Panoply™ Human SIGLEC15 Over-expressing Stable Cell Line

Siglec-15 is a novel tumor immune suppressor, but the regulatory mechanisms underlying its widespread upregulation in human cancers remain unclear. Here, researchers discovered that the long noncoding RNA (lncRNA) LINC00973 is highly expressed in Siglec-15-positive clear cell renal cell carcinoma (ccRCC) and that LINC00973 positively regulates Siglec-15 expression at the transcriptional level. This effect is clearly dependent on miR-7109-3p (hereafter miR-7109), providing evidence that Siglec-15 is a direct target of miR-7109. By recruiting miR-7109, LINC00973 acts as a competing endogenous RNA (ceRNA) to control Siglec-15 cell surface abundance, thereby contributing to cancer immunosuppression. Furthermore, expression of LINC00973 and miR-7109 in ccRCC antagonizes immune activation in co-cultured Jurkat cells. These studies highlight the importance of LINC00973-miR-7109-Siglec-15 in ccRCC immune evasion, which provides important opportunities for therapeutic intervention and diagnostic/prognostic studies.

Here, researchers established Siglec-15-overexpressing and knockdown cell lines in A704, A498, 769-p, and Caki-1 (Figures 1A-C). The immunosuppressive effect of Siglec-15 was confirmed by measuring IL-2 production in co-cultures with Jurkat ccRCC cells. Effector T cell specificity was established by transfecting Jurkat cells with a MART-I-specific 1D3 TCR, which potently recognized the MART-I peptide pre-loaded with ccRCC cells. In Siglec-15-overexpressing A704 and A498 cells, Jurkat cell IL-2 secretion was significantly reduced (Figure 1D). Conversely, Siglec-15 knockdown increased IL-2 production in the co-culture (Figure 1E). Notably, co-transfection of LINC00973 with a control miR significantly inhibited IL-2 production, a phenomenon readily reversed by co-transfection with a miR-7109 mimic (Figure 1F). In contrast, co-transfection of shLINC00973 and a control miR significantly stimulated IL-2 production in 769-p and Caki-1 cells, whereas addition of a miR-7109 inhibitor suppressed IL-2 production (Figure 1G). These findings clearly indicate that LINC00973-miR-7109 participates in immunosuppression in ccRCC by regulating Siglec-15, and this regulatory effect of Siglec-15 can be readily abolished by the addition of a Siglec-15 antibody to the co-culture system (Figure 1D, F, H).

Figure 1. LINC00973-miR-7109-Siglec-15 axis involved in immune activations in clear-cell renal cell carcinoma (ccRCC) cells. (Liu Y, et al., 2020)