ABSTRACT
Tumor heterogeneity is the concept that different tumor cells provide distinct biomorphological lesions, gene expressions, proliferation, microenvironment and graduated capacity of metastatic lesions. Brain tumor heterogeneity has been recently discussed about the interesting interaction of chronic inflammation, microenvironment, epigenetics and glioma steam cells. Brain tumors remain a challenge with regards to medication and disease, due to the lack of treatment options and unsatisfactory results. These results might be the result of the brain tumor heterogeneity and its multiple resistance mechanisms to chemo and radiotherapy.
Subject(s)
Neoplastic Stem Cells/cytology , Brain Neoplasms/genetics , Genetic Heterogeneity , Gene Expression Profiling , Glioma/genetics , Receptor Protein-Tyrosine Kinases/genetics , Drug Resistance, Neoplasm/genetics , Stem Cell Niche/genetics , Tumor Microenvironment , Clonal Evolution/genetics , Cellular Microenvironment/genetics , RNA-SeqABSTRACT
Objective To explore the relationship between the change of phenotype of glioma stem cells and expres-sion of RNA binding proteins in hypoxia. Methods Glioma stem cells(U87MG-SLC and GSC5) were cultured un-der hypoxia (1% O2) and normoxia(20% O2). Cell proliferation was measured by MTS assay and self-renewal ability was determined by tumorsphere formation assay. The expression of RNA binding protein and stemness mark-ers protein were examined by Western blot and statistics was carried out. Results The proliferation of glioma stem cells was inhibited and the self-renewal ability was promoted in hypoxia. Meanwhile,hypoxia significantly promoted the expression of HIF-1α and stemness markers.Under hypoxia, the expression of RNA binding protein was changed. The expression of hnRNPF, UNRIP and HuD increased. Meanwhile the expression of PCBP2 and UNR was downregulated. But,other RNA binding proteins(hnRNPK,ADAR1,PCBP1,CIRP,EBP1,eEF1A,PTBP1,PTBP2) had no significant change. Conclusions The change of phenotype of glioma stem cells in hypoxia is relat-ed with the RNA binding proteins (hnRNPF,UNRIP,HuD,PCBP2 and UNR).
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@#[Abstract] Objective: To study the effect and possible mechanism of TGF-β2 on the invasion of glioma stem cells (GSCs). Methods: Tumor tissues of 8 patients with glioblastoma multiforme, who underwent resection at Department of Neurosurgery of the FirstAffiliated Hospital of China Medical University duringApril 2016 toApril 2017, were collected. The primary culture of glioma cells were conducted with trypsin digestion. Partial primary glioma cells were seeded into serum-free DMEM/F12 culture medium containing EGF, bFGF and B27 to obtain suspension of tumor spheres. Immunoflurenscent staining and differentiation assay were used to detect whether the tumor spheres were GSCs. TGF-β2 secretion ability of GSCs was determined by ELISAassay.After transfection of TGF-β2 siRNA, the invasion ability of glioma stem cells was determined by Transwell assay. Western blotting was used to examine the effect of TGF-β2 on expression of matrix metalloproteinases (MMP) in glioma stem cells. Results: The suspended tumor spheres were proved to be GSCs by immunofluorescent staining and differentiation assay; the tumor spheres expressed the marker of GSCs（CD133）and had the ability to multi-differentiate (glia and neuronal cells). Compared with the primary glioma cells, Glioma stem cells exerted significantly improved TGF-β2 secretion ability ([74.13±3.63] vs [46.13±2.61] pg/ml, P<0.05); and TGF-β2 silencing significantly reduced the invasion ability of glioma stem cells ([105.71±8.69] vs [63.67±5.93], P<0.05) and inhibited MMP-2 and MMP-9 expressions. Conclusion: TGF-β2 can promote the invasiveness of glioma stem cells through MMP-2 and MMP-9 pathway.
