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. 2022 Jul 11;13(7):593. doi: 10.1038/s41419-022-05026-x

Fig. 4. CircFBXL4/miR-146a-5p/STAT1 axis constructed in GC.

Fig. 4

A Flow chart of LSECtin-regulated STAT1-associated ceRNA network construction. B Venn diagram of miRNAs that may correlate with STAT1, as identified in four databases. C QPCR (top) and Western blot analysis (bottom) measures of STAT1 expression following transfection with miR-146a-5p mimics, a miR-146a-5p inhibitor or miR-NC. D Hierarchical clustering revealed the circRNA expression profile in GC tissues and adjusted normal tissues (greater than 1.5 fold difference in expression; P < 0.05). E Circulation site and formation process of circFBXL4. CircFBXL4 derived from FBXL4 exons 2–6 was 1507 nt in length. F Divergent primers (⊲⊳) spanning the spliced junction were used to amplify circFBXL4, and convergent primers (▶◀) were used to amplify FBXL4 mRNA, and agarose gel electrophoresis was used to identify circFBXL4 expression products in cDNA and genomic DNA. G qRT-PCR analysis of circFBXL4 and FBXL4 mRNA after treatment with actinomycin D at the indicated time points. H qPCR assays showing that silencing or overexpression circFBXL4 decreased or increased steady level of miR-146a-5p. I RIP followed by RT-qPCR was used to detect circFBXL4 and miR-146a-5p endogenously associated with Ago2 in BGC-823 cells. J FISH analysis of GC cells showing that circFBXL4 was colocalized with miR-146a-5p in cytoplasm, and nuclei were stained with DAPI. K Dual-luciferase activity was measured for the targeted binding sites in circFBXL4 and miR-146a-5p in HEK293 cells. L Western blot experiments showed that circFBXL4 and miR-146a-5p coregulate STAT1 expression in GC cells. M EdU assays indicated that miR-146a-5p inhibitors abolished the promotion of proliferation induced by circFBXL4 knockdown. N Wound healing assays showed that miR-146a-5p inhibitors suppressed the migration induced by circFBXL4 knockdown. O Transwell assays showed that miR-146a-5p inhibitors suppressed the migration and invasion induced by circFBXL4 knockdown. Error bars indicate standard deviation (n = 3). Data, the means ± SD.