MiR-145-mediated SOX2-Wnt/?-catenin Enhances The Sensitivity Of Glioma Stem Cells To Demethoxycurcumin Chemotherapy

Objective:Glioma is known to be the most common type of intracranial tumor.It could not be global resected because of diffuse growth and lack of obvious boundary with surrounding brian tissues.It always recur and is difficult to cure although combine chemotherapy and radiotherapy after surgery.One of the key reason is the existence of glioma stem cells.Glioma stem cells are resistant to chemotherapy through changes in DNA damage response structure,hypoxic microenvironment,Notch signaling pathway and multidrug resistance mechanisms.Therefore,it is considered that glioma stem cells are a new target for the treatment of glioblastoma,and killing glioma stem cells is the key to improve therapeutic effect of glioblastoma.The effect of demethylcurcumin(DMC)on chemotherapy of glioma has been confirmed by many studies,especially the effect of DMC on glioma stem cells has been reported to be better than other chemotherapy drugs.miRNAs and its related signaling pathways are closely related to the occurrence and development of human tumors.Many studies have found that microRNAs are involved in regulating the biological functions of glioma stem cells.In recent years,more and more attention has been paid to the concept of drug effect miRNAs,so we want to find a certain miRNA as drug effect miRNA to enhance the sensitivity of glioma stem cells to DMC chemotherapy.Previous studies have found that the expression of miR-145 in glioma stem cells is low,and up-regulation of miR-145 can inhibit the proliferation of glioma stem cells and promote their invasion.The aim of this study is to enhance the chemosensitivity of glioma stem cells to DMC through the synergistic of DMC and miR-145,and to explore the molecular mechanism of their synergistic effect by detecting the activity of SOX2-Wnt/beta-catenin signaling pathway.This study provides a theoretical basis for improving the efficacy of chemotherapy on glioma stem cells.Methods:The expression of microfluorescence quantitative PCR was used to detect the expression of miR-145 in different grades of gliomas and normal brain tissues.The primary glioma cells were isolated and cultured from glioblastoma(WHO grade IV)tissues.CD133~+glioma stem cells were isolated by immunomagnetic beads method.Nestin and CD133 immunofluorescence staining were used to verify the model of primary glioma stem cells.The expression of miR-145 in glioma stem cells,standard glioma cell lines and astrocytes were compared.After miR-145 was up-regulated by lentiviral transfection,the proliferation and invasion of glioma stem cells were examined to verify the inhibitory effect of miR-145 on glioma stem cells.Different concentrations of DMC combined with miR-145 lentivirus transfection inhibit the proliferation and invasion of glioma stem cells,verify the synergistic effect of DMC and miR-145,and proved by animal models.And further experiments to investigate the molecular mechanism of DMC combined with TMZ against glioma stem cells.Result:(1)We successfully isolated and cultured primary glioma cells(pGSCs)in vitro,and verified them as CD133 and Nestin positive by immunohistochemistry.(2)The expression of miR-145 was high in normal brain tissues,and decreased in low-grade glioma tissues compared with normal brain tissues,but was significantly low in high-grade glioma tissues.The expression of miR-145 in primary glioma stem cells was lower than that in primary glioma cells,U373 cells and LN-229 cells,and lower than that in normal astrocytes.(3)miR-145 lentivirus transfected pGSCs,and the expression of miR-145 was significantly high;MTT assay indicated that over-expression of miR-145 significantly inhibited the proliferation of pGSCs,and TUNEL cell apoptosis assay indicated that over-expression of miR-145 did not affect the apoptosis of pGSCs.(4)Overexpression of miR-145 combined with DMC significantly increased the effect of inhibiting proliferation of glioma stem cells and inducing apoptosis of glioma stem cells,the effect was significantly better than that of use DMC alone;(5)In vivo experiments in nude mice showed that DMC and miR-145 lentiviral vector could inhibit tumor growth and promote tumor cell apoptosis,combined with DMC and miR-145 Lentiviral vectors played a stronger role.(6)DMC and miR-145 lentiviral vectors inhibit SOX2 expression in glioma stem cells,down-regulate the expression of?-catenin in the downstream Wnt/?-catenin signaling pathway,and down-regulate the expression of cyclin D1 and c-myc protein in the downstream,and combined intervention of DMC and miR-145 has synergistic effect.(7)Luciferase reporter gene suggests that miR-145 can directly act on SOX2,and up-regulation of SOX2 can reverse the inhibitory effect of DMC and miR-145 on glioma stem cells,and reverse the low expression of cyclin D1 and c-myc proteins downstream.Conclusion:The results of this study demonstrate that overexpression of miR-145 is effective in sensitizing DMC against glioma stem cells by mediating the SOX2-Wnt/?-catenin signaling pathway.Therefore,we proved that targeted regulation of miRNAs could enhance the sensitivity of glioma stem cells to chemotherapeutic agents,improve the efficacy of chemotherapy,and ultimately improve the overall prognosis of glioma patients.