Effect And Mechanism Of Centaureidin-3-O-glucoside On Proliferation,Stemness And Chemoresistance Of Glioma Cells

Objective:Gliomas are primary tumours of the brain that seriously affect the physical and mental health of patients.Existing clinical treatments for glioma include traditional therapies based on surgery,radiotherapy and chemotherapy,as well as emerging therapies such as molecular targeted therapy,tumour electric field therapy and immunotherapy.However,no significant progress has been made in the treatment of gliomas over time,so new therapeutic approaches are urgently needed.Centaureidin-3-O-glucoside（C3G）is one of the main active components of anthocyanins and plays an important role in anti-inflammatory and antioxidant activities.Recent studies have confirmed that C3G plays an anticancer role in a variety of malignancies and is involved in tumour proliferation,migration,invasion and apoptosis.However,the biological role of C3G in glioma cells is not fully understood.The aim of this study was to investigate the effects of C3G on glioma cell proliferation,stemness and chemoresistance,to confirm the mediating role of C3G-related mi RNAs andβ-catenin and related mechanisms,and to provide a theoretical basis for the clinical treatment of glioma.Methods:1.Five types of glioma cells（U87MG,SW1783,U251,LN18 and T98G）were used as screening subjects.The CCK-8 method was used to observe the inhibitory effect of C3G on cell proliferation,and the protein blotting assay was used to observe the differences inβ-catenin and Calpain-2 protein expression.After the initial identification of U87MG as the study target,protein blotting and immunofluorescence assays were used to investigate the effect of C3G onβ-catenin and Calpain-2 protein expression in U87MG cells.CHX tracking method and immunoprecipitation assay were performed to observe the effect of C3G onβ-catenin stability and ubiquitination in U87MG cells.U87MG cells were treated with C3G,and MG132 inhibited the proteasome when necessary,to observe changes in cellularβ-catenin phosphorylation and nuclearβ-catenin protein expression.RT-Fq PCR to examine the effect of C3G on mi R-214-5p and mi R-137-3p expression in U87MG cells.Transfection of mi R-214-5p mimic and mi R-137-3p mimic to validate the effect of mi R-214-5p and mi R-137-3p on the proliferation of U87MG cells.Prediction and validation of mi R-214-5p and mi R-137-3p target genes by bioinformatics and dual luciferase reporter gene assay.Protein blotting assay was used to observe the effect of transfection with mi R-214-5p mimic or/and mi R-137-3p mimic on the expression ofβ-catenin and Calpain-2protein in U87MG cells.CCK-8 method to observe the effect of transfection with mi R-214-5p mimic or/and mi R-137-3p mimic on the inhibition of U87MG cell proliferation by C3G.2.Glioma stem cell spheres were cultured in serum-free tumour sphere medium using U87MG cells as the study target.Immunofluorescence assays were performed to detect the expression of stem cell markers CD44 and SOX-2 protein.Cell sphere-forming assay and plate cloning assay were performed to investigate the effect of C3G on the sphere-forming ability and clone-forming ability of U87MG cells.Protein blotting and immunofluorescence assays were performed to observe the effect of C3G on CD44 and SOX-2 protein expression in U87MG cells.The effect of C3G on the tumorigenic ability of U87MG cells was examined in a subcutaneous transplantation tumor assay in nude mice.Immunohistochemical staining assay to observe the effect of C3G on CD44 and SOX-2 protein expression in transplanted tumor tissues.Protein blotting assay was performed to observe the effect of transfection with mi R-137-3p inhibitor+mi R-214-5p inhibitor on the induction ofβ-catenin,Calpain-2,CD44 and SOX-2 protein expression in U87MG cells by C3G.Sphere-forming assay and plate cloning assay were performed to examine the effect of transfection of mi R-137-3p inhibitor+mi R-214-5p inhibitor on the inhibition of sphere-forming and clone formation of U87MG cells by C3G.3.TMZ resistant cells LN-18/TR were constructed using LN-18 cells using gradually increasing drug concentration and gradient induction method.MTT assay was performed to examine the ability of LN-18/TR cells to resist TMZ.Protein blotting and immunofluorescence assays were performed to observe the differences in MGMT andβ-catenin protein expression in LN-18and LN-18/TR cells.MTT assay was performed to observe the effect of C3G on the proliferation ability of LN-18/TR cells.MTT assay to detect the effect of C3G on TMZ resistance in LN-18/TR cells.RT-Fq PCR was performed to examine the effect of C3G on mi R-214-5p expression in LN-18/TR cells.Protein blotting assay was performed to observe the effect of C3G on the expression of MGMT andβ-catenin proteins in LN-18/TR cells.Protein blotting assays were performed to observe the changes in MGMT andβ-catenin protein expression in LN-18/TR cells by transfecting mi R-214-5p mimic and mi R-214-5p inhibitor,respectively.Protein blotting assay was performed to observe the effect of transfection with mi R-214-5p inhibitor on the inhibition of MGMT andβ-catenin protein expression in LN-18/TR cells by C3G.Flow cytometry to observe the effect of C3G and mi R-214-5p mimic on TMZ-induced apoptosis in LN-18/TR cells.Flow cytometry to verify the effect of transfection with mi R-214-5p inhibitor on C3G-induced apoptosis in LN-18/TR