Fig 2: Mechanism of the regulatory network and function of the FN1 3′-UTR region.

Fig 3: FN1 3'-UTR serves as the core regulator of a network and sponges various microRNAs in GC. Flow chart showing the analyses of miRNA-seq and RNA-seq data (A). Venn plot of the number of enriched miRNAs in HGC27 and AGS cell lines, and the regulatory network among the FN1 3′-UTR and various miRNAs (B). Luciferase activity in GC cells co-transfected with the luciferase reporter containing the FN1 3′-UTR and the mimics of let-7i-5p, miR-423-5p, miR-629-5p, miR-296-3p, or mutant. Data are presented as the relative ratio of Renilla luciferase activity (C). The alterations of the migration and invasion abilities of FN1 3′-UTR overexpressed cells after the transfection of four miRNA mimics. Scale bars = 500μm (D). The adhesion ability at 30 min or 60 min (scale bars = 1000μm) (E) and surface mobilities (scale bars = 500μm) (F) of FN1 3′-UTR overexpressed cells after transfecting the four miRNA mimics. The data are presented as a histogram of the mean ± SEM of three independent experiments in C and compared using Student's t-test (*P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n = 3).

Fig 4: FN1 3'-UTR in GC cells may affect mesothelial cells in the peritoneal environment. Experimental model of cocultured GC cells and mesothelial cells (A). The ability of HGC27 and AGS cells adhering to HPMCs (scale bars = 500μm) (B) and invading HPMCs (scale bars = 1000μm) (C), which were cocultured with different GC cells and analyzed using ImageJ software. Tumor volume was monitored using the in vivo optical imaging system for 5 weeks after intraperitoneal injection (D) and representative tumor nodules were marked in the abdominal cavity of nude mice in different groups (E). The average ROI luminescence intensity obtained by in vivo imaging in the FN1 3′-UTR overexpressed group (F). Differences of the tumor nodule weights between the FN1 3′-UTR overexpressed group, the FN1 protein overexpressed group, and the negative control group (G). The data are presented as a histogram of the mean ± SEM of three independent experiments in B and C and compared using Student's t-test (*P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n = 3).

Fig 5: The FN1 3′-UTR-let-7i-5p-THBS1 axis exists in vivo and plays a role in promoting tumor metastasis. Immunohistochemical results of THBS1 expression in the metastatic nodules in lungs (50X: scale bars = 500μm; 200X: scale bars = 100μm) (A) and tumor nodules in the abdominal cavity (50X: scale bars = 500μm; 200X: scale bars = 100μm) (B). Immunofluorescence microscopy and analysis of the co-localization between FN1 3'-UTR and let-7i-5p in the metastatic nodules of lungs from the FN1 3′-UTR overexpression group and the negative control group. Scale bars = 50μm (C-D). Immunofluorescence microscopy and analysis of the co-localization between FN1 3'-UTR and let-7i-5p in the tumor nodules of the abdominal cavity from the FN1 3′-UTR overexpression group and the negative control group. Scale bars = 50μm (E-F). The average ROI luminescence intensity obtained by in vivo imaging and the number of metastatic nodules in the lungs of let-7i-5p overexpressed group in metastasis assays (G). The average ROI luminescence intensity obtained by in vivo imaging and the weights of tumor nodule of let-7i-5p overexpressed group in peritoneal implantation assays (H). The data are presented as a histogram of the mean ± SEM of six independent experiments in A-C and compared using Student's t-test (*P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n = 6).