Sp1 Promote Cell Proliferation,Migration And TMZ Resistance In Glioblastoma By Inhibiting MiRNA-130a-3p

Background and Objective: Glioma is the most common tumor of the central nervous system and is the most invasive primary intracranial tumor.It has a high incidence and lethality,and the prognosis of radiochemotherapy is still poor.In recent years,with the rapid development of molecular targeted therapy,the treatment of malignant tumors at the gene level has become the focus of scholars at home and abroad.Sp1 is a member of the Sp family and regulates a variety of gene transcription processes.Sp1 is highly expressed in many cancers and involves in tumorigenesis and poor prognosis.It is a potential tumor early diagnosis biomarker and therapeutic target.However,the expression and regulation of Sp1 in gliomas is still unknown.Therefore,the aim of this study is to detect the expression of Sp1 in gliomas by a series of molecular biology and cell function experiments.To find the miRNA that targets Sp1 by bioinformatics tools,gene silencing,overexpression,and dual luciferase reporting systems.To explore the molecular mechanism of Sp1 participating in glioma tumorigenesis and provide evidence for Sp1 to become a early diagnosis biomarker of gliomas.Method: We first explored the expression of Sp1 in tissues of different grades of gliomas and the relationship between Sp1 and the prognosis of patients with glioma by analyzing glioma gene expression profiles in TCGA,CGGA and other public data sets,and we compared the expression of Sp1 in glioma cells and HA1800 normal glial cells by qRT-PCR and Western blot.Then,to predict Sp1 targeted miRNA using online bioinformatics tools.Using gene silencing,over-expression,dual luciferase reporter system to verify the targeting relationship between Sp1 and miRNA.Thirdly,a series of cell functional experiments including MTT assay,colony formation assay,flow cytometry analysis,and Transwell assay,as well as molecular biology experiments were used to explore the effects of Sp1 and its targeted miRNA on glioma proliferation,migration,and TMZ resistance.Finally,by establishing a subcutaneous transplantation model of glioma cells in BALB/c nude mice to explore the effects of Sp1 and its targeted miRNA on glioma cell proliferation,migration and TMZ resistance in vivo.Results: In this study,using the GEPIA platform we analyzed the glioma gene expression profile in the TCGA public dataset,and it was found that the expression level of Sp1 in low-grade glioma and GBM tissues was higher than that in normal brain tissue.The results of qRT-PCR and Western blot showed that the mRNA and protein levels of Sp1 in glioma cell lines were significantly higher than those in normal glioma cells.The analysis of the CGGA dataset preliminarily confirmed that Sp1 expression was closely related to the prognosis of patients with low-grade gliomas.This study also analyzed the French public dataset through the R2 platform,and found that Sp1 expression level was related to the prognosis of IDH R132 wild-type and IDH R132 mutant glioma patients,and Sp1 low-expressed glioma patients both in IDH R132 wild-type and mutant group had a poor prognosis.Further verified that Sp1 was aberrantly highly expressed in gliomas and played important roles in gliomas development.Through Starbase V2.0 and TargetScan online prediction,we found that miR-130a-3p was complementary to the 3￠-UTR of Sp1 gene,indicating that Sp1 might be a potential target of miR-130a-3p.MiR-130a-3p mimic transfection significantly reduced the expression of Sp1 while miR-130a-3 inhibitor markedly increased the expression of Sp1,which verified that Sp1 is inversely related to miR-130a-3p expression in gliomas.In addition,miR-130a-3p expression level was much lower in glioma tissues and cells,preliminary verified the interaction between miR-130a-3p and Sp1.We also constructed the wild-type pmir GLO-WT vector containing the target sequence and mutant vector pmir GLO-MUT,and further confirmed the targeting relationship between Sp1 and miR-130a-3p by the dual luciferase reporter gene system.Then,the results of MTT,colony formation,Transwell assay and flow cytometry analysis demonstrated that miR-130a-3p inhibited the proliferation,migration,and TMZ resistance of glioma cells.Finally,we established a Sp1 overexpressing glioma cell line and demonstrated that overexpression of Sp1 in glioma cells attenuated the inhibitory effects of miR-130a-3p on cell proliferation,migration and TMZ resistance.These results suggested that miR-130a-3p regulated the malignant biological behavior of glioma cells by inhibiting Sp1 expression.Furthermore,we established a subcutaneous heterotopic xenograft tumor model in nude mice,and confirmed that Sp1 regulated the tumorigenicity of glioma cells by regulating miR-130a-3p in vivo.Conclusion:(1)Sp1 is highly expressed in glioma cell lines and tissues and is closely related to the prognosis of glioma patients,suggesting that Sp1 is involved in the development of gliomas.(2)Sp1 is a downstream target gene of miR-130a-3p.(3)MiR-130a-3p overexpression inhibits the proliferation,migration,and TMZ resistance of glioma cells.(4)Overexpression of Sp1 attenuates the inhibitory effect of miR-130a-3p on the malignant biological behavior of glioma cells.Therefore,Sp1 regulates glioma cell proliferation,migration and TMZ resistance through miR-130a-3p.