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Objective To investigate the effect of fibronectin type Ⅲ and SPRY domain containing 1 (FSD1) protein on the invasion of glioma stem cells (GSCs), so as to probe into the new biomarkers or potential therapeutic targets for gliomas. Methods The Cancer Genome Altas (TCGA) database data were used to analyze and compare the FSD1 gene expression (the FSD1 mRNA level) in the glioblatoma (also known as glioblastoma multiforme, GBM) and normal brain tissues as well as in the different grade glioma tissues, and the correlation of the FDS1 gene expression (mRNA level) with the survival prognosis of patients was also analyzed using the TCGA database data. The lentivirus was used to overexpress the FSD1 protein in the GSCs, T4121 and D456. The effect of the overexpressed FSD1 protein on the invasive ability of the GSCs, T4121 and D456 was evaluated by Transwell invasion assay. Results The FSD1 gene expression (mRNA level) was significantly lower in GBM than in normal brain (P＜0.01). The FSD1 gene expression (the mRNA level) in gliomas significantly decreased with the increase of the gliomas grade (gradeⅡvs Ⅲ, P＜0.05;gradeⅢvs Ⅳ, P＜0.01). The survival prognosis of patients with gilomas was well associated with the level of FSD1 gene expression (the FSD1 mRNA level), as indicated by the overall survival rate of the patients, which was significantly lower in the patients with the low FSD1 mRNA level than in the patients with the high FSD1 mRNA level (P＜0.01). In the Transwell invasion assay, the count of the invasive cell numbers significantly decreased in the FSD1 protein-overexpressed T421 and D456 groups than in the corresponding control group (P＜0.01 in both T4121 and D456 cell lines). Conclusion There is a clinical relevance of the FSD1 expression for the malignant progression of gliomas (the grade of gliomas). The low level FSD1 is favorable for keeping the invasive ability in GSCs.
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Objective To study the effect of Pirin(an iron-binding nuclear protein)on the proliferation and self-renewal of glioma stem cell(GSC)so as to provide a potential therapy target for malignant glioma.Methods PLKO.1-shPirin lentiviral plasmids were constructed to stably knock down Pirin in GSC by using lentivirus infection system.The interference efficiency of Pirin short hair?pin RNA(shRNA)was detected by Western blotting. The capacity of GSC was examined by the assessment of cell viability. Tumor sphere formation assay was used to detect the effect of Pirin on GSC self-renewal capacity. Results Pirin was highly expressed in GSC.Consistently,the protein level of Pirin in the conditioned medium from GSC was much higher than that from the corresponding non-stem tumor cell(NSTC).Gene-sequencing analysis demonstrated that PLKO.1-shPirin lentiviral plasmids were successfully con?structed.Pirin shRNA transfection significantly inhibited the expression of Pirin in GSC and suppressed the cell viability and ability of tumorsphere formation.Conclusion Knocking down Pirin significantly inhibites the cell proliferation and self-renewal of GSC.
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Objective To compare different Nrf2 expressions in glioblastoma cell lines and glioma stem cells (GSCs) from xenografts and to study the concentration of Nrf2 in nuclear. Methods GSCs were analyzed by immunofluorescence and different expressions of Nrf2 in glioblastoma cell lines and GSCs from xenografts were detected with real-time RCR and Western. Results GSCs were successfully isolated from xenografts of U251 and U87 cell lines. The percentage of tumor stem cells in total cells was 1.24%, and that was 1.63% in xenografts. Immunofluorescence indicated that Nrf2 was overexpressed in GSCs as compared with that in glioblastoma cell lines. Conclusion Nrf2 may be a potential biomarker and rational therapeutic target for GSCs.
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Objective:To investigate the expression and significance of ABCG2(the G2 member of ATP-binding cassette family)in U251and its stem cell.Methods:The U251 were cultured in serum-free medium with EGF,bFGF,LIF and B27.The tumor spheres formed and cultured continuously in the serum-free medium through the monoclonal formation experiment.The immunofluorescence staining was employed to identify the U251 stem cell.The expression of ABCG2 was detected by confocal microscopy and Western blot.Results:The stem cells from malignant glioma cell line U251 have been successfully isolated.The cells expressed CD 133.The ABCG2 protein positive stain was located in the cell membrane in both U251 cells and its stem cell with confocal microscopy method.Western blotting showABCG2 was expressed in U251 cells and in its stem cells.The expression level in the stem cells was significantly higher than U251 cells.Conclusion:ABCG2 is expressed in U251 cells and in U251 stem cells.The expression level in the latter was higher.