Zinc finger Asp-His-His-Cys palmitoyl -acyltransferase 19 accelerates tumor progression through wnt/β-catenin pathway and is upregulated by miR-940 in osteosarcoma

Abstract

Osteosarcoma (OS) is the most frequent malignant primary bone tumor in children and young adults. Zinc finger Asp-His-His-Cys palmitoyl-acyltransferase 19 (ZDHHC19) is a key enzyme in protein palmitoylation and plays crucial roles in tumor progression. However, its expression profile and biological function in OS have been unclear. In the present study, the expression level of ZDHHC19 in OS cell lines was determined by qRT-PCR and Western blot. The effect of ZDHHC19 in cell growth, invasion and migration was analyzed by CCK8, EDU, transwell, wound healing assay in vitro, and xenograft tumor model in vivo. In addition, bioinformatics analysis was used to explore the potential mechanism of ZDHHC19 in OS. Furthermore, the luciferase reporter assay was conducted to determine the direct binding between miR-940 and ZDHHC19. We discovered that ZDHHC19 was overexpressed in OS cells compared with the normal cells. The functional investigation demonstrated that ZDHHC19 silencing could inhibit proliferation, invasion and migration of OS in vitro and suppress tumorigenicity and lung metastasis in a xenograft model in vivo. Mechanistically, we identified that ZDHHC19 was a direct target of miR-940 and forced ZDHHC19 expressions partially rescue the suppression of proliferation, migration and invasion induced by miR-940. Moreover, bioinformatics analysis combined with validation experiments revealed that activating wnt/β-catenin pathway contributed to the pro-oncogenic effect induced by ZDHHC19. Furthermore, rescue experiments further verified that miR-940/ZDHHC19 axis regulated wnt/β-catenin pathway. Overall, these findings indicated that miR-940/ZDHHC19 axis played a significant role in OS progression and might be considered as a novel target for OS treatment.Abbreviations: OS, osteosarcoma; miRNAs, microRNAs; 3'-UTR, 3'- untranslated region; TARGET, Therapeutically Applicable Research To Generate Effective Treatments; qRT-PCR, quantitative real-time PCR; IHC, Immunohistochemistry; GSVA, Gene Set Variation Analysis; GSEA, Gene Set Enrichment Analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Keywords: ZDHHC19; miR-940; osteosarcoma; wnt/β-catenin pathway.

Figure 1.

ZDHHC19 was upregulated in OS and predicted poor prognosis. The expression status of ZDHHC19 in OS cell lines (U2OS, MG63 and 143B) and the normal human bone cells (HFOB and HOBC) at mRNA (a) and protein levels (b) were determined by RT-qPCR and Western blot, respectively. Kaplan-Meier survival curves of overall survival (c) and disease-free survival (d) between high ZDHHC19 expression group and low ZDHHC19 expression group in TARGET database. *P < 0.05; **P < 0.01.

Figure 2.

ZDHHC19 knockdown inhibited cell proliferation, invasion and migration in OS cells. (a, b) qRT-PCR and Western blot analysis of OS cells transfected with sh-ZDHHC19 or sh-NC. The ability of proliferation in OS cells transfected with sh-ZDHHC19 or sh-NC by CCK8 (c), EDU (d) and colony formation (e). (f) Analysis the effect of ZDHHC19 on the migration of 143B and MG63 cells by wound-healing assay. The ability of invasion and migration in OS cells transfected with sh-ZDHHC19 or sh-NC by transwell migration (g) and transwell invasion assay (h). (i) Expressions of migration-related proteins (MMP2, MMP7 and MMP9) in sh-ZDHHC19-transfected OS cells were detected by Western blot analysis. All data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05; **P < 0.01.

Figure 3.

ZDHHC19 knockdown suppress tumor growth in vivo. (a) The tumor representative image from mice injected with OS cells transfected with sh-ZDHHC19 or sh-NC. (b) Relative photon flux analysis between sh-ZDHHC19 group and sh-NC group. (c) Tumor weight analysis between sh-ZDHHC19 group and sh-NC group. (d) Tumor volume analysis between sh-ZDHHC19 group and sh-NC group. (e) The representative staining images and expression levels analysis of ZDHHC19 and Ki-67 in tumors from sh-ZDHHC19 group and sh-NC group. (f) The representative images and quantitative analysis of lung metastasis between sh-ZDHHC19 and sh-NC group. All data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05; **P < 0.01.

Figure 4.

ZDHHC19 promoted OS progression through wnt/β-catenin signaling pathway. (a) GSVA analysis showed the enrich pathways between ZDHHC19 high expression group and low expression group. (b) GSEA analysis suggested the significant correlation between wnt/β-catenin signaling and ZDHHC19 expression. (c) KEGG analysis showed enriched pathways in TARGET-OS cohort. (d) The relative wnt/β-catenin signaling activity analysis between sh-ZDHHC19 group and sh-NC group. (f) Western blot analysis of wnt/β-catenin signaling related proteins between sh-ZDHHC19 group and sh-NC group. All data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05; **P < 0.01.

Figure 5.

miR-940 directly bound to the 3’-UTR of ZDHHC19 and reversely regulated ZDHHC19 expression. (a) Bioinformatics analysis showed the miR-940 potential binding site located on the 3’-UTR of ZDHHC19. (b) The relative luciferase analysis based on a dual – luciferase reporter assay. (c) The expression status of miR-940 in OS cell lines (U2OS, MG63 and 143B) and the normal human bone cells (HFOB and HOBC). (d) The mRNA expression level of ZDHHC19 in OS cells transfected with inhibitor NC or miR-940 inhibitor. (e) The mRNA expression level of ZDHHC19 in OS cells transfected with mimics NC or miR-940 mimics. (f) The protein expression level of ZDHHC19 in OS cells transfected with miR-940 inhibitor or miR-940 inhibitor. All data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05; **P < 0.01.

Figure 6.

ZDHHC19 overexpression could partly reverse the inhibitory effect of miR-940 in OS progression. (a) The protein expression level of ZDHHC19 in OS cells transfected with mimics NC, miR-940 mimics or miR-940 mimics & ZDHHC19 plasmid. (b, c) The ability of cell proliferation in OS cells transfected with mimics NC, miR-940 mimics or miR-940 mimics & ZDHHC19 plasmid by CCK8 (b) and colony formation (c). (D, E, F) The ability of migration in OS cells transfected with mimics NC, miR-940 mimics or miR-940 mimics & ZDHHC19 plasmid determined by wound healing assay (d), transwell migration (e) and transwell invasion assay (f). All data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05; **P < 0.01.

Figure 7.

miR-940/ZDHHC19 axis regulates wnt/β-catenin pathway. (a) Western blot analysis of wnt/β-catenin signaling related proteins in OS cells transfected with mimics NC, miR-940 mimics or miR-940 mimics & ZDHHC19 plasmid. All data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05; **P < 0.01. (b) Summary of the mechanism by which miR-940/ZDHHC19 axis regulates the malignant phenotype of OS.