Research Paper Volume 16, Issue 11 pp 9846—9858

Figure 4. miR-223-3p targeted SOX6. (A) Venn diagram illustrates the intersection of downregulated genes from GEO datasets (GSE13355, GSE14905 and GSE50790) and TargetScan predicted targets of miR-223-3p. (B) qRT-PCR was performed to evaluate the effects of miR-223-3p mimic transfection on SOX6, F3 and ENPP5 mRNA level. GAPDH served as an internal reference. (C) Western blotting was used to detect the protein level of F3 and SOX6 in HaCaT keratinocytes with the transfection of miR-223-3p mimic/inhibitor. GAPDH served as an internal reference. (D) The binding site between miR-223-3p and SOX6. (E) HaCaT keratinocytes were co-transfected with miR-223-3p/NC mimic and SOX6 wide type (SOX6-WT)/SOX6 mutant (SOX6-MUT) plasmid for 48 hours. Dual-luciferase reporter assay was performed to verify the interaction between miR-223-3p and SOX6. (F) Expression correlation of miR-223-3p and SOX6 in dataset GSE142582. (G) qRT-PCR was used to detect the expression of SOX6 in HaCaT keratinocytes with the transfection of NC inhibitor/ miR-223-3p inhibitor. GAPDH served as an internal reference. (H) The efficiency of interference of si-SOX6 in HaCaT keratinocytes was detected. (I) The effects of SOX6 interference on cell viability were assessed using CCK-8 kit. (J) qRT-PCR was used to detect the expression of KRT6, KRT16 in HaCaT keratinocytes with the transfection of si-NC/si-SOX6. GAPDH served as an internal reference. (K) Western blotting was used to detect the protein level of Cyclin A2, CDK4, Cyclin D1 and KRT6 in HaCaT keratinocytes with the transfection of si-NC/si-SOX6. GAPDH served as an internal reference. (L) The rate of cell proliferation was assessed using EdU incorporation assays. Scale bar: 100 μm. (M) Flow cytometry was performed to analyze the effects on cell cycle distribution. *P<0.05, **P<0.01, ***P<0.001.