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Fig. 6 | Molecular Cancer

Fig. 6

From: The chromosome 11q13.3 amplification associated lymph node metastasis is driven by miR-548k through modulating tumor microenvironment

Fig. 6

MiR-548k modulates EGFR pathways by directly targeting KLF10 in ESCC. a, The predicted binding sequence of human hsa-miR-548k and its binding site in the 3′-untranslated region (3’-UTR) of KLF10 were presented for alignment. KLF10–3’-UTR-mut was the mutated sequences of 3’-UTR of KLF10 without the binding sites of miR-548k. b, Quantitative real time PCR results show that KLF10 could be downregulated by miR-548k in KYSE30 cells and KYSE510 cells (p < 0.001, t-test). Data were expressed as mean ± S.E.M of three independent experiments. c, Luciferase assay was used to confirm the interaction of miR-548k with KLF10. 3’-UTR of KLF10 containing the target binding site (KLF10–3’-UTR-wt) and the mutated sequences of 3’-UTR of KLF10 (KLF10–3’-UTR-mut) were cloned into downstream of a firefly luciferase gene. The plasmids were transfected into empty vector and miR-548k stably expressing cells (KYSE30-lenti-miR-548k). Renilla luciferase plasmid was co-transfected for normalisation. GV126-Control vector was co-transfected as positive control (*p < 0.05). d, Real time PCR examined the mRNA level of EGFR in miR-548k overexpression cells (KYSE30-Lenti-548 k and KYSE510-Lenti-548 k) and control cells. e, Western blot analysis the expression level of KLF10, EGFR and the phosphorylation protein level and total protein level of ERK1/2, Akt in miR-548k overexpression cells and control cells. f, Ectopic expressed KLF10 open reading frame plasmid without 3’-UTR (cannot be targeted by miR-548k) in miR-548k overexpression cells (KYSE30-Lenti-548 k and KYSE510-Lenti-548 k) and control cells. g, Growth curves of miR-548k overexpressing cells (KYSE30-Lenti-miR-548k) and control cells (KYSE30-Lenti-miR-NC) with or without ectopic expression KLF10 open reading frame. h, Transwell assays evaluated the migration and invasion capacities of miR-548k overexpressing cells (KYSE30-Lenti-miR-548k) and control cells (KYSE30-Lenti-miR-NC) with or without ectopic expression KLF10 open reading frame. Left, representative images; right, quantitative data. Error bars represent the mean ± S.E.M from three independent experiments, ns, no significance

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