Autor(es): Alves, Bárbara ; Pereira, Joana F. ; Ferrão, José ; Vieira, Luís ; Matos, Paulo ; Jordan, Peter ; Gonçalves, Vânia
Data: 2023
Identificador Persistente: http://hdl.handle.net/10400.18/8874
Origem: Repositório Científico do Instituto Nacional de Saúde
Assunto(s): interleukin 6; inflammation; Colorectal Cancer; RAC1B; RNAseq; Vias de Transdução de Sinal e Patologias Associadas
Introduction: An inflammatory microenvironment was identified as a critical tumor-promoting condition for cells harboring tumor-initiating mutations. Cancer cells respond to pro-inflammatory signals with changes in their transcriptome, namely upregulating the expression of pro-tumorigenic transcript variants. A paradigmatic example is the variant RAC1B. We found increased RAC1B levels in samples from inflammatory bowel disease patients or following experimentally-induced acute colitis in mouse models and showed it to be overexpressed in colorectal tumors. Recently, we found that the overexpression of RAC1B in polarized Caco-2 colorectal cancer (CRC) cells was triggered by the presence of pro-inflammatory interleukin (IL)-6. Here we describe the use of RNA-seq to characterize the transcriptome-wide changes induced in CRC cells by exposure to IL-6. Methods: Total RNA isolated from control and IL-6-stimulated cells was subjected to library preparation and paired-end sequencing. Sequencing fastq files were first processed and quality controlled. Salmon was used to align raw reads to the human reference transcriptome (hg38) and to perform transcript quantification. Differentially expressed genes (DEGs) were identified using DESeq2, by analyzing the summarized counts per gene comparing control and IL-6 stimulation conditions. We have also performed an analysis for biological process enrichments by Gene Ontology. Selected DEGs in significantly enriched categories were validated by semiquantitative (sq)RT-PCR. Results and discussion: We show that the most significant IL-6-induced transcriptional changes were associated with the upregulation of a cluster of 18 genes, mostly associated with the regulation of apoptosis and CEACAM-mediated cell-cell adhesion. Using sqRT-PCR, we validated the upregulation of three of these genes in IL-6 treated Caco2-cells, but, interestingly, found their expression to be instead downregulated in primary tumors of the CRC TCGA dataset. Conclusion: Exposure of polarized Caco-2 CRC cells to IL-6 causes upregulation of pro-tumorigenic genes, which, paradoxically, appear to be downregulated in primary CRC tumors. Further studies will be required to clarify the impact of these results in CRC development.
