GALE Promotes The Proliferation And Migration Of Glioblastoma Cells And Is Regulated By MiR-let-7i-5p

BackgroudGlioblastoma,as the most malignant type of human glioma,is one of the most deadly tumor types in humans.Despite substantial advances in imaging diagnosis,surgery,chemotherapy,and radiation therapy,glioma patients do not have an ideal prognosis.According to the World Health Organization(WHO)classification,gliomas are divided into four grades(?-?).Glioblastoma(WHO grade ?)leads to the worst prognosis,with a median survival time of only 12 to 15 months after a confirmed diagnosis.UDP-galactose-4-epimerase(GALE),a key enzyme of galactose metabolism,is overexpressed in some kinds of cancers,such as papillary thyroid carcinoma,and we also found GALE overexpression in glioblastoma.MicroRNAs(miRNAs)constitute a class of single-stranded noncoding RNA that are approximately 20 to 22 nucleotides(nt)in length and have been shown to inhibit gene expression at the posttranscriptional level.miRNAs have been confirmed to play important roles in cell development,progression and human cancer recurrence.Mutations in let-7,which was first discovered in Caenorhabditis elegans,can lead to abnormal larval growth and are therefore lethal to these organisms.Bioinformatics analysis have demonstrated that the microRNA let-7 is highly conserved in various animal species,supporting its important role in early developmental stages.Let-7 plays a vital role in the cell cycle and polarization.The expression of a few niR-let-7 family members has been discovered in many malignancies,including pancreatic cancer,human colon cancer,human lung cancers and breast cancer.However,the association of miR-let-7i-5p with glioma progression remains unknown.In this study,the specific functions of miR-let-7i-5p in human GBM were explored in multiple GBM cell lines.We used the Cox proportional hazards model to identify the gene GALE and the public online database was used to analyze relationship between GALE and the patients' overall survive time or the WHO classification of gliomas.This was also demonstrated by in vitro and in vivo experiments on GBM,and the result was that GALE promote the progression of GBM.We used a dual-luciferase gene reporter assay to confirm whether miR-let-7i-5p directly inhibits GALE expression,which would result in the subsequent inhibition of GBM cell proliferation and migration.In the current research,we examined GALE in human glioma to elucidate its association with tumor progression,and our findings reveal the tumor-suppressive effect of GALE regulated by miR-let-7i-5p for GBM therapies.ObjectiveThe expression differences of GALE gene in human normal brain tissue and glioblastoma at various levels were analyzed,and small interfering RNA against GALE was transfected into human glioblastoma cell lines for in vitro experiments and in vivo experiments in small animals to explore the relationship between GALE and glioblastoma proliferation and migration and other tumor characteristics.To explore the molecular mechanism of miR-let-7i-5p regulating GALE expression and its effect on malignant behavior of glioblastoma cells.Methods1.Cox proportional risk model was established by analyzing TCGA and other network databases to analyze the relationship between GALE gene and prognosis of human glioma patients.2.Pathological specimens of clinical glioma patients during surgery were collected and immunohistochemical staining was performed to analyze the differences in expression levels of GALE in normal brain tissues and in glioma tissues of various grades.3.Human glioblastoma cell lines U87 and U251 were cultured and tumor cells were transfected with small interfering RNA of the targeted GALE gene.4.CCK-8 and EdU experiments were conducted to evaluate the changes in cell activity of tumor cells.5.Cell colony formation experiment was conducted to evaluate the changes in the proliferation capacity of tumor cells.6.The changes in the invasion and migration ability of tumor cells were evaluated by the scratch healing experiment and Transwell experiment.7.The changes of GALE protein,n-cad,mmp-2 and other proteins related to tumor proliferation and migration between the GALE interference group and the NC group were confirmed by Western blot.8.Flow cytometry was used for cell cycle assay to evaluate the difference of GBM cell cycle between the GALE interference group and the NC group.9.Cell apoptosis was measured by flow cytometry to evaluate the difference of GBM cell apoptosis between the GALE interference group and the NC group.10.The relationship between miR-let-7i-5p and GALE was predicted by Bioinformatics analysis.11.The relationship between miR-let-7i-5p and GALE was confirmed by the dual-luciferase reporting test.12.Human glioblastoma cell lines U87 and U251 were cultured and transfected with miR-let-7i-5p mimics or miR-let-7i-5p inhibitor.13.Changes in cell activity of tumor cells were evaluated by EdU assay.14.Cell colony formation experiment was conducted to evaluate the proliferation ability of tumor cells.15.The changes of invasion and migration ability of tumor cells were evaluated by the scratch healing experiment and Transwell experiment.16.Western blot experiments confirmed the changes of GALE proteins,n-cad and other proteins related to tumor proliferation and migration between miR-let-7i-5p mimics group and NC group.17.Flow cytometry was used for cell cycle assay to evaluate the difference of GBM cell cycle between