cells.Results:1.The proliferation of U87MG cells was significantly inhibited by 20μM C3G（p<0.01）;the proliferation of SW1783,U251 and LN18 cells was significantly inhibited by 80μM C3G（p<0.01）;C3G had almost no effect on the proliferation of T98G cells.Calpain-2 andβ-catenin protein expression were significantly higher in U87MG cells compared to SW1783,U251,T98G and LN18 cells（p<0.01）.Using U87MG cells as the study target,C3G was found to significantly down-regulate Calpain-2 andβ-catenin protein expression in U87MG cells（p<0.01）.C3G significantly reduced the half-life ofβ-catenin protein,i.e.reduced protein stability（p<0.01）;whereas MG132restored the effect of C3G onβ-catenin protein stability（p<0.01）.C3G significantly enhanced the ubiquitination of p-β-catenin protein（p<0.01）.MG132 inhibited the proteasome and C3G significantly induced down-regulation of p-β-catenin^Y86 and p-β-catenin^Y654 protein expression（p<0.01）.C3G also significantly induced down-regulation of nucleusβ-catenin protein expression.In addition,mi R-214-5p and mi R-137-3p were observed to be significantly elevated in U87MG cells after C3G treatment（p<0.01）.Exogenous transfection of mi R-214-5p mimic or mi R-137-3p mimic significantly inhibited U87MG cell proliferation（p<0.01）.Bioinformatic analysis revealed that mi R-214-5p and mi R-137-3p had complementary binding sites to theβ-catenin and Calpain-2 m RNA 3’UTR,respectively,and were validated by a dual luciferase reporter gene.Transfection of mi R-214-5p and mi R-137-3p alone or cotransfected inhibitedβ-catenin protein expression（p<0.01）;transfection of mi R-137-3p inhibited Calpain-2 protein expression（p<0.01）.Transfection of mi R-214-5p inhibitor,mi R-137-3p inhibitor and mi R-137-3p inhibitor+mi R-214-5p inhibitor all inhibited the C3G-induced decrease in proliferation viability of U87MG cells（p<0.01）.2.U87MG cells were used for stemness-related studies,and Stem XVivo serum-free tumour sphere medium was used to grow cells in spherical suspension and express the stem cell markers CD44 and SOX-2 protein.C3G significantly inhibited the sphere-forming and clone-forming ability of U87MG cells（p<0.01）.Molecular level studies revealed that C3G treatment significantly down-regulated CD44 and SOX-2 protein expression in U87MG cells（p<0.01）.C3G significantly inhibited subcutaneous graft tumour formation in U87MG cells in nude mice（p<0.01）.At the same time,C3G significantly down-regulated CD44 and SOX-2 protein expression in U87MG cell nude mice subcutaneous graft tumor tissues（p<0.01）.Co-transfection with mi R-137-3p inhibitor and mi R-214-5p inhibitor reversed the C3G-induced down-regulation of Calpain-2,β-catenin,CD44 and SOX-2protein expression（p<0.01）.Co-transfection with mi R-137-3p inhibitor and mi R-214-5p inhibitor significantly inhibited C3G-induced inhibition of sphere-formation and clonogenesis（p<0.01）.3.TMZ-resistant cells constructed with LN-18 cells LN-18/TR cells were significantly resistant to TMZ;the IC₅₀ of TMZ was significantly increased in LN-18/TR cells（p<0.01）.β-catenin and MGMT were significantly upregulated in LN-18/TR cells compared to LN-18 cells（p<0.01）.0-90μM C3G had no significant effect on the proliferation viability of LN-18 and LN-18/TR cells,whereas 90μM C3G significantly inhibited cell proliferation viability（p<0.01）.For this reason,we used 90μM C3G as the highest concentration,and 60μM and 30μM C3G were used for subsequent experiments.The cytotoxic effect of TMZ on LN-18/TR cells was enhanced by different concentrations of C3G,and the IC₅₀ of TMZ on LN-18/TR cells was significantly reduced（p<0.01）.C3G significantly induced an increase in mi R-214-5p levels in LN-18/TR cells（p<0.01）.C3G also induced a down-regulation ofβ-catenin and MGMT protein expression in LN-18/TR cells（p<0.01）.Transfection with mi R-214-5p mimic significantly down-regulatedβ-catenin and MGMT expression in LN-18/TR cells,while transfection with mi R-214-5p inhibitor significantly up-regulatedβ-catenin and MGMT protein expression（p<0.01）.Moreover,transfection of mi R-214-5p inhibitor reversed the C3G-induced down-regulation ofβ-catenin and MGMT protein expression in LN-18/TR cells（p<0.01）.C3G significantly enhanced TMZ-induced apoptosis in LN-18/TR cells（p<0.01）.Transfection with mi R-214-5p mimic also significantly enhanced TMZ-induced apoptosis in LN-18/TR cells（p<0.01）.Transfection of mi R-214-5p inhibitor reversed the enhancement of TMZ-induced LN-18/TR apoptosis by C3G（p<0.01）.Subcutaneous graft tumor efficacy assays in nude mice showed that both C3G and mi R-214-5p agomir treatment enhanced the inhibitory effect of TMZ on graft tumor growth（p<0.01）.In addition,histological results showed that C3G and mi R-214-5p agomir significantly inhibited MGMT expression in LN-18/TR transplanted tumor tissues（p<0.01）.Conclusions:1.C3G can inhibitβ-catenin protein accumulation and activation by downregulating Calpain-2 expression through mi R-137-3p,and also directly inhibitβ-catenin expression through mi R-214-5p,ultimately inhibiting U87MG cell proliferation.2.C3G inhibitsβ-catenin and down-regulates CD44 and SOX-2 protein expression through the direct and indirect effects of mi R-214-5p and mi R-137-3p,ultimately inhibiting the stemness of U87MG cells.3.C3G inhibited TMZ-resistant glioma cells LN-18/TR against TMZ,and this effect was associated with targeted inhibition ofβ-catenin/MGMT signaling pathway by upregulating mi R-214-5p.