Investigating the expression of CTLA-4 and CD28 genes as immune regulators in patients with colorectal polyps

Colorectal cancer (CRC) develops through a multistep process originating from precancerous polyps. Elucidating the mechanisms that regulate immune homeostasis in precancerous lesions and in the systemic circulation prior to malignant transformation is of critical importance. This study aimed to evaluate alterations in the expression levels of the CTLA-4 and CD28 genes in patients with hyperplastic and adenomatous colorectal polyps.

Peripheral blood samples were collected from 40 patients diagnosed with adenomatous or hyperplastic colorectal polyps and 30 healthy controls. The relative expression levels of CTLA-4 and CD28 were quantified using real-time quantitative PCR (qRT-PCR). Transcriptomic data for microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) were retrieved from the TCGA-COAD database. Differential expression analysis was performed using the edgeR package, with thresholds set at a false discovery rate (FDR) < 0.05 and |log2 fold change (FC)| > 1.5. Subsequently, miRNA-mRNA and lncRNA-miRNA interactions were integrated to construct a lncRNA-miRNA-mRNA regulatory network. Immune cell infiltration analysis was conducted using the TIMER platform to evaluate the association between gene expression levels and immune cell infiltration in colon adenocarcinoma (COAD).

The results demonstrated that CTLA-4 and CD28 expression levels were upregulated in patients with colorectal polyps, although the magnitude of upregulation varied between polyp subtypes. Receiver operating characteristic (ROC) curve analysis yielded area under the curve (AUC) values of 0.66 and 0.54 for CTLA-4 and CD28, respectively. A significant positive correlation was observed between CTLA-4 and CD28 expression levels. In COAD tissues, 245 differentially expressed miRNAs and 868 differentially expressed lncRNAs were identified from the TCGA dataset. Construction of the lncRNA-miRNA-mRNA network identified KCNQ1OT1 as a hub lncRNA, potentially regulating CD28 and CTLA-4 indirectly through interactions with multiple miRNAs. Furthermore, TIMER analysis revealed significant associations between CD28 and CTLA-4 expression and the infiltration of immune cells, including neutrophils, macrophages, CD8⁺ T cells, and CD4⁺ T cells, within the COAD cohort.

Our findings indicate that CTLA-4 and CD28 are upregulated in patients with colorectal polyps and exhibit a positive correlation in expression. The regulatory network analysis highlights KCNQ1OT1 as a potential upstream modulator of these genes via miRNA-mediated mechanisms. Moreover, the strong association between CTLA-4 and CD28 expression and immune cell infiltration underscores their potential role in immune dysregulation during the early stages of colorectal tumorigenesis. Collectively, these findings suggest that CTLA-4 and CD28 may contribute to polyp progression through modulation of the immune microenvironment.