Fig. 5.

circLIFR inhibits cell migration in vitro and tumor metastasis in vivo. A Agarose gel electrophoresis and Sanger sequencing of semi-quantitative RT-PCR products demonstrated that circLIFR was correctly circularized and successfully overexpressed in KYSE30 and SK-Hep-1 cells. B Transwell migration assays showed that circLIFR overexpression inhibited the migratory ability of KYSE30 and SK-Hep-1 cells. C Wound healing assays showed that circLIFR overexpression inhibited cell migration in both KYSE30 and SK-Hep-1 cells. D Decreased tumor metastasis formed in the lungs of mice via tail vein injection of circLIFR-overexpressing KYSE30 cells, as indicated by representative bioluminescent images of lung metastasis at 6 weeks (left) and H&E staining of lung metastatic lesions (right) of mice for each group (n = 5 mice/group). E Decreased tumor metastasis formed in the livers of mice via spleen injection of circLIFR-overexpressing SK-Hep-1 cells, as indicated by representative bioluminescent images of liver metastasis at 8 weeks (left) and H&E staining of liver metastatic lesions (right) of mice for each group (n = 5 mice/group). F Volcano plot of differentially expressed genes affected by circLIFR in KYSE30 cells. G,H Gene Ontology (G) and GSEA analysis (H) showed that dysregulated genes upon circLIFR overexpression was involved in cell adhesion. NES, normalized enrichment score; FDR, false discovery rate. p-values are calculated by permutation test. I,J Quantification of metastasis-related genes by RT-qPCR in KYSE30 control cells and circLIFR-overexpressing cells. Data represented mean ± S.D.; the p-values were determined by a two-tailed unpaired Student’s t-test