK signaling pathwayObjective:To explore the effect of IGFBP-3on glioma cell proliferationand its probablely molecular mechanism.Methods: Selection of cell line with low expression of IGFBP-3in gliomaU87MG, the research groups were divided into three groups: untreated group(Control), pCMV-myc transfected group (NC), pCMV-IGFBP-3transfectedgroup (I), all of the three groups were transfected into U87MG cell lines. After48h incubation in cell culture box, we extracted the total RNA and totalcellular protein, then, detected the intracellular mRNA and protein expressionlevels of ERK1/2and phosphorylation levels of ERK1/2through RT-qPCRtechnology and Western blot method respectively; the expression ofanti-apoptotic protein Bcl-XL was also detected in our research. At the sametime, we used MTT assay to detect cell proliferation after U87MG cell lineswas stimulated by IGFBP-3and cell apoptosis of transected pCMV-IGFBP-3plasmid by AnnexinV-FITC/PI double staining on flow cytometry.Results: The over-expression IGFBP-3group indicated that ERK1/2mRNA and protein levels did not change significantly, but phosphorylationincreased significantly; the expression of downstream regulatory gene Bcl-XLwas upregulated. MTT displayed that proliferation of cells increasedsignificantly at a concentration of50ng/ml and500ng/ml IGFBP-3(p<0.05and p<0.01,respectively), and the extend of ERK1/2phosphorylation wasincreased with time, anti-apoptotic protein Bcl-XL expression also increasedgradually. Apoptosis was not observed any change in cell apoptosis assay.Conclusion:IGFBP-3can promote ERK signaling pathway activation andup-regulate the expression of downstream target gene Bcl-XL in intracellular and extracellular, thereby promoting the proliferation of glioma cells. Theresults show that in glioma cells IGFBP-3has its own unique biologicalfunctions.Part Ⅱ: The effect of GalNAc-T14on U87MG cell apoptosis throughERK signaling pathwayObjective:Study the effect of GalNAc-T14on apoptosis of U87MG celland its probablely molecular mechanism.Methods: This study has successfully constructed the eukaryotic expressionvector of pcDNA3.1-GalNAc-T14.The U87MG cells were divided into threegroups: untreated group (Control), pcDNA3.1(+) plasmid transfectedgroup(NC), pcDNA3.1-GalNAc-T14plasmid transfected group (T14).PcDNA3.1(+) plasmid and pcDNA3.1-GalNAc-T14plasmid were transientlytransfected into the U87MG cell lines, and the cell total RNA and total cellularprotein were extracted after48h incubation. We used RT-qPCR technologyand Western blot method to detect levels of mRNA, protein expression,phosphorylation of ERK1/2, and the expression level of anti-apoptotic proteinBcl-XL. At the same time, AnnexinV-FITC/PI double staining was applied tomeasure cell apoptosis by flow cytometry.Results:The ERK1/2mRNA and protein levels in over expression ofGalNAc-T14group decreased significantly, but its phosphorylation level hadlittle changes, and the expression of Bcl-XL was decreased greatly. Flowcytometric results showed that the apoptosis rate in Control group and NCgroup were3.48%and4.42%respectively, while the apoptosis rate in the T14group was40.83%.Conclusion:GalNAc-T14can inhibit the expression of ERK1/2,down-regulate the expression levels of its downstream target gene Bcl-XL,thereby promot glioma cell apoptosis.Further,the molecular mechanism ofGalNAc-T14which plays a biological function in tumor cells is improved.Part Ⅲ:The study on biological functions of IGFBP-3regulated byGalNAc-T14in U87MG cell linesObjective:To discuss the biological effect of GalNAc-T14on IGFBP-3in brain glioma.Methods: The U87MG cells were divided into four groups: pCMV/pcDNAgroup(Control),pCMV-IGFBP-3/pcDNA3.1(+) group(BP-3/pcDNA), pcDNA3.1-GalNAc-T14/pCMV-myc group(T14/pCMV), pCMV-IGFBP-3/pcDNA3.1-GalNAc-T14group(BP-3/T14). The plasmids were transfected into theU87MG cell lines respectively, and were incubated with0,12h,24h,48h,72h,96h. MTT method was used to detect the cells proliferation. AnnexinV-FITC/PI double staining was applied to measure cell apoptosis transfected withpCMV-IGFBP-3/pcDNA3.1-GalNAc-T14by flow cytometry.Results:Compared with control group, IGFBP-3had the capacity tofacilitate the cell of proliferation; GalNAc-T14owned the function of toobviously promote cell apoptosis. Compared with IGFBP-3group, the cellactivity was reduced greatly in IGFBP-3/GalNAc-T14cotransfection group.The apoptosis was not increased by flow cytometry after co-expression ofIGFBP-3and GalNAc-T14in U87MG.Conclusion:GalNAc-T14can attenuate the proliferation function ofIGFBP-3and regulate the biological functions of IGFBP-3in glioma cells.