RESUMO
Tumor hypoxia is one of the main causes of progression and metastasis of colorectal cancer. Changes in the expression of miRNA responsible for post-translation regulation of gene expression is an important molecular mechanism of cell response to hypoxia. We performed sequencing of miRNA and mRNA of human colorectal adenocarcinoma HT-29 cells treated with two chemical agents mimicking hypoxia: cobalt (II) chloride and oxyquinoline. Bioinformatics analysis revealed differentially expressed miRNA isoforms (hsa-miR-210-3p|0, hsa-miR- 22-3p|0, hsa-let-7a-3p|0, hsa-miR-615-3p|0, and hsa-miR-4521|0) and their targets that changed their expression in both models of hypoxia. Thus, we identified new regulatory mechanisms of cell response to hypoxia.
Assuntos
Perfilação da Expressão Gênica , MicroRNAs , Células HT29 , Humanos , Hipóxia/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Isoformas de Proteínas/genéticaRESUMO
In the present study, we assessed the role of annexin 13 membrane-binding protein (ANXA13) in the intracellular transport of vesicles containing type II ribosome-inactivating proteins (RIP-IIs). A modified human intestinal epithelial cell line HT29 was used, in which the expression of ANXA13 was significantly reduced. The cytotoxic effect of ricin and viscumin was evaluated by modification of 28S ribosome RNA. The observed differences in the activity of toxins on the parental and modified HT29 lines indicate that ANXA13 plays a different role in the intracellular transport of vesicles containing the RIP-IIs.
Assuntos
Anexinas/metabolismo , Substâncias para a Guerra Química/farmacologia , Neoplasias do Colo/patologia , Proteínas Inativadoras de Ribossomos Tipo 2/farmacologia , Proteínas Inativadoras de Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Ricina/farmacologia , Toxinas Biológicas/farmacologia , Transporte Biológico , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Células HT29 , HumanosRESUMO
The role of proteasome proteins and proteins of the ERAD system in the cytotoxicity of type II ribosome-inactivating proteins ricin and viscumin was investigated. For this, the cell line of colorectal adenocarcinoma HT29, as well as the HT29-sh002 line obtained on its basis, were used. On the basis on the proteome analysis of these lines and the estimation of the proportion of inactivated ribosomes, it was shown that the contribution of the proteasome to the degradation of the catalytic subunits of toxins is different. The role of the Cdc37 co-chaperone in maintaining the stability of A subunit of viscumin in the cytoplasm is shown.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/biossíntese , Proteínas Inativadoras de Ribossomos Tipo 2/farmacologia , Ricina/farmacologia , Toxinas Biológicas/farmacologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Proteínas de Ciclo Celular/genética , Chaperoninas/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Citoplasma/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ribossomos/metabolismo , Células Tumorais CultivadasRESUMO
Metastatic cascade is associated with the process of epithelial-mesenchymal transition accompanied by changes in cell proliferation, migration, adhesion, and invasiveness mediated by the insulin-like growth factor (IGF) signal pathway. IGFBP6 protein binds IGF and prevents its interaction with receptors. IGFBP6 gene knockdown through RNA-interference inhibits cell migration and increased the rate of proliferation of breast cancer MDA-MB-231 cells. IGFBP6 knockdown cells are characterized by increased expression of MIR100 and MIRLET7A2 genes encoding hsa-miR-100-3p, hsa-miR-100-5p, hsa-let-7a-5p, and hsa-let-7a-2-3p miRNA. The target genes of these microRNAs are IGF2, IGF1R, INSR, and CCND1 associated with IGF signaling pathway and proliferative and migratory activity during the metastatic cascade. A significant decrease in the expression of INSR and CCND1 genes was demonstrated by PCR and microarray analysis.
Assuntos
Antígenos CD/metabolismo , Ciclina D1/metabolismo , MicroRNAs/metabolismo , Receptor de Insulina/metabolismo , Receptores de Somatomedina/metabolismo , Antígenos CD/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Ciclina D1/genética , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Humanos , MicroRNAs/genética , Receptor IGF Tipo 1 , Receptor de Insulina/genética , Receptores de Somatomedina/genéticaRESUMO
The effects of laminins 332 and 411 (LM-332 and LM-411) on the epithelial-mesenchymal transformation of colorectal cancer cells (lines HT-29, HCT-116, and RKO) with different metastatic potential were studied. Culturing of RKO cells on both laminins was associated with modification of the cell shape, which became more spindle-like or stellate, and with higher expression of EMT-associated transcription factors SNAI1 and ZEB1. In addition, culturing on LM-332 led to a decrease in the expression of laminin α5 chain (LAMA5), while culturing on LM-411 led to an increase in the expression of a cell-cell junction component (DSP). Culturing of HT-29 cells on LM-332 was associated with the formation of more close contacts between the cells and by a higher expression of epithelial markers (CDH1 and DSP genes) and a decrease in SNAI1 expression. Culturing of HCT-116 cells on both laminins led to a decrease in FN1 expression, on LM-332 - to an increase in laminin α4 chain (LAMA4) expression, and on LM-411 - to a lesser expression of LAMA4 and transcription factors SNAI2 and ZEB1. These data indicated that colorectal cancer cell adhesion to laminins contributed to the probability of epithelial-mesenchymal transformation of cells. The direction of this transformation seemed to depend on the initial characteristics of the cells.
Assuntos
Materiais Revestidos Biocompatíveis/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Laminina/farmacologia , Plásticos/farmacologia , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desmoplaquinas/genética , Desmoplaquinas/metabolismo , Transição Epitelial-Mesenquimal/genética , Fibronectinas/genética , Fibronectinas/metabolismo , Perfilação da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Laminina/genética , Laminina/metabolismo , Especificidade de Órgãos , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Propriedades de Superfície , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
Protein IGFBP6 plays an important role in the pathogenesis of many malignant tumors, including breast cancer. The relationship between IGFBP6 protein and the expression of genes associated with the epithelial-mesenchymal transition is studied. Gene IGFBP6 knockdown does not trigger the epithelial-mesenchymal transition in MDA-MB-231 cells, but modifies significantly the expression of many genes involved in this process. A decrease of IGFBP6 expression can involve a decrease in the expression of N-cadherin and transcription factor Slug.
Assuntos
Neoplasias da Mama/metabolismo , Caderinas/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Neoplasias da Mama/genética , Caderinas/genética , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Modelos BiológicosRESUMO
IGFBP6 gene plays an important role in the pathogenesis of breast cancer. In this work, we performed knockdown of IGFBP6 gene in MDA-MB-231 cells and obtained a stable cell line. Knockdown of IGFBP6 gene was confirmed by the real-time PCR. The influence of IGFBP6 gene on migration and proliferation of breast cancer cells was studied. Knockdown of IGFBP6 gene reduced migration activity of MDA-MB-231 cells and increased their proliferation rate. This in vitro cell model can be used for the further analysis of the role of IGFBP6 gene in the pathogenesis of breast cancer.