engleski

Hypomethylation of ASC promoter region in tumor tissue of patients with NSCLC

Introduction: DNA methylation, an epigenetic alteration, can affect gene expression without changing the DNA itself. Abberant methylation present in CpG islands of gene promoter regions can contribute to tumor promotion and is now recognized as a hallmark of lung cancer. Many epidemiological studies have linked chronic inflammation to cancer initiation, progression and dissemination. Upon insult, either endogenous or exogenous, cells act together to trigger inflammation via the activation of pattern recognition receptors (PRRs). Subgroups of PPRs, such as NLRs or ALRs, can recognize their ligands and form multiprotein platforms called inflammasomes. Activation of the inflammasome is required for the activation of caspase-1 and the subsequent secretion of proinflammatory cytokines IL-18 and IL-1β. A critical adaptor molecule in inflammasome activation is apoptosis-associated speck-like protein containing a CARD/target of methylation-induced silencing-1 (ASC/TMS1). Since the activation and controlling mechanisms of inflammation and infection are regulated by NF-κB and rely on PRRs, the suppression of these proinflammatory pathways may provide opportunities for both prevention and treatment of cancer. Aim: The aim of the study was to evaluate the methylation status of the ASC/TMS1 promoter region and to correlate methylation status with its mRNA and protein expression. Material and Methods: To define the methylation status of the ASC/TMS1 gene promoter region in tumor and paired lung tissue, the pyrosequencing approach was used. In order to correlate the methylation status of the promoter region with protein expression, western blotting was performed and the results were quantified by densitometry. Further, the correlation of the hypomethylation of the ASC/TMS1 promoter and the overall survival and tumor grade of NSCLC patients was assessed. To determine the expression of genes involved in TLR and NOD signaling, RT-qPCR was performed. Results: It was found that the promoter region of ASC/TMS1 in tumor tissues in patients diagnosed with lung cancer exhibited a reduced methylation status compared to healthy tissue. The reduced methylation status of the ASC/TMS1 promoter in tumor tissues was correlated with higher protein expression and vice versa. In addition, the expression of ASC mRNA in tumor tissues was up-regulated compared to healthy lung tissues. A tumor-specific cytokine profile was found on the mRNA level as well: up regulation of cytokines such as IL-18 and IL-1β and down-regulation of cytokines such as IL- 12A, IL33 and IL-6. According to survival analysis, out of 11 CpGs tested in the ASC/TMS1 promoter region, only hypomethylated CpG4 was found to be associated with decreased overall survival, while higher methylation of CpG8 of the ASC/TMS1 gene was associated with TNM stage 2. Conclusion: Hypomethylation of the ASC/TMS1 promoter region in tumor tissues of patients with non-small cell lung cancer (NSCLC) contributes to higher ACT/TMS1 mRNA levels and protein expression compared to healthy tissue and is associated with tumor grade and overall survival of patients with NSCLC.

lung carcinoma, methylation, promoter region, ASC

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