ABSTRACT
MicroRNA (miR)-125b has been shown to play a potential role in the development of glioma stem cells. However, the relationship between miRNA and glioma stem cells is still elusive. This study was designed to elucidate this potential relationship. We established a highly invasive glioma stem cell and progenitor (GSCP) cell line SU3. SU3 cell suspensions were injected into nude mice brains in situ, and the invasiveness of graft tumors was analyzed using hematoxylin and eosin staining as well as immunohistochemistry. Real-time polymerase chain reaction (PCR) was used to measure the expression levels of miR-125b in SU3 and other cells. In vitro, SU3 cells expressed CD133 and nestin as well as differentiation markers glial fibrillary acidic protein (GFAP) and β-tubulin III, which were consistent with the characteristics of glioma stem cells. Scratch assays indicated that the migration ability of SU3 cells was stronger than that of U251 stem cells (U251s). In vivo, SU3 cells invaded into each part of the mouse brain from the caudate nucleus in a diffuse pattern and highly expressed invasive and proliferative cell markers matrix metalloprotease 2 (MMP2), MMP9, and Ki-67. Real-time PCR results revealed that the levels of miR-125b and MMP9 were significantly higher in SU3 and SU2, also a highly invasive GSCP cell line we established before, than in U251s. High expression of miR-125b both in newly established GSCPs, SU3, and long-term cultured GSCPs, SU2 suggests that miR-125b exhibits oncogene-like behavior. This behavior should be considered in further studies of miR-125b in cancer stem cells. Furthermore, MMP9, which plays a role in cancer stem cell invasion, may be a target gene of miR-125b.
Subject(s)
Animals , Humans , Mice , AC133 Antigen , Antigens, CD , Metabolism , Biomarkers, Tumor , Metabolism , Brain Neoplasms , Metabolism , Pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Glial Fibrillary Acidic Protein , Metabolism , Glioblastoma , Metabolism , Pathology , Glycoproteins , Metabolism , Intermediate Filament Proteins , Metabolism , Ki-67 Antigen , Metabolism , Matrix Metalloproteinase 2 , Metabolism , Matrix Metalloproteinase 9 , Metabolism , Mice, Nude , MicroRNAs , Metabolism , Neoplasm Transplantation , Neoplastic Stem Cells , Metabolism , Pathology , Nerve Tissue Proteins , Metabolism , Nestin , Peptides , Metabolism , Tubulin , MetabolismABSTRACT
<p><b>OBJECTIVE</b>Based on the distribution of genetic polymorphisms regarding phase I metabolic enzyme cytochrome P450 1A1 (CYP1A1) and phase II metabolic enzymes glutathione S-transferase GSTM1 and GSTT1 genotypes in acute leukemia patients and health controls among general population of Hunan in China, this study was to explore the relationship between these gene polymorphisms and the susceptibility to acute leukemia.</p><p><b>METHODS</b>Using case-control methodology, we studied 204 healthy controls and 232 patients with acute leukemia, of which 112 patients were suffering acute lymphoblastic leukemia (ALL) and 120 with acute non-lymphoblastic leukemia (ANLL). The frequencies of the genotypes were detected by PCR and PCR-RFLP techniques.</p><p><b>RESULTS</b>The variation frequencies of CYP1A1 gene (Msp I polymorphisms, site 3801T-C variation) in ALL and ANLL groups were 74.1% and 70.8% respectively which were higher than 63.3% appeared in the healthy controls. However, the differences between patients (ALL or ANLL) and healthy controls were not statistically significant (P > 0.05 for both). The null genotype of GSTM1 (GSTM1 -/-) in ALL group was 60.7%, which was not significantly different from the controls (55.4%). However, GSTM1 -/- genotype in ANLL group was 68.3%, significantly different from the controls (P < 0.05). The null genotypes among GSTT1 (GSTT1 -/-) in ALL, ANLL and control group were 50.9%, 55.0% and 49.0% but their differences were not statistically significant (P > 0.05). The incidences of GSTM1 -/- and GSTT1 -/- combined genotype in ALL, ANLL and control group were 33.0%, 40.0% and 27.5%, of which the difference between ANLL group and control group was statistically significant (P < 0.05) and CYP1A1 gene heterozygous mutation type or homozygous mutation type combined with GSTM1 -/- and GSTT1 -/- increased the risk of ANLL (OR value 1.890, 95% CI: 1.084-3.295).</p><p><b>CONCLUSION</b>These results indicated that both the variation of CYP1A1 gene or GSTT1 -/- genotype alone might not be associated with the susceptibility of acute leukemia while GSTM1 -/- genotype alone or combined with GSTT1 -/- or the 3801 T-C variation of CYP1A1 gene were correlated with ANLL. These findings suggest that GSTM1 - / - genotype alone or in combination with other defective genotypes might serve as risk factors to the etiology of ANLL.</p>
Subject(s)
Humans , Case-Control Studies , China , Cytochrome P-450 CYP1A1 , Genetics , Gene Frequency , Genetic Predisposition to Disease , Genotype , Glutathione Transferase , Genetics , Leukemia, Myeloid, Acute , Genetics , Polymerase Chain Reaction , Polymorphism, Genetic , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Genetics , Risk FactorsABSTRACT
<p><b>OBJECTIVE</b>To identify hemin-induced gene expression in K562 cells.</p><p><b>METHODS</b>Poly A(+) RNAs were isolated from hemin-induced (tester) and non-induced K562 cells (driver) respectively, and double-strand cDNAs were synthesized by reverse transcription. The forward subtracted cDNA library was constructed by using suppression subtractive hybridization (SSH) techniques. The recombinant plasmids were extracted and the positive clones were identified by EcoR I digestion after the amplification and screening of the library. The inserts were amplified by PCR. The upregulated cDNA transcripts were identified by reverse dot blot hybridization, DNA sequencing and homology analysis with GenBank database "blast" respectively.</p><p><b>RESULTS</b>Fifteen upregulated clones were identified and most of them were homologous to the mRNA sequences of protein with known function, including globin epsilon1, glutathione S-transferase like glutathione transferase Omega (GSTTLp28), selenoprotein X1 (SEPX1), triosephosphate isomerase (TPI1), ribosomal protein L7 (RPL7), ribosomal protein S13 (RPS13), ferritin light polypeptide, globin A gamma, RAD 51 homolog C(RAD51C), ferritin heavy polypeptide, X-box binding protein (XBP1). A part of the hemin-induced cDNA clones exhibited sequence similarities to that of the GenBank registered mRNA with unknown function of their expressed proteins, including the cDNA clones of DKFZp434I116, hypothetical protein HSPC014 and NOL1R2 proteins.</p><p><b>CONCLUSIONS</b>Hemin mainly induces the genes expression related to erythroid differentiation, protein synthesis and metabolism in K562 cell. There results provide comprehensive information useful for the differential gene expression in hemin-induced erythroid differentiation and for further function study of genes involved in hematopoiesis.</p>