Panoply™ Human SIGLEC15 Knockdown Stable Cell Line

Pulmonary metastasis is a major cause of poor prognosis in osteosarcoma. Sialic acid-binding immunoglobulin lectin 15 (Siglec-15) has been shown to be closely associated with pulmonary metastasis in osteosarcoma patients. However, the effect of Siglec-15 on autophagy in osteosarcoma remains unclear, and the role and mechanism of Siglec-15-related autophagy in lung metastasis remain unclear. Here, researchers examined the expression of Siglec-15 and Beclin-1 in osteosarcoma lung metastases and in patients with lung metastases. Immunoprecipitation experiments revealed that Siglec-15 directly interacts with the important autophagy protein Beclin-1. Furthermore, Siglec-15 depletion significantly inhibited autophagy and reduced Beclin-1/ATG14 expression. The decreased invasion and migration ability caused by Siglec-15 silencing could be reversed by Beclin-1 overexpression. Furthermore, autophagy can promote epithelial-mesenchymal transition (EMT) and influence cytoskeletal rearrangements, as demonstrated by Beclin-1 overexpression or silencing.

Transwell assays and wound healing assays indicate that Siglec-15 depletion significantly decreased the migration and invasive capacity of KHOS and U2OS cells (Figure 1b). Since epithelial-mesenchymal transition (EMT) is also a key step in tumor metastasis, the researchers examined the effects of Siglec-15 knockdown on EMT by Western blotting. Compared to the control group, the Siglec-15 knockdown cell showed increased expression of the epithelial marker E-cadherin (Figure 1a). In the Siglec-15 knockdown cell, expression of N-cadherin and vimentin (mesenchymal markers) was decreased (Figure 1a). Matrix metalloproteinase-9 (MMP-9) was also decreased in the Siglec-15-deficient group (Figure 1a). The cytoskeleton of KHOS and U2OS cells was visualized using confocal microscopy. Results showed that distinct lamellipodia formed in the submembranous region of control cells, but the opposite phenomenon was observed in the Siglec-15 knockdown cell, with both KHOS and U2OS cells displaying rearranged cytoskeletons and evenly distributed F-actin (Figure 1c). Because RhoA regulates the phosphorylation of LIMK and cofilin, changes in RhoA expression following Siglec-15 knockdown were examined. GTPase assays revealed that RhoA activity was reduced in the Siglec-15 knockdown group compared to the control group (Figure 1c). These results indicate that Siglec-15 expression can significantly affect cytoskeletal rearrangements in osteosarcoma cells.

Figure 1. Siglec-15 knockdown suppresses the migration and invasion of osteosarcoma cells in vitro. (Zheng B, et al., 2022)