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
Detection of colorectal cancer biomarkers (CRC) remains an urgent task for the diagnosis and prediction of the disease course. A promising approach is the study of cancer stem cell markers. The cell surface glycoprotein CD44 is very important for CRC and its stem cells. Alternative splicing of 9 variable exons of CD44 mRNA leads to the formation of various isoforms of the protein with different roles in the progression of cancer. Studies of the functions of CD44 isoforms require adequate models considering the distribution of CD44 isoforms in real tumor samples. In the present study, the expression profile of CD44 isoforms in CRC was assessed based on the publicly available mRNA sequencing data of patient tumors from the TCGA-COAD database. It was shown that normal tissues predominantly expressed isoforms 3 and 4 at nearly equal levels, whereas tumors mainly expressed isoforms 2, 3, and 4; isoform 3 was expressed at the highest level. Further, the most relevant cell lines for studying the role of CD44 in CRC were identified based on the analysis of mRNA sequencing data of 55 CRC cell lines form CCLE database.
Assuntos
Neoplasias Colorretais , Receptores de Hialuronatos , Processamento Alternativo , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Humanos , Receptores de Hialuronatos/biossíntese , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Real-time reverse-transcription polymerase chain reaction (RT-PCR) is currently the most popular method for early COVID-19 diagnostics. However, loop-mediated isothermal amplification (LAMP) is superior to real-time RT-PCR in rapidity and simplicity, since it does not require expensive laboratory equipment and trained personnel. LAMP-based diagnostic kits for COVID-19 testing already exist, but corresponding tests are not yet widely available. The method has great potential for mass application. Here, we discuss the technical and methodological aspects of its widespread adoption.
RESUMO
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
Received August 28, 2018; revised October 10, 2018; accepted November 6, 2018 The loss of apical-basal cell polarity is a necessary stage of the epithelial-mesenchymal transition (EMT). Polarized epithelial cells interact with the basement membrane (BM) and, in particular, with laminins, the major components of BM. Here, we examined the effect of the transition of colon cancer cells from 2D polarized state to non-polarized 3D state on the expression of EMT associated genes, as well as the role of laminins 332 and 411 (LM-332 and LM-411) in this process. The three studied cell lines, HT-29, HCT-116 and RKO, were found to have different sensitivity to cultivation conditions (2D to 3D changes) and to addition of laminins. One of the possible reasons for this maybe a difference in the initial 2D state of the cells. In particular, it was shown that the cell lines were at different EMT stages. HT-29 exhibited more epithelial expression profile, RKO was more mesenchymal, and HCT-116 was in an intermediate state. The most laminin-sensitive cell line was HCT-116. The magnitude and the specificity of cell response to LM-332 and LM-411 depended on the expression pattern of laminins' receptors. EMT gene expression profile was not substantially changed neither during the transition from 2D to 3D state, nor the presence of laminins' isoforms. However, we detected changes in expression of SNAI1 and ZEB1 genes encoding transcription factors that control the EMT process. Notably, in all three studied cell lines, the expression of SNAI1 was enhanced in response to laminin treatment.
Assuntos
Biomarcadores Tumorais/metabolismo , Moléculas de Adesão Celular/farmacologia , Técnicas de Cultura de Células/métodos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Laminina/farmacologia , Linhagem Celular Tumoral , Humanos , CalininaAssuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/patologiaAssuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Procedimentos Analíticos em Microchip/métodos , Paclitaxel/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Humanos , Concentração Inibidora 50RESUMO
Extracellular concentration of heat shock protein (Hsp) with a molecular weight of 70 kDa (Hsp70) rapidly increases in the serum in response to stress and returns to the basal level during recovery. Further regulation of its blood concentration is unclear. A possible regulator is HspBP1, a protein binding Hsp70. Binding to ATPase domain of Hsp70, HspBP1 inactivates it, thus acting as a factor of nucleotide exchange. Blood sera from athletes were examined at the beginning and end of the last mesocycle of the training period by two-staged immunoaffinity test system. The concentration of HspBP1 increased with decreasing Hsp70 concentration under conditions of long-term training. Presumably, the dynamics of Hsp70 and HspBP1 concentrations can serve as the test for evaluating the adaptation potential.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/sangue , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Aptidão Física/fisiologia , Adolescente , Teste de Esforço , HumanosRESUMO
The mechanisms of early activation of natural killers were studied in volunteers of different athletic qualification. The initial levels of expression of KIR2DL2, PRF1, and GZMB genes regulating the functions of natural killer differ in athletes and untrained subjects. Moderate exercise stimulates transcription activities of these genes. In athletes, the expression increases more intensely than in controls. Stimulation of inhibitory (KIR2DL3) and activation (KIR2DS2) receptors was revealed. This indicated nonspecific stimulation of natural killers, probably mediated by an increase in serum concentration of heat shock protein with a molecular weight of 70 kDa.
Assuntos
Exercício Físico , Receptores KIR2DL3/genética , Receptores KIR/genética , Receptores de Células Matadoras Naturais/metabolismo , HumanosRESUMO
The mechanisms of acetylcholine release in presynaptic terminals of motoneurons induced by mutant alpha-latrotoxin (LT(N4C)) were analyzed. In contrast to wild-type alpha-latrotoxin that causes both continuous and splash secretion of acetylcholine and necessarity block neuromuscular transmission, LT(N4C) causes only splash release lasting over many hours. Thus, activation of alpha-latrotoxin receptors controls long-lasting enhanced secretion of acetylcholine.
Assuntos
Acetilcolina/metabolismo , Receptores de Peptídeos/metabolismo , Receptores de Peptídeos/fisiologia , Sinapses/metabolismo , Animais , Eletrofisiologia , Exocitose , Camundongos , Mutação , Venenos de Aranha/genética , Venenos de Aranha/metabolismoRESUMO
Intensive exercise triggers the cascade processes of body adaptation, including modulation of splisosome functioning, and can lead to modification of its activity and choice of alternative exons. We studied the effect of exercise of the maximum aerobic power on activation of transcription of genes involved in the splicing process. Short-term exercise resulted in a significant increase of mRNA expression of genes encoding proteins involved in the formation of precatalytic splisosome: DDX17, DDX46, HNRNPR, PRPF4B, and SRPK2. The role of the detected regulators in initiation of splisosome assembly under conditions of maximally intensive exercise is discussed.
Assuntos
Exercício Físico/fisiologia , Regulação da Expressão Gênica , Splicing de RNA/genética , Adolescente , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/fisiologia , Perfilação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/fisiologia , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/fisiologia , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U4-U6/fisiologia , Adulto JovemRESUMO
Muscular dystrophy is a common multisystem disease, which results from the impairment of alternative splicing. An increase in the number of unstable CTG and CCTG repeats in untranslated regions of the DMPK and ZNF9 genes is followed by dysregulation of RNA-binding proteins. Further changes are followed by dysfunction of insulin receptors, membrane Cl- channels, and other proteins. We developed a new mathematical model for the regulation of splicing of exon 11 in the IR gene, which describes the effect of various factors on alternative splicing.
Assuntos
Processamento Alternativo/genética , Modelos Genéticos , Distrofia Miotônica/genética , Receptor de Insulina/genética , Humanos , Modelos BiológicosRESUMO
Studying the copy number of ribosomal protein L7/L12 was performed using monoclonal antibody 3G9 to the linear epitope on the C-terminal domain of this protein from Escherichia coli. Immunohistochemical study showed that Agrobacterium tumefaciens ribosomes include 6 copies of protein L7/L12. Our results suggest that the copy number of this protein has an evolutionary role.