Integrated bioinformatics and tissue-based validation reveal the oncogenic role of hsa_circ_0043256 and hsa_circ_0004789 in gastric cancer

Circular RNAs (circRNAs) play a key role in gastric cancer (GC) pathogenesis. This study hsa_circ_0043256 and hsa_circ_0004789, and their interactions with miR-28–5p/Cyclin B1 (CCNB1) and miR-5683/CCNB1, via bioinformatic and experimental methods. We retrieved expression data for circRNAs, miRNAs, and mRNAs in GC from Gene Expression Omnibus and The Cancer Genome Atlas. Using online databases and R tools, we identified downstream miRNAs and target mRNAs to build a competing endogenous RNA (ceRNA) network. After identification of hub genes and performing functional enrichment, we defined two regulatory axes: hsa_circ_0043256/miR-28–5p/CCNB1 and hsa_circ_0004789/miR-5683/CCNB1. We studied 32 paired tumor and adjacent tissues to assess all genes and CCNB1 protein expression, along with correlations, histopathological associations, ROC curves, and survival outcomes. We identified 58 circRNAs, 123 miRNAs, and 2126 mRNAs, and, further, by novelty checking and downstream RNA analysis, identified two axes. The expression of hsa_circ_0043256, hsa_circ_0004789, and CCNB1 mRNA and protein levels was elevated, while miR-28–5p and miR-5683 levels were reduced. Correlations observed among axis components supported the ceRNA hypothesis. The hsa_circ_0004789/miR-5683/ CCNB1 axis showed an AUC value for the combined ROC curve near 1, suggesting strong diagnostic potential. Lower CCNB1 and higher miR-5683 levels were correlated with better survival. Both circ_0043256 and circ_0004789 were associated with histological grade, lymphatic invasion, perineural invasion, and lymph node involvement. This study highlights circ_0043256/miR-28–5p/CCNB1 and circ_0004789/miR-5683/CCNB1 as promising axes for GC diagnosis and treatment strategies. ( A ) A schematic representation of the proposed mechanism, where hsa_circ_0043256 and hsa_circ_0004789 act as sponges for hsa-miR-28–5p and hsa-miR-5683, preventing their access to CCNB1 mRNA. As a result, CCNB1 mRNA is not degraded and is translated, leading to cell cycle progression. ( B ) The lower part of the figure schematically shows all the study steps. Bioinformatics analysis suggested the circ_0043256/miR-28–5p/CCNB1 and circ_0004789/miR-5683/CCNB1 axes. This study included 32 pairs of tumor and margin tissues. RNA was extracted and gene expression analysis was done by RT-qPCR. The level of CCNB1 protein was evaluated by Western blot after protein extraction. • Upregulated hsa_circ_0043256 and hsa_circ_0004789 in GC tissues correlate positively with CCNB1 and inversely with hsa-miR-28–5p and hsa-miR-5683, respectively. • The combined ROC curve for hsa_circ_0004789/miR-5683/CCNB1 axis was close to 1, indicating its potential for GC diagnosis. • Hsa_circ_0043256 and hsa_circ_0004789 associate with histopathological features such as grade, lymphatic/perineural invasion and lymph node involvement. • Hsa_circ_0043256 and hsa_circ_0004789 show promise as GC biomarkers and therapeutic targets.