ABSTRACT
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.
Subject(s)
Gene Expression Profiling , MicroRNAs , HT29 Cells , Humans , Hypoxia/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Protein Isoforms/geneticsABSTRACT
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.
Subject(s)
Annexins/metabolism , Chemical Warfare Agents/pharmacology , Colonic Neoplasms/pathology , Ribosome Inactivating Proteins, Type 2/pharmacology , Ribosome Inactivating Proteins/metabolism , Ribosomes/drug effects , Ricin/pharmacology , Toxins, Biological/pharmacology , Biological Transport , Cell Survival/drug effects , Colonic Neoplasms/drug therapy , Colonic Neoplasms/metabolism , HT29 Cells , HumansABSTRACT
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.
Subject(s)
Cell Cycle Proteins/metabolism , Chaperonins/metabolism , Colorectal Neoplasms/drug therapy , Proteasome Endopeptidase Complex/biosynthesis , Ribosome Inactivating Proteins, Type 2/pharmacology , Ricin/pharmacology , Toxins, Biological/pharmacology , Adenocarcinoma/drug therapy , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Cell Cycle Proteins/genetics , Chaperonins/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Cytoplasm/metabolism , Humans , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Ribosomes/metabolism , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
Antigens, CD/metabolism , Cyclin D1/metabolism , MicroRNAs/metabolism , Receptor, Insulin/metabolism , Receptors, Somatomedin/metabolism , Antigens, CD/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Movement/physiology , Cell Proliferation/genetics , Cell Proliferation/physiology , Cyclin D1/genetics , Gene Expression Regulation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic/physiology , Gene Knockdown Techniques , Humans , MicroRNAs/genetics , Receptor, IGF Type 1 , Receptor, Insulin/genetics , Receptors, Somatomedin/geneticsABSTRACT
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.
Subject(s)
Coated Materials, Biocompatible/pharmacology , Epithelial-Mesenchymal Transition/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Laminin/pharmacology , Plastics/pharmacology , Antigens, CD/genetics , Antigens, CD/metabolism , Cadherins/genetics , Cadherins/metabolism , Cell Adhesion/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Desmoplakins/genetics , Desmoplakins/metabolism , Epithelial-Mesenchymal Transition/genetics , Fibronectins/genetics , Fibronectins/metabolism , Gene Expression Profiling , HCT116 Cells , HT29 Cells , Humans , Laminin/genetics , Laminin/metabolism , Organ Specificity , Snail Family Transcription Factors/genetics , Snail Family Transcription Factors/metabolism , Surface Properties , Zinc Finger E-box-Binding Homeobox 1/genetics , Zinc Finger E-box-Binding Homeobox 1/metabolismABSTRACT
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.
Subject(s)
Breast Neoplasms/metabolism , Cadherins/metabolism , Epithelial-Mesenchymal Transition/physiology , Insulin-Like Growth Factor Binding Protein 6/metabolism , Breast Neoplasms/genetics , Cadherins/genetics , Cell Line, Tumor , Epithelial-Mesenchymal Transition/genetics , Female , Gene Expression Regulation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic/physiology , Humans , Insulin-Like Growth Factor Binding Protein 6/genetics , Models, BiologicalABSTRACT
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.
