Indeed, when miR-337-3p mimic was transfected into A549 and H1975, JAK2 protein level was greatly reduced, luciferase assay confirmed the regulatory role of miR-337-3p on JAK2 is through directly binding to JAK2 3UTR, indicating JAK2 is the target of miR-337-3p. sub-G1 phase, impaired cell migration and colony formation. Bioinformatic analysis discovered that miR-337-3p was a direct target of circ_ZNF124. In contrast to circ_ZNF124, miR-337-3p expression was significantly downregulated in NSCLC cells. Biotin labeled circ_ZNF124 immunoprecipitation and luciferase assay showed that miR-337-3p could directly bind to and affect circ_ZNF124 activity. The regulation of circ_ZNF124 on miR-337-3p was also investigated. Further analysis showed that despite STAT3 (signal transducer and activator of transcription 3), JAK2 was also a target of miR-337-3p, overexpression of miR-337-3p greatly downregulated JAK2, STAT3 and JAK2/STAT3 downstream regulated oncogenes HIF1a (Hypoxia-inducible factor 1-alpha), BCL2 (B cell lymphoma 2) and c-FOS expression, however, the roles of miR-337-3p in JAK2/STAT3 signaling pathway were greatly inhibited in the presence of circ_ZNF124. Conclusion In NSCLC, highly expressed circ_ZNF124 promoted the activation of JAK2/STAT3 signaling pathway by acting as a sponge of miR-337-3p, thus promoting the occurrence and development of NSCLC. Circ_ZNF124 could be a potential biomarker or target for the treatment of NSCLC patients in the future. non-small cell lung cancer. *P? ?0.05, **P? ?0.01 To investigate the expression of circ_ZNF124 in NSCLC. Normal immortalized epithelial cell line BEAS-2B and four lung cancer cell lines were used for our study. qRT-PCR was used to detect the expression SRT 1460 of circ_ZNF124. As indicated, circ_ZNF124 expression was much higher in these lung cancer cell lines than normal epithelial cell BEAS-2B (Fig.?1c, **P? ?0.01). Knockdown of circ_ZNF124 induces cell cycle arrest and decreases cell growth, colony formation and migration To further characterize the roles of circ_ZNF124 in NSCLC, A549 and H1975 were randomly selected for downstream study. SRT 1460 siRNA that specifically target the junction of the covalently joined 3 and 5 ends was used to inhibit circ_ZNF124 expres-sion. siRNA transfection efficiently downregulated circ_ZNF124 expression in A549 and H1975 (Fig.?2a, b, **P? ?0.01), A549 and H1975 cells cycle were also arrested in sub-G1 when circ_ZNF124 was knocked down (Fig.?2c, **P? ?0.01). In addition, cell growth assay demonstrated that silencing circ_ZNF124 greatly decreased A549 and H1975 growth rate even at the early times after seeding the cells. (Figure?2d, e, **P? ?0.05, **P? ?0.01). Cell colony formation and migration assay suggested that inhibition of circ_ZNF124 could also impair A549 and H1975 colony formation and cell migration ability (Fig.?2fCk, *P? ?0.05, **P? ?0.01). These results revealed that circ_ZNF124 plays an important role in the proliferation of NSCLC. Open in a separate window Fig.?2 Circ_ZNF124 promoted NSCLC cells proliferation, migration and colony formation. a, b qRT-PCR results of circ_ZNF124 expression after siRNA knock down in A549 and H1975. c Cell cycle detect after knock down of Rabbit Polyclonal to STAT5B circ_ZNF124. d, e Cell growth was impaired after interfering circ_ZNF124 expression compared with scramble control. f Representative images of cell colony formation. g, h Statistic results of colony number in scramble and after circ_ZNF124 knock down. i Representative images SRT 1460 of cell migration with or without circ_ZNF124 knock down. j, k Statistic results of cell migration. At least 3 replicates were used for analysis. *P? ?0.05, **P? ?0.01 miR-337-3p is a target of circ_ZNF124 in vivo Studies have shown that one of the important roles of circRNA is to remove the inhibitory effects of miRNA on downstream target genes by adsorbing miRNA . To find which SRT 1460 miRNA is the target of circ_ZNF124, we investigated the circinteractome database. Bioinformatics prediction showed that miR-337-3p was scored the highest in all the circ_ZNF124-miRNA matches. The potential binding site of miR-337-3p in circ_ZNF124 as indicated (Fig.?3a). RIP assay with anti-Ago2 antibody showed that both miR-337-3p and circ_ZNF124 can be efficiently pulled down by Ago2 antibody compared with the control SRT 1460 IgG (Fig.?3b, **P? ?0.01). To test whether circ_ZNF124 can directly interact with miR-337-3p in vivo, we synthesized biotin-labeled WT circ_ZNF124 or miR-337-3p binding site mutated circ_ZNF124 (Fig.?3c) and used them to precipitate miR-337-3p from cell lysates. As expected, WT circ_ZNF124 efficiently pulled down miR-337-3p, while mutated circ_ZNF124 failed to pull down miR-337-3p (Fig.?3d, **P? ?0.01). Luciferase assay with WT or miR-337-3p binding sites mutated circ_ZNF124 ligated into PGL3-Luc vector suggested that overexpression of miR-337-3p suppressed the luciferase activity of the circ_ZNF124 WT construct, but not the miR-337-3p binding sites mutated circ_ZNF124 construct in A549 cells (Fig.?3e, **P? ?0.01). Thus, these results indicated that miR-337-3p is a target of circ_ZNF124 in vivo. Open in a separate window Fig.?3 miR-337-3p.
Indeed, when miR-337-3p mimic was transfected into A549 and H1975, JAK2 protein level was greatly reduced, luciferase assay confirmed the regulatory role of miR-337-3p on JAK2 is through directly binding to JAK2 3UTR, indicating JAK2 is the target of miR-337-3p