Effects Of IGFBP-3 And GalNAc-T14 On Glioma Cell Proliferation And Cycle And Their Mechanisms Of Action

Objective:To observe the effects of IGFBP-3 and GalNAc-T14 on glioma cell proliferation and cycle and explore their signaling mechanismsMethods:1.One hundred and eight samples of neurosurgically resected glioma tissues with paired adjacent tissues were collected at the Second Affiliated Hospital of Hebei Medical University.Immunohistochemical staining was used to detect IGFBP-3 protein expression in glioma and adjacent tissues and cyclin E protein expression in glioma tissues.The relationship between IGFBP-3 and cyclin E protein expression was analyzed,in addition to the survival rate of patients with glioma.2.U87MG and U251MG glioma cell lines were treated with recombinant human IGFBP-3(rhIGFBP-3),transfected with IGFBP-3 plasmid,or co-transfected with IGFBP-3 and GalNAc-T 14 plasmids,and fluorescence microscopy was used to observe the fluorescence intensity.Cell proliferation was assessed using MTT assay.A plate cloning assay was performed to detect cell clone formation.Flow cytometry was performed to assess the effects on cell cycle.Western blotting was performed to detect IGFBP-3 and GalNAc-T14 expression,and their effects on cyclin E,CDK2,and p-ERK1/2 protein levels.Results:1.IGFBP-3 expression was higher in glioma tissues than in adjacent tissues(P<0.01).Cyclin E was upregulated in glioma tissues with elevated IGFBP-3 expression compared with those without elevated IGFBP-3 expression(P<0.01).There was a strong positive correlation between IGFBP-3 expression and cyclin E protein levels in glioma tissues(r=0.7353,P<0.01).Increased expression of IGFBP-3 and cyclin E protein was associated with reduced survival rate(P<0.05).2.rhIGFBP-3 treatment significantly increased U87MG and U251MG cell proliferation rates in a time-dependent manner(P<0.05),and it significantly enhanced the clone-forming ability of U87MG and U251MG cells(P<0.05).Treatment with rhIGFBP-3 for 48 h significantly reduced the proportion of U87MG and U251MG cells in G1 phase,and it simultaneously significantly increased the proportion of cells in S phase(P<0.01).rhIGFBP-3 treatment increased the expression of p-ERK1/2,cyclin E,and CDK2 in U87MG and U251MG cells in a time-dependent manner(P<0.01).U87MG and U251MG cells transfected with IGFBP-3 plasmid displayed green fluorescence with strong intensity,and IGFBP-3 protein expression was significantly upregulated in these cells(P<0.05).IGFBP-3 overexpression significantly enhanced the clone-forming ability of U87MG and U251MG cells,increased p-ERK1/2,cyclin E,and CDK2 expression in these cells,and significantly decreased and increased the proportion of U87MG and U251MG cells in G1 and S phases,respectively.3.U87MG and U251MG cells co-transfected with IGFBP-3 and CalNAc-T14 plasmids displayed green and red fluorescence with strong intensity;the expression of IGFBP-3 and CalNAc-T14 proteins was significantly upregulated.Compared with cells overexpressing IGFBP-3,the clone-forming ability of U87MG and U251MG cells was significantly reduced by the overexpression of IGFBP-3 and CalNAc-T14.The proportion of cells in G1 phase was significantly increased(P<0.01),and the proportion of cells in S phase was significantly reduced;p-ERK1/2,cyclin E,and CDK2 protein expression was significantly downregulated.Conclusions:1.IGFBP-3 expression is upregulated in glioma tissues and correlated with poor patient prognosis.Cyclin E expression is upregulated in glioma tissues with elevated IGFBP-3 expression,which is also correlated with poor patient prognosis.2.IGFBP-3 can phosphorylate and activate ERK1/2 and upregulate the expression of cyclin E and CDK2 proteins,thereby promoting G1/S phase transition and proliferation of U87MG and U251MG glioma cells.3.GalNAc-T14 inhibited the expression of p-ERK1/2,cyclin E,and CDK2 in IGFBP-3-treated U87MG and U251MG cells,thereby inhibiting Gl/S phase transition and proliferation in the cells.