Avaliação de mediadores da via de sinalização do fator de necrose tumoral alfa e de microRNAs em câncer gástrico e efeito do silenciamento de seus receptores
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Data
2019-07-25
Autores
Freire, Ana Flávia Teixeira Rossi
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Universidade Estadual Paulista (Unesp)
Resumo
Introdução: Câncer gástrico (CG) apresenta altas taxas de incidência e mortalidade no mundo, sendo a infecção persistente pela bactéria Helicobacter pylori (H. pylori) um dos seus principais fatores etiológicos, uma vez que promove inflamação crônica e a expressão de mediadores inflamatórios, como o fator de necrose tumoral (TNF)- α. Esta citocina é um mediador chave entre inflamação e câncer, induzindo múltiplas respostas celulares por meio de dois receptores: TNFR1 e TNFR2. Ambos os receptores podem desencadear a sobrevivência, mas apenas TNFR1 é capaz de induzir morte celular. A regulação desta via é complexa e pode ser alterada por microRNAs (miRNAs) podendo favorecer o processo tumorigênico. Objetivos: a) avaliar a expressão de genes e miRNAs envolvidos na via de sinalização de TNF-α e suas relações em amostras de CG; b) construir uma rede de interação miRNAsRNAm; c) avaliar o efeito do silenciamento dos receptores TNFR1 e TNFR2 e do tratamento com extrato da H. pylori (eHP), na expressão dos mesmos genes e miRNAs, bem como nos processos celulares de proliferação, ciclo celular e apoptose em linhagem de câncer gástrico. Materiais e Método: Total de 30 amostras provenientes de indivíduos com CG foram submetidas a PCR quantitiva (qPCR) com ensaios TaqMan®, para quantificação relativa (RQ) do RNAm de genes da via de sinalização de TNF-α (TNFA, TNFR1, TNFR2, TRADD, TRAF2, CFLIP, NFKB1, NFKB2, CASP8, CASP3) e de miRNAs que apresentam alguns destes RNAm como alvo (miR-19a, miR-34a, miR-103a, miR-130a, miR-181c). Odiagnóstico molecular da H. pylori foi realizado por nested PCR para o gene HSP60. Linhagens de adenocarcinoma gástrico AGS estáveis contendo os receptores TNFR1 (AGS-shR1) e TNFR2 (AGS-shR2) silenciados por shRNAs específicos e linhagem AGS não silenciada foram tratadas com 30% v/v de eHP por 6 horas. Posteriormente, foi realizada a quantificação dos mesmos RNAm e miRNAs por qPCR, ensaio MTT para avaliação da proliferação celular e citometria de fluxo para análise do ciclo celular e apoptose. Resultados: Nas amostras de CG houve aumento na expressão de genes pró-sobrevivência (TNF, TNFR2, TRADD, TRAF2, CFLIP e NFKB2), CASP8 e do miR-34a, enquanto a expressão de mediadores pró-apoptóticos (TNFR1 e CASP3) estava reduzida. Infecção pela H. pylori não influenciou a expressão dos RNAm e miRNAs nas amostras de CG. A rede de interação miRNA:RNAm mostrou possíveis interrelações entre os miRNAs analisados e os genes da via de sinalização de TNF-α, destacando os miR-19a e miR-103a que podem regular o maior número de genes. Nos experimentos in vitro, AGS-shR1 apresentou redução significante na expressão de genes de sobrevivência celular (TNFA, NFKB1 e NFKB2) e aumento na expressão de CASP3, embora não alterou a expressão de miRNAs e nem os processos celulares. Por outro lado, silenciamento parcial de TNFR2 reduziu significantemente a expressão de TRADD e TRAF2, assim como aumentou a expressão de miRNAs, suprimiu a apoptose e desencadeou parada do ciclo celular na fase G2/M. Tratamento com eHP na linhagem não silenciada, além de aumentar o número de células em necrose, aumentou a expressão dos genes TNFR1, NFKB1, NFKB2 e CFLIP, e reduziu a expressão de TNFR2, embora expressão reduzida dos receptores tenha diminuido a intensidade da resposta ao tratamento. Conclusão: Portanto, nossos resultados destacam o importante papel da via de sinalização de TNF-α mediada tanto por TNFR2 como por TNFR1 em câncer gástrico. Em amostras de tecido, o efeito pró-tumoral de TNF-α deve-se a ativação da via de sinalização mediada principalmente por TNFR2. Na linhagem celular, silenciamento parcial de TNFR1 e TNFR2 reduziu a expressão de genes pró-sobrevivência e anti-apoptóticos e afetou processos celulares e expressão de miRNAs, de modo que silenciamento simultâneo dos receptores pode ter um efeito anti-tumoral em câncer gástrico. Além disto, a resposta de infecção por H. pylori nesta via de sinalização é mediada principalmente por TNFR1, direcionando para a via de sobrevivência celular.
