Apelin promotes osteosarcoma metastasis by upregulating PLOD2 expression via the Hippo signaling pathway and hsa_circ_0000004/miR-1303 axis

Figures

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  Figure 1. Clinicopathological characteristics of APLN in human osteosarcoma tissue. (A-B) Normal bone and osteosarcoma specimens were subjected to IHC staining with APLN antibody (scale bar 100 µm), then quantified. (C) APLN mRNA expression in normal bone and osteosarcoma tissue were examined by qPCR. (D) APLN mRNA expression in primary osteoblast and osteosarcoma tissue was analyzed in samples from the GEO GSE12865dataset.

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  Figure 2. A positive correlation between APLN and PLOD2 expression in human osteosarcoma tissue. (A-B) Normal bone and osteosarcoma specimens were subjected to IHC staining with PLOD2 antibody (scale bar 100 µm), then quantified. (C) PLOD2 mRNA expression in normal bone and osteosarcoma tissue were examined by qPCR. (D) APLN mRNA expression in normal and osteosarcoma tissue was analyzed in samples from the GEO GSE16008 dataset. (E) Analysis of correlations between APLN and PLOD2 expression in osteosarcoma tissue.

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  Figure 3. Apelin promotes osteosarcoma cell migration by increasing PLOD2 expression. (A-C) Osteosarcoma cells were treated with different concentrations of APLN (0, 0.3, 1, or 3 ng/mL) for 24 h. The Transwell assay, qPCR and Western blot examined in vitro migratory activity, and mRNA and protein expression of PLOD2, respectively. (D) Cells were pretreated with a PLOD2 inhibitor (minoxidil, 0.5 mM) or transfected with PLOD2 siRNA, then stimulated with APLN (3 ng/mL) for 24 h. The Transwell assay quantified in vitro migration. (E) Osteosarcoma cells were transfected with PLOD2 siRNA for 24 h and levels of PLOD2 mRNA were assessed by qPCR. *p < 0.05 compared with controls; # p < 0.05 compared with the APLN-treated group.

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  Figure 4. The MST1/MOB1 pathway is involved in APLN-induced osteosarcoma cell migration and increases PLOD2 expression. (A-E) Osteosarcoma cells were incubated with a MST1 inhibitor (XMU MP1, 3 µM) for 30 min, or transfected with MST1 or MOB1 siRNAs for 24 h, then treated with APLN (3 ng/ml) for 24 h. The Transwell, qPCR and Western blot assays examined in vitro migratory activity and levels of PLOD2 expression. (F-G) 143B and MG-63 cell lines were transfected with MST1 or MOB1 siRNAs for 24 h, then MST1 and MOB1 expression was examined by Western blot. (H) The schema illustrates the involvement of MST1 and MOB1 in APLN-induced PLOD2 expression and the migration of osteosarcoma cells. * p < 0.05 compared with controls; # p < 0.05 compared with the APLN-treated group.

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  Figure 5. The YAP pathway is related to APLN-induced osteosarcoma cell migration and increases PLOD2 expression. (A-D) Osteosarcoma cell lines were treated with YAP inhibitors (verteporfin 0.5 µM or (R)-PFI 2 hydrochloride 10 µM) for 30 min or transfected with a YAP siRNA for 24 h, then stimulated with APLN (3 ng/mL) for 24 h. The Transwell assay, qPCR and Western blot determined in vitro migration, PLOD2 mRNA and protein expression, respectively. (E) 143B and MG-63 cell lines were transfected with a YAP siRNA for 24 h, then YAP expression was examined by Western blot. (F) 143B cells were treated with APLN (3 ng/mL) for the indicated times, and phosphorylation of YAP was examined by Western blot. (G) The immunoprecipitation assay determined the interaction between YAP and 14-3-3 in the 143B cell line treated with APLN (3 ng/mL) for the indicated times. (H) ChIP and qPCR were performed to verify the regulatory interaction of YAP and the PLOD2 promoter. (I) The schema illustrates how APLN-induced YAP nuclear localization promoted PLOD2 expression and increased osteosarcoma cell migration. Data are represented as means ± SD. * p < 0.05 compared with controls; # p < 0.05 compared with the…

