Fig 2: miR-223-3p Suppresses CDH6 Expression by Directly Targeting Its 3' UTR(A–D) Immunoblot analysis of the indicated osteosarcoma cell lines transfected with miR-223-3p (A and B) or anti-miR-223-3p (C and D). The histograms on the left of the immunoblots show the corresponding miR-223-3p mRNA expression levels. (E) miRNA luciferase reporter assays in 143B and U2OS cells co-transfected with wild-type or mutated CDH6 reporters and miR-223-3p are shown. The top panel indicates wild-type and mutant forms of putative miR-223-3p target sequences in the 3' UTR of CDH6. Bold and italicized fonts indicate putative miR-223-3p-binding sites in the 3' UTR of human CDH6. Underlining indicates mutations introduced into the 3' UTR of CDH6. (F) miRNA luciferase reporter assays in 143B and U2OS cells co-transfected with wild-type or mutated CDH6 reporters and anti-miR-223-3p are shown. Each bar represents the mean ± SD of at least three independent experiments performed in triplicate. *p < 0.05; **p < 0.01.

Fig 3: miR-223-3p Expression in Human Osteosarcoma Samples and the Correlation between miR-223-3p and CDH6(A) Expression of miR-223-3p in osteosarcoma tissues and matched adjacent normal tissues (n = 133) was compared using the Mann-Whitney U test. U6 small nuclear RNA was used as the internal control. (B and C) Kaplan-Meier survival curves and log rank tests were used to compare (B) DFS and (C) OS of osteosarcoma patients with low and high expression levels of miR-223-3p. (D) The relationship between miR-223-3p and CDH6 expression was assessed by Spearman’s rank correlation analysis in the osteosarcoma samples. The symbols represent individual samples.

Fig 4: miR-223-3p Inhibits Osteosarcoma Cell Invasion, Migration, Growth, and Proliferation through the Suppression of CDH6 Production(A and B) Invasion of 143B cells transfected with miR-223-3p or miR-223-3p plus CDH6 (A) or miR-223-3p inhibitor (B) was evaluated using a Matrigel invasion chamber. The invaded cells were fixed and stained with crystal violet (A and B left images). The scale bar represents 100 µm. (C and D) Wound healing assays were conducted in 143B cells transfected with miR-223-3p or miR-223-3p and CDH6 (C) or miR-223-3p inhibitor (D). Cell migration was measured 24 hr after the cell layers were scratched. (E and F) 143B cells expressing miR-223-3p or miR-223-3p and CDH6 (E) and 143B cells transfected with miR-223-3p inhibitor (F) were cultured in regular medium. At the specified times, cell numbers were determined with the CCK-8 assay. The representative immunoblot shows CDH6 expression. (G and H) 143B cells transfected with miR-223-3p (G) or miR-223-3p inhibitor (H) were plated and assayed for colony formation. Representative images show colonies in plates (left panels). Each bar represents the mean ± SD of at least three independent experiments performed in triplicate. *p < 0.05; **p < 0.01.

Fig 5: Validation of the human BNB transcriptome. Merged digital photomicrographs of sural nerve endoneurial microvessels (UEA-1 FITC-positive, green) in situ show BNB endothelial cell expression (yellow-green, yellow or orange co-localization dependent of the relative fluorescent intensity of protein marker in red) of transporters (ABCA8, ABCB1, AQP1, SLC1A1, SLC2A1, SLC3A2, SLC5A6, SLC16A1 and SLC19A2), cytoskeletal proteins (ACTG1, CALD1, FLNA, MYO10), cell membrane proteins (CAV1, CD44, CD63, ESAM), junctional complex proteins (CDH5, CDH6, CLDN4, CLDN5, CTNNA1, GJA1, LIN7A, MPDZ, PCDH1, TJP1, VEZT, ZYX), vascular endothelial cell secreted protein CTGF, and chemokine receptor CXCR4. Positive expression of AQP1, CALD1, CAV1, CD44, CD63, FLNA, MYO10 and SLC2A1 (red) by cells that surround and are in direct contact with endothelial cells (most likely pericytes), and ABCA8 by cells close to but not in direct contact with endothelial microvessels (most likely Schwann cells) are also observed. Nuclei are identified in blue. Original magnification 400X.