The Molecular Mechanism Of Non-coding RNA Regulating Malignant Transformation Of Stromal Cells In Microenvironment Of Glioma Stem Cells

Part Ⅰ The molecular mechanism of lncRNA HOTAIRM1 regulating malignant transformation of fibroblasts in GSC microenvironmentObjective:Studies reported that tumor-associated fibroblasts(TAFs)played a pivotal role in the occurrence and development of solid tumors such as liver cancer and breast cancer.Previous studies have shown that malignant transformation could occur over fibroblasts in glioma stem cell(GSCs)microenvironment,but the relevant molecular mechanism is unknown.Our study aims to explore the molecular mechanism of long non-coding RNA(lncRNA)related to the malignant transformation of fibroblasts(FBs)in GSCs microenvironment.Methods:GSCs SU3-RFP expressing red fluorescent protein(RFP)were inoculated into the intracranial of Balb/c nude mice expressing green fluorescent protein(GFP)to establish a dual-color fluorescence tracer orthotopic transplanted tumor model in vivo.The transplanted tumor was digested into single cells and primarily cultured then observed under fluorescence microscopes.GFP positive cells were mono-cloned by microtubule siphon method and subcultured continuously.CCK-8 assay was used to detect the proliferation ability of GFP positive cells to verify whether they underwent malignant transformation.Fibroblasts markers in GFP positive cells was detected by immunocytochemistry.The expression difference of lncRNAs between malignant fibroblasts and normal fibroblasts were compared by lncRNAs microarray,and the most significant expression difference of lncRNAs were verified by qRT-PCR.The expression of the target lncRNA in glioma cell lines,tissues and transformed fibroblasts was detected by bioinformatics analysis,qRT-PCR and immunofluorescence experiments.After regulating the expression of the target lncRNA,the proliferation,invasion,migration and tumorigenesis of malignant fibroblasts were analysed with EdU assay,Transwell assay,wound healing assay and tumorigenicity assay.Bioinformatics analysis,luciferase assay,qRT-PCR and Western Blot were used to verify the relationship between the lncRNA and its target miRNA,as well as the direct interaction between the miRNA and its target gene.Furthermore,EdU assay,Transwell assay and wound healing assay were used to study whether regulating expression of miRNA or target gene could restore the effect of lncRNA or miRNA on the proliferation,invasion and migration of transformed fibroblasts.Results:RFP positive cells(derived from GSCs)and GFP positive cells(derived from stromal cells)were observed under fluorescence microscopes in the intracranial transplanted tumor undergoing primarily cultured.GFP positive cells were mono-cloned and some of them could proliferate indefinitely.CCK-8 assay showed that two of the mono-cloned GFP positive cells had strong proliferation ability.Immunocytochemical staining showed that two GFP positive cells with high proliferation ability were fibroblast markers positive and were named malignant transformed fibroblasts(t-FBs).LncRNAs microarray and qRT-PCR showed that lncRNA HOTAIRM1 was highly expressed in t-FBs.Bioinformatics analysis and qRT-PCR assay showed that HOTAIRM1 was highly expressed in glioma tissues and cells.Down-regulating the expression of HOTAIRM1 in t-FBs,the proliferation,invasion,migration and tumorgenicity of t-FBs were significantly inhibited.Overexpression of HOTAIRM1 in t-FBs,the proliferation,invasion,migration and tumorgenicity of t-FBs were significantly enhanced.Bioinformatics analysis,qRT-PCR and luciferase assay showed that HOTAIRM1 could directly target miRNA-133b-3p,and miRNA-133b-3p could directly target TGFβ.Down-regulation of miRNA-133b-3p in t-FBs could restore the inhibitory effect of HOTAIRM 1 knockingdown on proliferation,invasion and migration.Overexpression of miRNA-133b-3p could inhibit the proliferation,invasion and migration of t-FBs.Overexpression of TGFβ,could restore the inhibitory effect of