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  Figure 6. APLN increases PLOD2 expression, which promotes osteosarcoma cell migration by downregulating miR-1303 expression. (A) An analysis of 6 miRNA prediction databases pedicted 5 miRNAs that bind with PLOD2 (B) The 143B cell line was treated with APLN (3 ng/mL) and miRNAs were screened using a qPCR assay. (C) 143B and MG-63 cell lines were treated with different concentration of APLN (1, 0.3, 1, 3 ng/mL) for 24 h and miR-1303 expression was examined by qPCR. (D-E) Cells were transfected with miR-1303 mimic or control mimic for 24 hours, the treated with APLN (3 ng/mL) for 24 h. Transwell assay and Western blot examined the in vitro migration and PLOD2 protein expression. (F) The wild-type and mutant-PLOD2 3'-UTRs contained the miR-1303 binding site. (G) 143B cells were transfected with the WT-PLOD2 3'-UTR plasmid or MUT-PLOD2 3'-UTR plasmid for 24 h, then stimulated with APLN (3 ng/mL) for 24 h, and relative luciferase activity was measured. (H) Levels of miR-1303 expression in normal bone tissue and human osteosarcoma tissue were examined by qPCR. * p < 0.05 compared with controls; # p < 0.05 compared with the APLN-treated group.

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  Figure 7. APLN enhances osteosarcoma cell migration and increases PLOD2 expression by upregulating circ_0000004 sponging of miR-1303. (A) circRNAs predicted by the Circular RNA Interactome to act as sponges of miR-1303. (B) The 143B cells were treated with APLN (3 ng/mL) and circRNAs were screened by qPCR. (C) 143B cells were transfected with 3 siRNAs of hsa_circ_0000004 for 24 h and hsa_circ_0000004 expression was examined by qPCR. (D-F) Osteosarcoma cells were transfected with circ_0000004 siRNA 1 for 24 h, then treated with APLN (3 ng/mL) for 24 h. The Transwell assay and Western blot examined in vitro cell migration, and PLOD2 mRNA and protein expression. (G) Schematic presents the hsa_circ_0000004 sequence containing the miR-1303 binding site. (H) 143B cell lines were transfected with WT-hsa_circ_0000004 plasmid or MUT-hsa_circ_0000004 plasmid, then transfected with control mimic or miR-1303 mimic for 24 h and relative luciferase activity was measured. (I) hsa_circ_0000004 was pulled down with biotin-labeled miR-1303 by the RNA pull-down assay. (J) Levels of miR-1303 expression in normal bone tissue and human osteosarcoma tissue were examined by qPCR. * p < 0.05 compared with controls.

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  Figure 8. Knockdown of APLN suppresses lung metastasis. 143B/Luc cells stably expressing control-shRNA or APLN-shRNA were established. (A-B) APLN and PLOD2 protein expression was determined by Western blot. (C) Migration of 143B/Luc and 143B/shAPLN-Luc cells was examined by the Transwell assay. (D) 143B/Luc or 143B/shAPLN-Luc cells were injected into the lateral tail veins of Balb/c nude mice, and lung metastasis was monitored by bioluminescence imaging. (E) Quantification of in vitro bioluminescence imaging. (F-G) At 9 weeks after injection, the mice were sacrificed and the lung tissues were excised, photographed, and quantified for 143B/Luc and 143B/shAPLN-Luc expression. (H) IHC staining of APLN and PLOD2 expression in lung tumors. L, lung tissue; T, tumor. (I-J) Quantification of APLN and PLOD2 expression in IHC images. * p < 0.05 compared with 143B/Luc-control cells.

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  Figure 9. Schema illustrates the involvement of APLN in osteosarcoma metastasis. APLN facilitates PLOD2-dependent migratory activities of osteosarcoma cells through the MST1, MOB1 and YAP signaling cascades and the miR-1303/hsa_circ_0000004 axis.