Fig. 4: The tRF-Ser/CNBP axis transcriptionally represses HSPA8 to inhibit GC progression.

A, B Transcriptomic profiling of MKN-45 cells with tRF-Ser overexpression and negative control. Heatmap (A) and volcano plot (B). C, D qRT-PCR validation of the top 11 candidate downstream genes in MKN-45 cells with tRF-Ser overexpression and knockdown. E WB analyses of HSPA8 protein expression following tRF-Ser modulation in GC cells. F Dose-dependent suppression of HSPA8 expression by increasing concentrations of tRF-Ser mimics in GC cells, as shown by WB. G, H Rescue experiments showed that HSPA8 overexpression attenuated the anti-proliferative (CCK-8, G) and cell cycle-arresting (flow cytometry, H) effects of tRF-Ser overexpression in MKN-45 cells. I, J Positive correlation between CNBP and HSPA8 expression in GC from public database (GEPIA, I) and our cohort of 30 patients (J). K, L qRT-PCR (K) and WB (L) analyses of HSPA8 expression upon CNBP modulation in GC cells. M Motif sequence specifically recognized by CNBP. N The one binding site of CNBP to the promoter region of HSPA8 and the point mutations specifically designed to disrupt the predicted CNBP binding motif (WT-site and MT-site) were shown. O ChIP-qPCR analyses showing CNBP enrichment on the HSPA8 promoter in GC cells. P Dual-luciferase reporter assays indicating CNBP-mediated activation of the HSPA8 promoter in GC cells. Q-S Rescue experiments suggested that HSPA8 knockdown reversed the pro-proliferative (CCK-8, Q, R) and pro-invasive (Transwell, S) effects of CNBP overexpression in GC cells. Data are expressed as mean ± SD. (Student′s t-test, **p < 0.01, and ***p < 0.001). ns means no significant.