overexpressed miRNA-133b-3p on proliferation,invasion and migration in t-FBs.Conclusions:Fibroblasts can undergo malignant transformation in GSCs microenvironment.The high expression of lncRNA HOTAIRM 1 in malignant fibroblasts can promote the malignant biological phenotype of malignant fibroblasts by adsorbing miRNA-133b-3p and targeting TGFβ.Part Ⅱ The molecular mechanism of miR-146b-5p regulating malignant transformation of GSC/MSC fusion cellsObjective:Mesenchymal stem cells(MSCs)were commonly used in tumor targeted therapy because of their chemotaxisrecommonly used carriers for tumor targeted therapy because of their tumor tendency.Previous studies have found that MSCs underwent malignant transformation in glioma stem cell(GSCs)microenvironment,but the regulation of non-coding RNA(ncRNA)in it and the relevant molecular mechanism are unknown.The purpose of this study was to study the biological phenotype changes after spontaneous fusion of MSCs and GSCs and the regulatory role of ncRNA in it.Methods:The glioma tissues derived from patients with glioblastoma(GBM)were primarily cultured for establishment of human GSCs line SU4,which were transfected with red fluorescent protein(RFP)lentivirus to construct GSCs SU4-RFP.Primarily cultured bone marrow MSCs derived from Balb/c nude mice expressing green fluorescent protein(GFP),and co-cultured with GSCs SU4-RFP directly in vitro,to establish a dual-color fluorescence tracer model in vitro.The cells fluorescence expression was observed under fluorescence microscopes.Mono-cloned RFP and GFP double positive cells were subcultured by microtubule siphon method.Then fluorescence in situ hybridization(FISH),Western blotting,immunocytochemical staining and chromosome karyotype analysis were used to verify whether the double fluorescence cells were fusion cells.The biological phenotype of fusion cells was analysed by CCK-8 assay,colony formation assay,EdU assay,cell cycle assay and Transwell assay.MiRNAs microarray was used to compare the expression difference of miRNAs between fusion cells and normal MSCs,and qRT-PCR was used to verify the most significant differentially expressed miRNAs.CCK-8 assay,colony formation assay,EdU assay,Transwell assay and wound healing assay were used to study the effect on proliferation,invasion and migration ability of fusion cells after regulating the target miRNA expression.Bioinformatics analysis and Western blot were used to verify the target genes of miRNA,and luciferase assay was used to confirm whether the two directly bind.Bioinformatics analysis and immunofluorescence experiment were used to detect the expression level of target gene in glioma.Finally,CCK-8 assay,colony formation assay,EdU assay,Transwell assay and wound healing assay were used to analyse whether changing the expression of target gene could restore the function of the miRNA on fusion cells,and further verified in vivo.Results:Primary cultured the GBM tissue specimen,exhibiting sphere-like growth.The markers of neural stem cells were detected and found positive expression of Nestin,CD 133 and SOX2 in the cells.After contiueously subculture,GSCs SU4 was harvested and transfected with RFP.Co-culture of GSCs SU4RFP and MSCs derived from GFP nude mice in vitro,RFP and GFP double positive cells were found under fluorescence microscopes.It was further verified that RFP and GFP double positive cells co-expressed double fluorescent protein gene,and co-expressed GSCs marker Nestin and MSCs marker CD 105,CD90,and co-existence of the characteristic chromosome of GSCs and MSCs,suggesting that RFP and GFP double positive cells were GSC/MSC fusion cells(F-GSC/MSC).Cell biological function assay showed that the proliferation and invasion ability of GSC/MSC fusion cells were both stronger than those of GSCs and MSCs.MiRNA microarray and qRT-PCR showed that the expression of miR-146b-5p in F-GSC/MSC was significantly down-regulated.Overexpression of miR-146b-5p can significantly inhibit the proliferation,invasion and migration of F-GSC/MSC.It