Introduction: Gastric cancer (GC) has high rates of incidence and mortality in the world and persistent infection by the bacterium Helicobacter pylori (H. pylori) is one of the main etiological factors since it promotes chronic inflammation and inflammatory mediators’ expression, such as tumor necrosis factor (TNF)-α. This cytokine is a key mediator between inflammation and cancer, inducing multiple cellular responses through two receptors: TNFR1 and TNFR2. Both receptors can trigger survival, but only TNFR1 is capable of inducing cell death. This pathway’s regulation is complex and can be altered by microRNAs (miRNAs) which may favor the tumorigenic process. Objectives: a) to evaluate the expression of genes and miRNAs involved in TNF-α signaling pathway and their relationships in GC samples; b) to build a miRNA:mRNA interaction network; c) to evaluate the effect of TNFR1 and TNFR2 receptors silencing and treatment with H. pylori extract (eHP) on gastric cancer lineage in the expression of same genes and miRNAs, as well as in the cellular processes of proliferation, cell cycle and apoptosis. Materials and Method: total 30 samples from GC subjects were submitted to quantitative PCR (qPCR) with TaqMan® assay, for the relative quantification (RQ) of the TNF-α signaling pathway mRNA (TNFA, TNFR1, TNFR2, TRADD, TRAF2, CFLIP, NFKB1, NFKB2, CASP8, CASP3) and miRNAs that presented some of these mRNAs as targets (miR-19a, miR-34a, miR-103a, miR-130a, miR-181c). Molecular diagnosis of H. pylori was performed by nested PCR for gene HSP60. Stable AGS gastric adenocarcinoma lineage containing the TNFR1 (AGS-shR1) and TNFR2 (AGS-shR2) receptors downregulated by specific shRNAs and non-silenced lineage were treate with 30% v/v eHP for 6 hours. Subsequently, the same mRNA and miRNAs were quantified by qPCR, MTT assay for cell proliferation evaluation and flow cytometry for cell cycle analysis and apoptosis. Results: In GC samples, it was verified up-regulation of prosurvival genes (TNF, TNFR2, TRADD, TRAF2, CFLIP and NFKB2), CASP8 and miR34a, whereas proapoptotic mediators’s expression (TNFR1 and CASP3) were downregulated. H. pylori infection did not influence the expression of mRNAs and miRNAs in GC samples. miRNA:mRNA interaction network showed several possible relationships between the analyzed miRNAs and the TNF-α signaling pathway genes, highlighting miR-19a and miR-103a which can regulate the greatest number of genes. In vitro experiments, AGS-shR1 presented significant decreased in the expression of survival genes (TNFA, NFKB1 and NFKB2) and increased CASP3 expression, although no changes in cellular processes and miRNAs expression were observed. In contrast, TNFR2 downregulation significantly decreased TRADD and TRAF2 expression, as well as increased the miRNAs expression, supressed apoptosis and promoted cell-cycle arrest at G2/M. The treatment with eHP in nonsilenced lineage, besides increasing the amount of necrosis cells, increased expression of genes TNFR1, NFKB1, NFKB2 and CFLIP, and decreased of TNFR2, however the receptors downregulation decreased treatment response intensity. Conclusion: Therefore, our results highlight the important role of the TNF-α signaling pathway mediated by both TNFR2 and TNFR1 in gastric cancer. In gastric samples, the pro-tumor effect of TNF-α is mainly due to activation of the TNFR2-mediated signaling pathway. In AGS cell line, TNFR1 and TNFR2 downregulation decrease expression of pro-survival and anti-apoptotic genes and affected cellular processes and miRNA expression, so that simultaneous silencing of both TNFR1 and TNFR2 may have an antitumor effect on gastric cancer. In addition, H. pylori infection response in this signaling pathway is mediated mainly by TNFR1, directing to the cellular survival pathway.
