Fig 1: KLF9 targets miR-338-3p directly in osteosarcoma cells. (A) qRT-PCR assay was used to assess the expression of the five candidate TFs screened by overlapping DEGs (137) with predicted targets (67). (B) The role of elevated KLF9 expression in NRCAM in osteosarcoma cells was explored by applying western blot assay. (C) The function of raised KLF9 expression on miR-338-3p expression in osteosarcoma cells was investigated by employing qRT-PCR analysis. (D, E) Elevated KLF9 expression could suppress the proliferation of osteosarcoma cells, and the effect may be reversed via diminished miR-338-3p expression. Scale bars = 100µm. (F, G, H) Raised KLF9 expression could significantly inhibit migration and invasion of osteosarcoma cells, which could be rescued by miR-338-3p decline. Scale bars = 100µm. (I) A schematic exhibited the proximal region of the miR-338-3p promoter that was targeted by KLF9. (J) ChIP assay was adopted to validate the binding correlation between KLF9 and the miR-338-3p promoter in osteosarcoma cells. (K) Luciferase activity was increased in the WT miR-338-3p promoter with KLF9 elevation that was clearly rescued via miR-338-3p diminishment. No significant change was unveiled in luciferase activity when KLF9 targeting sites at -1315 to -1303bp were mutated. Data was presented as mean ± SD from three independent experiments. *, P <0.05; ns, not significant.
Fig 2: Overexpression of NRCAM could rescue the enhanced effect of miR-338-3p on osteosarcoma cells. (A-C) The role of miR-338-3p and NRCAM in the proliferation of osteosarcoma cells was measured across cell viability as well as colony formation. (D) EdU incorporation assays were performed to assess the role of miR-338-3p and NRCAM on cell proliferation ability. Scale bars = 100μm. (E, F) The function of miR-338-3p and NRCAM in the invasion and migration of osteosarcoma cells was analyzed by employing transwell assay. (G) The function of miR-338-3p and NRCAM on osteosarcoma cells mobility were evaluated by wound-healing assay. Scale bars = 100μm. Data was presented as mean ± SD from three independent experiments. *, P <0.05.
Fig 3: NRCAM is the target gene of miR-338-3p in osteosarcoma. (A) Nine common target genes were discovered across the DEGs' overlapping analysis from TCGA and GSE12865 with predicted target genes. (B, C) The nine DEGs' heatmap were showed in GSE12865 set as well as TCGA database, respectively. (D) The six target genes that were obviously raised in both sets were assessed by qRT-PCR assay in MNNG/HOS, MG-63 and hFOB1.19 cells. (E) Western blot assay for NRCAM with miR-338-3p mimics. (F) The descriptive diagram for the binding site of miR-338-3p to NRCAM's 3'-UTR. (G) pMIR-REPORT luciferase vector comprising of NRCAM 3'UTR or a mutated type was co-transfected in osteosarcoma cells with miR-338-3p mimics or inhibitors or miR-NC. Data was presented as mean ± SD from three independent experiments. *, P <0.05; ns, not significant.
Supplier Page from Abcam for Anti-NrCAM antibody [EPR15654]