Tumor-specific co-expression of ACTB and miR-138-5p in colorectal adenocarcinoma

Abstract

Abstract Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and the second leading cause of cancer-related mortality worldwide. The ACTB gene, traditionally considered a reference gene, exhibits transcriptional instability in colorectal cancer tissues. The primary aim of this study was to confirm ACTB instability in different tissue compartments (tumor, peritumoral region, and controls). A secondary research aim was to investigate cross-sectional associations between ACTB expression and variables associated with disease stage and to assess its tumor-specific coexpression with the tumor suppressor miR-138-5p to uncover potential alterations in regulatory networks in the CRC microenvironment. Tissue from 79 colorectal cancer (CRC) patients (tumor, peritumoral, and control) was analysed. Initial transcriptomic analysis was performed on Affymetrix microarrays for mRNA (n = 5) and miRNA (n = 4). Subsequently, qPCR was used to determine relative ACTB expression (normalized to GAPDH) and validate miR-138-5p levels in a representative subcohort (n = 35; using global mean normalization). Statistical analysis included nonparametric tests and Spearman correlation to assess clinicopathological associations and mRNA-miRNA coexpression, and multivariate Generalized Linear Models (GLM). Microarray analysis revealed significant differences in ACTB expression between control and CRC tissue (FC = − 2.84, FDR p-value = 0.046). qPCR identified the lowest expression in the surrounding tissue (mean RQ value = 0.361), which was significantly lower compared to control tissue ( p = 0.0003) and tumor tissue ( p = 0.0019). Elevated ACTB levels in the surrounding tissue were also associated with distant metastases (cM1 vs. cM0; p = 0.017) and positively correlated with disease stage (cT: r = 0.423, cM: r = 0.371, pM: r = 0.283; p < 0.05). Furthermore, microarray screening and subsequent qPCR validation (N = 35) revealed a significant reduction in miR-138-5p expression in CRC tissues compared to healthy controls ( p = 0.014). Interestingly, ACTB expression positively correlated (r = 0.486; p < 0.05) with miR-138-5p levels only in the tumor microenvironment, a finding not seen in healthy tissues. Crucially, multivariate GLM identified miR-138-5p as the sole independent predictor of ACTB variance in the tumor ( p = 0.002), while metastasis status (cM) independently drove ACTB dysregulation in the peritumoral margin ( p = 0.005). ACTB exhibits significant transcriptional instability in colorectal cancer (CRC) and its microenvironment, suggesting it may be unsuitable as a sole reference gene. Tumor-specific coexpression of ACTB and the tumor-suppressive miR-138-5p indicates a novel, cancer-associated reprogramming of regulatory networks in CRC. Secondarily, multivariate exploratory cross-sectional analyses revealed that its expression patterns in the tumor environment are independently associated with stage-related clinical variables, although its clinical utility as a biomarker requires further longitudinal validation.