Knockdown of CD44 inhibits proliferation, migration and invasion of osteosarcoma cells accompanied by downregulation of cathepsin S

Abstract

Background: Osteosarcoma (OS) is a malignant bone tumour of mesenchymal origin. These tumours are characterised by rich vascularisation, therefore promoting rapid proliferation and facilitating metastasis. CD44 has been reported to be involved in OS, but its role and molecular mechanisms in the pathogenesis of the disease are not fully determined.

Methods: In this study, we investigated the antitumor effect of CD44 on the development of OS and further explored the molecular mechanisms. The expression of CD44, cathepsin S and MMP-9 was detected by Western blot (WB) and reverse transcription-polymerase chain reaction (RT-qPCR) in different cell lines (MG63, U2OS OS and hFOB 1.19). To elucidate the role of CD44 in OS, MG63 and U2OS cells were treated with small interference RNA (siRNA) to knock down CD44, and the knockdown efficiency was validated with GFP and RT-qPCR. Furthermore, cell proliferation was assayed using Cell Counting Kit‑8 (CCK-8) and colony formation assays, and cell migration and invasion were assayed by transwell and wound-healing assays.

Results: We found that CD44 expression in the MG63 and U2OS OS cell lines was markedly increased compared to that of the human osteoblast hFOB 1.19 cell line. Knockdown of CD44 inhibited proliferation, migration and invasion of MG63 and U2OS cells. Cathepsin S expression in the MG63 and U2OS OS cell lines was increased compared to that of the human osteoblast hFOB 1.19 cell line. When CD44 was knocked down, its expression level went down.

Conclusion: Taken together, our data reinforced the evidence that CD44 knockdown inhibited cell proliferation, migration and invasion of OS cells accompanied by altered expression of cathepsin S. These findings offer new clues for OS development and progression, suggesting CD44 as a potential therapeutic target for OS.

Keywords: CD44; Cathepsin S; Invasion; Migration; Osteosarcoma; Proliferation.

Fig. 1

CD44 and cathepsin S are upregulated in OS cell lines. A, B CD44 protein levels in MG63, U2OS, and hFOB 1.19 cell lines. **P < 0.01 versus hFOB group. C CD44 mRNA levels in MG63, U2OS, and hFOB 1.19 cell lines. **P < 0.01 versus hFOB group. D Cathepsin S mRNA levels in MG63, U2OS, and hFOB 1.19 cell lines. **P < 0.01 versus hFOB group

Fig. 2

CD44 knockdown in MG63 and U2OS cells in vitro. A Transfection efficiency of MG‑63 and U2OS cells was assessed by fluorescence microscopy. GFP, green fluorescent protein. Magnification, × 200. B Reverse transcription‑quantitative PCR analysis was used to assess the mRNA expression levels of CD44 in MG‑63 and U2OS cells after transfection for 24 h. **P < 0.01 versus si-NC group. C Western blot was used to assess CD44 expression levels in MG‑63 and U2OS cells 48 h after transfection

Fig. 3

CD44 knockdown inhibited the proliferation of MG63 and U2OS cells. A CCK8 assay demonstrated that silencing of CD44 inhibited the cell proliferation capability on the indicated time points after transfection with CD44 siRNA (si-CD44). *P < 0.05, **P < 0.01 versus si-NC group (the significant differences between the si-CD44-1, si-CD44-2 group and the si-NC group were consistent, with a common label). B Colony formation in MG-63 and U2OS cells. All data are presented as the mean ± SD of n = 3 experiments

Fig. 4

CD44 knockdown inhibited the migration and invasion of MG63 and U2OS cells. A A wound-healing assay was performed to detect the migration of MG-63 and U2OS cells. B Transwell assay was performed to detect migration and invasion of MG-63 and U2OS cells. Magnification, × 200. C Expression levels of migration and invasion-related proteins (MMP-9) were detected by Western blotting

Fig. 5

CD44 knockdown downregulated the expression of cathepsin S in MG63 and U2OS cells. A mRNA expression levels of CD44, cathepsin S, and β-actin were detected by RT-qPCR. B Protein expression levels of CD44, cathepsin S, and β-actin were detected by Western blotting. **P < 0.01 versus control group