Evaluation of tumor necrosis factor alpha signaling pathway mediators and microRNAs in gastric cancer and effect of their receptors silencing

Author(s)

Ana Flávia Teixeira Rossi Freire

• Document type: Doctoral Thesis
• Press: São José do Rio Preto. 2019-08-12.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências Letras e Ciências Exatas. São José do Rio Preto
• Defense date: 2019-07-25
• Advisor: Ana Elizabete Silva

Abstract

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. (AU)

• FAPESP's process: 15/23392-7 - Assessment of mediators of tumor necrosis factor-± signaling pathway and microRNAs in gastric carcinogenesis associated with Helicobacter pylori
• Grantee: Ana Flávia Teixeira Rossi Freire
• Support Opportunities: Scholarships in Brazil - Doctorate