was further found that miR-146b-5p could directly target SMARCA5 and inhibit its expression.SMARCA5 was significantly overexpressed in gliomas,and overexpressed SMARCA5 could restore the inhibitory effect of miR-146b-5p on the proliferation,invasion and migration of F-GSC/MSC.In vivo experiments showed that overexpression of miR-146b-5p could inhibit the subcutaneous tumorigenicity of F-GSC/MSC,and overexpressed SMARCA5 could restore the inhibitory effect of miR-146b-5p on the tumorigenesis of F-GSC/MSC.Conclusions:GSCs can spontaneously fuse with MSCs in microenvironment,and GSC/MSC fusion cells have higher malignant biological phenotype,and miR-146b-5p inhibits the malignant biological phenotype of GSC/MSC fusion cells by targeting SMARCA5.These results preliminarily reveal the biological phenotypic changes of MSCs in GSCs microenvironment and provide a reference basis for the treatment of MSCs-related gliomas.Part Ⅲ The Molecular mechanism of MiR-223-3p regulating malignant transformation of macrophages in GSC microenvironmentObjective:Tumor-associated macrophage(TAMs),as the main immune cells in glioma immune microenvironment,promotes the malignant progression of glioma by cooperating with glioma stem cell(GSCs)through multiple mechanisms.Previous studies have found that macrophages underwent malignant transformation in GSCs microenvironment,but the molecular mechanism related to ncRNAs has not been elucidated.The purpose of this study was to investigate the molecular regulation mechanism of malignant phenotype of macrophages in GSCs microenvironment mediated by ncRNA.Methods:GSCs SU4 was transfected with RFP lentivirus for expressing RFP stably.Premarily cultured bone marrow derived cells(BMDCs)of GFP nude mice and stimulated by M-CSF to harvest macrophages(Mφ).Co-culture of GSCs SU4-RFP and Mcp and mono-cloned GFP+cells with microtubule siphon method and subcultured continuously.CCK-8 assay was used to detect the proliferation of GFP+cells to verify whether they can grow infinitely.The expression of macrophages markers in GFP positive cells was detected by flow cytometry assay.MiRNAs microarray was used to compare the expression difference of miRNAs between malignant Mφ and normal Mφ,and qRT-PCR was used to verify the most significant differentially expressed miRNAs.The expression of target miRNA in glioma and malignant macrophages was detected by bioinformatics analysis,qRT-PCR assay.The effects of different expression levels of the target miRNA on the apoptosis of malignant macrophages were analysed by flow cytometry assay.Bioinformatic analysis,qRT-PCR,Western Blot and luciferase assay confirmed the relationship between the miRNA and target gene.Furthermore,flow cytometry assay was used to verify whether regulating the expression level of target gene could restore the effects of the miRNA on the apoptosis of malignant macrophages.Results:GFP+cells were mono-cloned and some of them could proliferate unlimitedly.CCK-8 assay showed that monocloned GFP+cells had unlimited proliferation ability.Flow Cytometry assay showed that the GFP+cells with high proliferation ability were positive for macrophages markers F4/80 and CD11b and named transformed macrophages(t-Mφ).MiRNA microarray and qRT-PCR showed that the expression of miR-223-3p in t-Mcp was significantly up-regulated.Bioinformatics analysis and qRT-PCR assay showed that miR-223-3p was significantly overexpressed in glioma and t-Mφ.Down-regulating the expression of miR-2 23-3p,the apoptosis of t-Mφ was significantly increased.Overexpression of miR-223-3p,the apoptosis of t-Mφ was significantly decreased.Bioinformatics analysis,qRT-PCR assay and luciferase assay showed that miR-223-3p could directly target RIPK3.Downregulation of RIPK3 can restore the promoting effect of miR-223-3p knockdown on the apoptosis of t-Mφ.Conclusions:Macrophages could undergo malignant transformation in the co-culture model of GSCs/Mφ.The high expression of miR-223-3p inhibits the apoptosis of malignant macrophages by targeting RIPK3.