Abstract
Background: Globally, gastric cancer (GC) ranks as the fourth most deadly and fifth most prevalent kind of cancer. Appropriate treatment methods, precise etiology, and molecular processes of GC are still unclear. Methods: In silico and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)-based expression of miRNA-27b was quantified in GC cell lines (AGS, MKN-28, MKN-45, NCI-N87, SNU-1), and ROC curve analysis was done to evaluate their diagnostic efficiency. In silico target prediction through miRDB and TargetScan followed by in vitro validation was done using luciferase assays. Expression analysis of MAPK/ERK target genes including GRB2, SOS1, KRAS, BRAF, MAP2K1, and MAPK1 was done using qRT-PCR and Western blot analysis, followed by ROC curve analysis to evaluate their diagnostic efficiency. GC cell lines were treated with Aloin (ALO), followed by cell viability, wound healing, and apoptosis assays. Furthermore, the expression of MAPK/ERK pathway genes in GC cell lines was evaluated by qRT-PCR following ALO treatment. Results: The in silico analysis identified specific binding sites for miRNA-27b within the 3'UTRs of key components in the MAPK/ERK signaling pathway, including GRB2, SOS1, KRAS, BRAF, MAP2K1, and MAPK1. Luciferase reporter assays confirmed the direct interaction of miRNA-27b with these target genes, showing significantly reduced luciferase activity in cells transfected with wild-type 3'UTRs compared to controls. Expression analysis revealed that miRNA-27b was significantly downregulated in GC patients and cell lines when compared to normal controls. The downregulation of miRNA-27b was further validated through qRT-PCR in a variety of GC cell lines. ROC curve analysis demonstrated an AUC of 100 for miRNA-27b, suggesting its strong potential as a diagnostic biomarker for GC. In contrast, the expression of MAPK/ERK pathway genes was significantly upregulated in GC cell lines, with ROC analysis revealing high diagnostic accuracy for several genes, including GRB2, SOS1, and KRAS. Protein expression analysis via Western blot confirmed the upregulation of these pathway components in GC cells. Further investigation into the effects of ALO treatment showed a dose-dependent reduction in cell viability, migration, and colony formation in GC cell lines. ALO treatment also induced apoptosis, as evidenced by the upregulation of apoptotic markers and the downregulation of the anti-apoptotic molecule Bcl-2. Conclusion: MiRNA-27b and MAPK/ERK pathway genes (GRB2, SOS1, KRAS, BRAF, MAP2K1, and MAPK1) could serve as efficient diagnostic, prognostic, and therapeutic targets for GC patients. Furthermore, this study's findings shed light on ALO's anti-tumor capabilities by demonstrating that it inhibits GC cell migration and proliferation while restoring the expression status of MAPK/ERK pathway genes.