GOLM1 Silencing Inhibits The Proliferation,migration And Invasion Of Human Glioblastoma Cells Via The Wnt/beta-catenin Signaling Pathway

Part 1: the expression of GOLM1 in human glioblastoma samples and serumObjective:To investigate the protein and mRNA expressions of GOLM1 in normal brain and glioblastoma tissues.And to determine circulating GOLM1 protein levels in patients with glioblastoma and healthy individuals.Methods:1.The expressions of GOLM1 gene in 207 normal brain tissues and 163 glioblastoma samples were analyzed by the GEPIA database.2.The GOLM1 mRNA expressions in 8 glioblastoma samples and 2 normal brain tissues were detected by q RT-PCR.3.Immunohistochemical staining was used to detect the expressions of GOLM1 protein in 8 normal tissues and 14 glioblastoma tissues.4.The ELISA assay was used to determine circulating GOLM1 protein levels in18 patients with glioblastoma and 6 healthy individuals.Results:1.The GEPIA database showed that the expressions of GOLM1 gene were higher in glioblastoma samples than in normal brain tissues(P<0.05).2.The results of q RT-PCR showed that the GOLM1 mRNA expressions in glioblastoma were higher than that in normal brain tissues(P<0.05).3.Immunohistochemistry showed that the protein expressions of GOLM1 in glioblastoma specimens were much higher than that in normal brain tissues(P=0.001).4.The ELISA assay showed that the serum GOLM1 levels in patients with glioblastoma were much higher than that in healthy donors(P<0.01).Conclusion:The expressions of GOLM1 protein and mRNA in glioblastoma samples were higher than that in normal brain tissues,and the serum GOLM1 levels in patients with glioblastoma were much higher than that in healthy donors.Part 2: the effect of GOLM1 silencing on glioblastoma cells proliferation,migration and invasionObjective:Interfering GOLM1 expression in glioblastoma by siRNA to investigate the effect of GOLM1 on cell proliferation,migration and invasion.Methods:1.The GOLM1 expressions in glioblastoma U87 and U251 cells were silenced by small interfering RNAs.And one siRNA with the greater silencing efficiency was selected by q RT-RCR and western blot to transfect the experimental group,and the control group was transfected with si NC.2.The proliferation of the two groups was detected by the Edu assay.3.The cell cycle changes were analyzed by flow cytometry.4.The proliferation abilities of the two groups were tested by CCK-8 assay under the serum-free conditions to exclude the effects of proliferation on the results in the scratch-wound and Transwell assays.5.The migration abilities of the two groups were detected by the scratch-wound assay.6.The Transwell assay was used to detect the migration and invasion abilities of the two groups.Results:1.Both Western Blot and q RT-RCR showed that siRNA2 had higher interference efficiency at protein and mRNA levels(P<0.0001),so siRNA2 was selected as the experimental group for subsequent experiments.2.The Edu assay showed that the Edu-positive cells percentage of the experimental group was lower than that of the control group in U87 and U251 cells(P<0.001).3.The results of flow cytometry showed that the cell cycle progression of the experimental group was inhibited,especially in the G1/S phase(P<0.01).4.Under serum-free conditions,the proliferations of U87 and U251 cells in the experimental group and the control group at 24 hours and 36 hours were not obvious,and there was no statistical difference between the two groups.5.The scratch-wound assays revealed significantly slower wound healing in the experimental group than in the control group for both cell lines 24 h after scratching(P<0.05,P<0.01).6.The Transwell invasion and migration assays showed that the number of cells passing through the chamber in the experimental group was less than that in the control group after 36 hours(P<0.01,P<0.001).Conclusion:Silencing the gene expression of GOLM1 may inhibit the proliferation of glioblastoma cells and inhibit their cell cycle progressions,especially in the G1/S phase.And the migration and invasion abilities of glioblastoma U87 and U251 cells were all inhibited after knockdown of GOLM1.Part 3: GOLM1 affects the development of glioblastoma through the Wnt/beta-catenin pathwayObjective:To investigate the mechanism of interfering GOLM1 expression-mediated U87 and U251 proliferation and motility,and the possible interaction mechanism with the Wnt/beta-catenin pathway.Methods:1.The effects of interfering GOLM1 expression on the expressions of Cyclin D1,Cyclin E1,c-Myc,p-AKT,Snail and MMP2 were detected by Western Blot.2.Western Blot was used to detect the effects of interfering GOLM1 expression on the expressions of the Wnt/beta-catenin pathway-related proteins,such as beta-catenin,p-GSK3 beta and nuclear beta-catenin.3.The expressions and distributions of GOLM1 and beta-catenin in the experimental group and the control group were detected by immunofluorescence assay after interfering with the expression of GOLM1.Results:1.After interfering GOLM1 expression,the expression levels of cell proliferation-related proteins such as Cyclin D1,Cyclin E1,c-Myc,and p-AKT were decreased in the experimental group.The expression levels of cell invasion-and migration-related proteins such as Snail and MMP2 were also decreased(P<0.05).2.After interfering GOLM1 expression,the expressions of Wnt/beta-catenin pathway-related proteins,such as beta-catenin and p-GSK3 beta,were decreased,and the expression of nuclear beta-catenin was decreased more significantly(P<0.05).3.Immunofluorescence assay showed that the fluorescence intensity of GOLM1 and beta-catenin protein were decreased in the experimental group after interfering GOLM1 expression,and the fluorescence intensity of beta-catenin in the nucleus decreased more significantly(P<0.05).Conclusion:GOLM1 silencing may affect the Wnt/beta-catenin pathway by inhibiting nuclear beta-catenin translocation,then affect the proliferation,migration and invasion of glioblastoma cells U87 and U251.Part 4: the effect of silencing GOLM1 in vivo on tumor formation in nude miceObjective:To investigate the effect of GOLM1 silencing on xenograft tumor growth in vivo.Methods:1.Xenograft tumor growth assays were conducted by injecting U87 cells subcutaneously into the left armpits of the nude mice.After subcutaneous tumor formation,the nude mice were randomly divided into two groups.In vivo-siRNA or in vivo-si NC were injected into the tumor mass once every 3 days for 3 weeks.Finally,the tumor pieces were removed and weighed.2.The expressions of GOLM1,Ki67 and Snail in the tumors of the two groups were detected by immunohistochemistry.Results:1.After interfering the expression of GOLM1,the tumor volumes and weights of the experimental group were significantly smaller than those of the control group(P<0.0001).2.Compared with the control group,the expression levels of GOLM1 in the experimental group decreased significantly,indicating that the knockdown efficiency was higher,and the proliferation index Ki67 and the invasion and migration index Snail expressions were also decreased.Conclusion:Subcutaneous tumor growth in nude mice slowed down after silencing GOLM1.