miR-let-7i-5p mimics group and NC group.18.Flow cytometry was used for cell apoptosis assay to evaluate the difference of GBM cell apoptosis between miR-let-7i-5p mimics group and NC group.19.Luciferase-labeled sh-nc and sh-GALE GBM cells were implanted into the intracranial of 4-week-old male nude mice by stereotaxic injection machine.The animals were imaged every 5 days by live small animal imager to pay attention to the changes of tumor growth in animals.20.According to the code of ethics,when the final image showed a sufficiently significant difference between the two groups of mice on day 20,the mice were sacrificed,the brain tissue was removed,and the sections were fixed for HE staining.21.Immunohistochemical staining was performed on tumor sections to detect the expression differences of Ki67,mmp-2 and bcl-2 to investigate the effect of GALE on the proliferation and invasion ability of glioblastoma.Results1.Bioinformatics analysis was used to predict the expression differences of GALE in human normal brain tissue and gliomas.1.1 Analysis in TCGA database showed that GALE mRNA level in human brain glioblastoma(GBMs)was significantly higher than that in low-grade glioma(LGGs)and normal brain tissue.1.2 Kaplan-meier survival curve suggested that GALE expression was associated with overall survival rate(OS)and prognosis in glioma patients.In TCGA database(n=703),LGG(P<0.001)and GBM(P<0.05)patients,the higher the GALE expression was,the worse the prognosis was,with statistical significance.Therefore,GALE may be a new biomarker for glioma prognosis.2.Tumor tissue samples of clinical glioma patients were stained with GALE immunohistochemical staining.Immunohistochemistry was used to detect GALE protein levels in human glioma(32 cases)and normal brain tissue(4 cases)from qilu hospital in jinan city.Consistent with the mRNA expression results in the database,the expression level of GALE protein was higher in GBMs than in LGGs or normal brain tissue.Therefore,in the open database and in our cohort of primary tumor specimens,GALE was positively correlated with an increase in tumor grade.3.GALE knockdown can reduce the proliferation and migration ability of GBM cells3.1 CCK-8 experiment confirmed that the OD450 value in U87 and U251 cells decreased after the GALE expression was down-regulated,which was statistically significant.3.2 EdU experiment confirmed that the percentage of EdU-positive cells in GBM cells decreased after the expression of GALE was down-regulated,which was statistically significant.3.3 The results of colony formation test showed that the colony formation of GBM cells significantly inhibited after GALE was knocked down.3.4 In the wound healing test,the healing speed of GALE knockout group was significantly decreased.3.5 The number of cell migration was significantly reduced in the GALE knockout group in transwell test.3.6 Western blot results showed that after GALE knockout,the expression level of tumor-related genes/pathway proteins such as n-cad,mmp-2 was significantly reduced.4.GALE knockdown induced GBM cell eycle arrest and apoptosis4.1 The results of flow cytometry cell cycle analysis showed that GALE knockout could increase the number of U87 and U251 cells in G1 phase.4.2 GALE knockout can promote the apoptosis of U87 and U251 cell lines.5.Bioinformatics predicted the relationship between miR-let-7i-5p and GALEWe use bioinformatics prediction software Targetscan(http://www.targetscan.org/)analysis of GALE upstream regulatory factors,selected miR-let-7i-5p.6.Fluorescein assayWe conducted a dual-luciferase reporting test,which confirmed the targeting relationship between miR-let-7i-5p and GALE,and miR-let-7i-5p inhibited GALE expression.7.miR-let-7i-5p over-expression can reduce the proliferation and migration ability of GBM cells7.1 EdU assay confirmed that the percentage of EdU-positive cells in GBM cells decreased after miR-let-7i-5p over-expression,with statistical significance.7.2 The cell colony formation test results showed that the colony formation of GBM cells was significantly inhibited after the over-expression of miR-let-7i-5p.7.3 The healing speed of miR-let-7i-5p group was significantly decreased in the wound healing experiment.7.4 Cell migration was significantly reduced in the miR-let-7i-5p over-expression group in transwell.7.5 Western blot results showed that the over-expression of miR-let-7i-5p significantly reduced the expression levels of GALE and tumor-related genes/pathway proteins such as n-cad and mTOR.8.GALE knockdown inhibited the proliferation and invasion ability of GBM tumors in vivo experiments.8.1 Compared with the control group,the size of tumor formation in GALE knockout group was significantly reduced and the boundary was clearer.8.2 Immunohistochemistry confirmed that the expression level of GALE protein in the sh-GALE group was decreased,the expression level of bcl-2 and mmp-2 were decreased,and the proliferation index ki-67 was also decreased.Conclusion1.GALE expression was positively correlated with glioma grade and negatively correlated with prognosis.2.Knockout GALE could significantly inhibit the proliferation and migration of glioblastoma in vitro experiments.3.miR-let-7i-5p targeting GALE and inhibit the expression of GALE.4.Over-expression of miR-let-7i-5p significantly inhibited the proliferation and invasion and migration of glioblastoma.5.Knockout GALE could significantly inhibit the proliferation and migration of glioblastoma in vivo experiments.