RESUMO
Death receptors (DRs) and the participants of DR-mediated signaling are characterized by a large number of mRNA isoforms generated by alternative splicing. Due to their high labor intensity and high cost, conventional methods (RT-PCR and RT-PCR in real time) are ineffective when the simultaneous detection of a plurality of mRNA isoforms is needed. In this regard, the use of DNA biochips is has prospective applications in analyzing the expression of many genes simultaneously. In this paper, we suggest an optimal strategy of probes selection aimed at detecting the maximum number of mRNA splice variants generated by major participants of DR-signaling. The objects of the study were 185 genes that form 1134 mRNA isoforms. As a result, a biochip design was developed that enables the detection of 499 mRNA isoforms (44% of total mRNA splice variants). The proposed strategy combines a high degree of modularity, the use of modern high-performance computers, and broad opportunities for setting up the selection criteria in accordance with the objectives of the study.
Assuntos
Processamento Alternativo , Sondas de DNA/metabolismo , Sondas Moleculares/metabolismo , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Receptores de Morte Celular/genética , Algoritmos , Apoptose/genética , Sondas de DNA/química , Éxons , Humanos , Íntrons , Sondas Moleculares/química , Análise de Sequência com Séries de Oligonucleotídeos , Precursores de RNA/genética , RNA Mensageiro/genética , Receptores de Morte Celular/metabolismo , Transdução de Sinais/genéticaRESUMO
Analysis of frequency of the occurrence of membrane and soluble forms of the mRNA DR3/LARD in blood in herpesviral infection of various etiology was studied. Four forms of the mRNA DR3/LARD were detected with various frequencies in blood cells of healthy volunteers. Patients with herpesviral infection of various etiology were studied using RT-PCR. Two forms encoded membrane molecules (mRNA LARD 1a, mRNA DR3beta) and two other forms accorded soluble forms of receptor (mRNA LARD 3, mRNA soluble DR3beta). It was revealed that the frequency of the occurrence of mRNA soluble DR3beta form decreased in patients with the varicella zoster virus (VZV) infection in comparison with healthy volunteers. However, the patients with the Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infection did not display significant change in occurrence of mRNA soluble DR3beta form. As a whole, changes in frequency of occurrence of spliced variants of mRNA DR3/LARD are directed toward modulation of apoptosis and restraint antiviral immune response.
Assuntos
Infecções por Citomegalovirus/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpes Zoster/imunologia , RNA Mensageiro/imunologia , Membro 25 de Receptores de Fatores de Necrose Tumoral/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo , Estudos de Casos e Controles , Citomegalovirus/imunologia , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/virologia , Feminino , Expressão Gênica , Herpes Zoster/genética , Herpes Zoster/virologia , Herpesvirus Humano 3/imunologia , Herpesvirus Humano 4/imunologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas , RNA Mensageiro/genética , Membro 25 de Receptores de Fatores de Necrose Tumoral/genética , SolubilidadeRESUMO
There are a lot of "death receptor" DR3/LARD mRNA variants that are formed during the alternative splicing. Membrane and soluble forms of the receptor are encoded with various types of the DR3/LARD mRNA and perform different functions. Using RT-PCR, the DR3/LARD mRNA spliced variants' frequency was measured in colon cancer patients' samples and cancer cell lines. In samples under investigation, four forms of the DR3/LARD mRNA were found with various frequencies. Two of them encoded the membrane receptors (LARD la mRNA and DR3beta mRNA) and other two expressed the soluble molecules (LARD 3 mRNA and soluble DR3beta mRNA). In blood of healthy volunteers 11 combinations (spectrums) of the DR3/LARD mRNA forms were revealed, and the "full" spectrum including all four variants of DR3/LARD mRNA dominated. In blood of colon cancer patients and tumour tissue samples, 6 DR3/LARD mRNA spectrums were found. The diversity of the DR3/LARD mRNA spectrums was decreased in colon cancer patients because of the frequency reduction of soluble DR3beta mRNA. Reduction of a variety of spectrums in cells of the patients was caused by decrease in occurrence of mRNA of the soluble DR3beta form. In samples of the tumor centers the spectrum with absence only mRNA of the soluble DR3beta form dominated. In blood of patients two spectrums prevailed: "full" range and presented mRNA LARD la and mRNA LARD 3. Only these two spectrums of mRNA DR3/LARD were also found in the tumor cell lines. Distinctions in occurrence of spectrums of DR3/LARD mRNA at healthy volunteers and colon cancer patients can define a different susceptibility of immunocompetent and tumor cells for apoptosis signals.