Introduction: Gastric cancer (GC) has high rates of incidence and mortality in the world and persistent infection by the bacterium Helicobacter pylori (H. pylori) is one of the main etiological factors since it promotes chronic inflammation and inflammatory mediators’ expression, such as tumor necrosis factor (TNF)-α. This cytokine is a key mediator between inflammation and cancer, inducing multiple cellular responses through two receptors: TNFR1 and TNFR2. Both receptors can trigger survival, but only TNFR1 is capable of inducing cell death. This pathway’s regulation is complex and can be altered by microRNAs (miRNAs) which may favor the tumorigenic process. Objectives: a) to evaluate the expression of genes and miRNAs involved in TNF-α signaling pathway and their relationships in GC samples; b) to build a miRNA:mRNA interaction network; c) to evaluate the effect of TNFR1 and TNFR2 receptors silencing and treatment with H. pylori extract (eHP) on gastric cancer lineage in the expression of same genes and miRNAs, as well as in the cellular processes of proliferation, cell cycle and apoptosis. Materials and Method: total 30 samples from GC subjects were submitted to quantitative PCR (qPCR) with TaqMan® assay, for the relative quantification (RQ) of the TNF-α signaling pathway mRNA (TNFA, TNFR1, TNFR2, TRADD, TRAF2, CFLIP, NFKB1, NFKB2, CASP8, CASP3) and miRNAs that presented some of these mRNAs as targets (miR-19a, miR-34a, miR-103a, miR-130a, miR-181c). Molecular diagnosis of H. pylori was performed by nested PCR for gene HSP60. Stable AGS gastric adenocarcinoma lineage containing the TNFR1 (AGS-shR1) and TNFR2 (AGS-shR2) receptors downregulated by specific shRNAs and non-silenced lineage were treate with 30% v/v eHP for 6 hours. Subsequently, the same mRNA and miRNAs were quantified by qPCR, MTT assay for cell proliferation evaluation and flow cytometry for cell cycle analysis and apoptosis. Results: In GC samples, it was verified up-regulation of prosurvival genes (TNF, TNFR2, TRADD, TRAF2, CFLIP and NFKB2), CASP8 and miR34a, whereas proapoptotic mediators’s expression (TNFR1 and CASP3) were downregulated. H. pylori infection did not influence the expression of mRNAs and miRNAs in GC samples. miRNA:mRNA interaction network showed several possible relationships between the analyzed miRNAs and the TNF-α signaling pathway genes, highlighting miR-19a and miR-103a which can regulate the greatest number of genes. In vitro experiments, AGS-shR1 presented significant decreased in the expression of survival genes (TNFA, NFKB1 and NFKB2) and increased CASP3 expression, although no changes in cellular processes and miRNAs expression were observed. In contrast, TNFR2 downregulation significantly decreased TRADD and TRAF2 expression, as well as increased the miRNAs expression, supressed apoptosis and promoted cell-cycle arrest at G2/M. The treatment with eHP in nonsilenced lineage, besides increasing the amount of necrosis cells, increased expression of genes TNFR1, NFKB1, NFKB2 and CFLIP, and decreased of TNFR2, however the receptors downregulation decreased treatment response intensity. Conclusion: Therefore, our results highlight the important role of the TNF-α signaling pathway mediated by both TNFR2 and TNFR1 in gastric cancer. In gastric samples, the pro-tumor effect of TNF-α is mainly due to activation of the TNFR2-mediated signaling pathway. In AGS cell line, TNFR1 and TNFR2 downregulation decrease expression of pro-survival and anti-apoptotic genes and affected cellular processes and miRNA expression, so that simultaneous silencing of both TNFR1 and TNFR2 may have an antitumor effect on gastric cancer. In addition, H. pylori infection response in this signaling pathway is mediated mainly by TNFR1, directing to the cellular survival pathway.
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Palavras-chave
Câncer gástrico, Via de sinalização de TNF-α, Silenciamento de TNFR1, Silenciamento de TNFR2, MiRNAs, Helicobacter pylori, Gastric cancer, TNF-α signaling pathway, TNFR1 knockdown, TNFR2 knockdown