To investigate the effect of Sialic acid-binding Ig-like lectin 15 (SIGLEC-15) gene on the proliferation, migration and invasion of triple negative breast cancer (TNBC) cells.

Three single-guide RNA (sgRNA) sequences specifically targeting human and murine SIGLEC-15 genes were designed. Stable SIGLEC-15 knockout MDA-MB-231 monoclonal cells (sg-control, sg-SIGLEC-15-1, sg-SIGLEC-15-2, and sg-SIGLEC-15-3 groups) and stable SIGLEC-15 knockout mouse TNBC 4T1 cells [normal control (NC group), KO1, KO2, and KO3 groups] were obtained using the CRISPR/Cas9 technology. The qRT-PCR and Western blot analysis were conducted to detect the mRNA and protein expression levels of SIGLEC-15 gene in eight monoclonal cell groups to evaluate the efficiency of SIGLEC-15 gene knockout. The effect of SIGLEC-15 gene knockout on the proliferation, migration and invasion abilities of MDA-MB-231 cells were assessed by EdU proliferation assay, wound healing assay, Transwell migration and invasion assay. The SIGLEC-15 gene-knocked-out 4T1 cells were injected subcutaneously into the adipose pads of female BALB/c mice, and the tumor volume was assessed every two days. After 24 days, the mice were euthanized, and the tumor volume and weight were measured. For quantitative data such as mRNA expression levels, protein expression levels, cell proliferation rates, cell scratch healing rates, number of migrating cells, number of invasive cells, volume and weight of xenograft tumors, comparisons between two groups were conducted using independent sample t-tests. Comparisons among multiple groups were performed using one-way ANOVA and repeated measures ANOVA, with pairwise comparisons among multiple groups conducted using Tukey’s method.

The mRNA expression levels of SIGLEC-15 in the NC, KO1, KO2 and KO3 groups were 1.00±0.06, 0.19±0.02, 0.15±0.01 and 0.16±0.02, respectively, indicating a significant difference between groups (F=405.807, P<0.001). The expression levels of SIGLEC-15 protein also indicated a significant difference among four groups(t=74.832, 103.210, 71.850, P<0.001). The mRNA expression levels of SIGLEC-15 in sg-control, sg-SIGLEC-15-1, sg-SIGLEC-15-2 and sg-SIGLEC-15-3 groups were 1.00±0.02, 0.20±0.02, 0.14±0.01 and 0.21±0.01, respectively, indicating a significant difference between groups (F=1 836.010, P<0.001). The expression levels of SIGLEC-15 protein also indicated a significant difference among four groups(t=23.810, 24.370, 23.960, P<0.001). There was no significant difference in the proliferation rate between sg-control and sg-SIGLEC-15-2 groups [(42.88±0.90)% vs (42.35±0.92)%, t=0.713, P=0.515]; however, significant differences were observed in the wound healing rate (62.33±0.72)% vs (31.89±0.29)%, t=68.150, P<0.001), the number of migrating cells (225.70±4.50 vs 104.70±5.69, t=28.880, P<0.001) and the number of invading cells (157.00±2.00 vs 57.33±4.16, t=37.380, P<0.001). There were significant differences in tumor volumes among the WT (injected with untreated 4T1 cells), NC and KO(KO2) groups of mice with breast cancer xenografts (group comparison, F=13 859.000, P<0.001; different time points, F=3444.021, P<0.001; interaction, F=351.700, P<0.001). The tumor volume in the KO group was significantly lower than that in the WT and NC groups (both P<0.001). The tumor weights were (750.00±21.99) mg, (758.60±18.70) mg and (443.80±14.52) mg in the WT, NC and KO groups, respectively, with a significant difference between groups (F=462.000, P<0.001).

In triple negative breast cancer cells, the SIGLEC-15 gene may be associated with the migration and invasion abilities of tumor cells, and its mechanism of action warrants further study.