Siglec-15 as a New Perspective Therapy Target in Human Giant Cell Tumor of Bone

Abstract

The main features of a giant cell tumor of bone (GCTB) are frequent recurrence and aggressive osteolysis, which leads to a poor prognosis in patients. Although the treatment methods for a GCTB, such as scraping and resection, effectively inhibit the disease, the tendency toward malignant transformation remains. Therefore, it is important to identify new treatment methods for a GCTB. In this study, we first found high Siglec-15 expression in GCTB tissues, which was significantly associated with Campanacci staging and tumor recurrence. In Spearman's analysis, Siglec-15 expression was significantly correlated with Ki-67 levels in tumor tissues. In vitro, the mRNA and protein levels of Siglec-15 were high in GCTB stromal cells (Hs737. T), and Siglec-15 knockdown inhibited the biological characteristics of GCTB stromal cells. The RNA sequencing results enabled a prediction of the downstream genes by using the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and MCODE analyses, and the findings showed that CXCL8 was significantly regulated by Siglec-15 and might be a promising downstream target gene of Siglec-15. Therefore, Siglec-15 may be a potential immunotherapy target for a GCTB.

Keywords: CXCL8; GO analysis; KEGG analysis; Siglec-15; functional assays; giant cell tumor of bone.

Figure 1

Working model illustrates the association between Siglec-15 and clinical parameters, the influence of high Siglec-15 for GCTB stromal cells, as well as the prediction of downstream pathways of Siglec-15 in GCTB stromal cells.

Figure 2

Expression of Siglec-15 in human GCTB tissues and stromal cells. (A) Siglec-15 was expressed in the cytoplasm and membrane of GCTB tissues as shown by immunohistochemistry assay. (B) Expression of Siglec-5 in recurrent GCTB cases. Case 1: recurrence once, Case 2: recurrence twice, and Case 3: recurrence three times. (C) Siglec-15 protein expression in the cytoplasm and membrane of GCTB stromal cells as shown by immunofluorescence assay. Scale bars, 200 µm and 400 µm.

Figure 3

Knockdown of Siglec-15 expression in GCTB stromal cells. (A) The transfection efficiency was observed under a fluorescence microscope. (B) The silencing efficiency was determined by RT-PCR assay. (C,D) The silencing efficiency was determined by Western blotting. *** p < 0.01. Scale bars, 200 µm.

Figure 4

The effect of Siglec-15 silencing on the proliferation and colony formation of GCTB stromal cells. (A) The viability of Hs737.T cells transfected with siRNA were detected by the MTS method. (B,C) Clone experiments showed colony formation of Hs737. T cells. The numbers of Hs737. T cell clones in the siSiglec-15 group were significantly fewer than those in the NC group. * p < 0.05, *** p < 0.001.

Figure 5

The effect of Siglec-15 silencing on the migration and invasion of GCTB stromal cells. (A) Scratch assay showed the mobility of Hs737. T cells transfected with siSiglec-15 compared with the NC group. (B) The remaining area was obviously wider in the siSiglec-15 group than in the NC group at 48 h and 72 h. (C,D) Transwell migration assays showed decreased migration of Hs737 T cells transfected with siSiglec-15 at 48 h compared with the NC group. (E,F) Transwell invasion assays showed the decreased invasion ability of Hs737. T cells transfected with siSiglec-15 at 24 h and 48 h compared with the NC group. Scale bars, 200 µm, * p < 0.05, ** p < 0.01, *** p < 0.001. ns means no significance.

Figure 6

RNA-seq analysis in GCTB stromal cells with Siglec-15 knockdown. (A) The RNA intensities for samples were nearly the same after normalization by box plots. (B) The differentially expressed genes between the siRNA group and NC group are shown by a histogram. (C) The distribution of differentially expressed genes between the NC group and siRNA group is shown by a volcano diagram. (D) Highly consistent transcriptional changes were exhibited by heatmaps with three replicates of the NC group and siRNA group.

Figure 7

GO enrichment analysis and KEGG pathway analysis. (A,B) Biological processes, cellular components, and molecular functions were involved in the biological functions of GCTB stromal cells with Siglec-15 knockdown in GO enrichment analysis. (C) Twenty signaling pathways linked to cancer were shown by KEGG pathway analysis.

Figure 8

CXCL8 as a potential gene downstream of Siglec-15 in GCTB stromal cells. (A) The protein-protein interaction network by STRING. (B–D) There were 40 genes in module 1, 11 genes in module 2, and 11 genes in module 3 with the density of nodes. (E) CXCL8 was downregulated in Hs737. T cells with Siglec-15 knockdown by RT-PCR. *** p < 0.001.