Stable MAGEC1/CT7 Inhibition by Short Hairpin RNA (shRNA) Does Not Affect Tumor Cell Proliferation and Invasive Potential in Sko-007 Multiple Myeloma Cell Line.

Abstract 4888 Introduction MAGEC1/CT7 gene encodes for a cancer testis (CT) antigen frequently expressed in multiple myeloma (MM) and may be a potential target for immunotherapy in this still incurable disease. This CT gene expression is restricted to malignant plasma cells and may be related with tumor progression, since it seems to play a role as a “gatekeeper” gene for other CT antigens and can be associated with a more aggressive phenotype. However, the exact function of this protein for tumor biology is not yet understood. Objective The aim of this study is to elucidate the role of MAGEC1/CT7 in the control of cellular proliferation, DNA synthesis and invasive potential of Sko-007 myeloma cell line. Material and Methods We used a short hairpin RNA (shRNA) specific for MAGEC1/CT7 gene that was previously inserted in the pRS (pRETROSUPER) retroviral vector. The pRS-shRNA- MAGEC1/CT7 construct was co-transfected with pCL-amphotropic packing vector in 293T cells to produce virus particles. Sko-007 myeloma cell line was transduced and selected for stable expression of shRNA- MAGEC1/CT7. Expression of MAGEC1/CT7 was analyzed by Western Blot and Real Time PCR (RQ-PCR). Functional studies included cell proliferation, 3 H thymidine incorporation and invasion potential, using matrigel-coated transwell filters. Results Sko-007 was chosen to perform the initial functional studies since the basal MAGEC1/CT7 gene expression level was higher in this cell line when compared with the other myeloma cell lines (U266 and SK-MM-2). Sko-007wt (wild type) cell was divided into three derivatives, Sko-007ev (empty vector), Sko-007sh MAGEC1/CT7 Δ (antisense strand deleted – GC bases) and Sko-007sh MAGEC1/CT7. MAGEC1/CT7 expression was approximately five times down regulated in Sko-007sh MAGEC1/CT7 when compared with Sko-007wt, Sko-007ev, and Sko-007sh MAGEC1/CT7 Δ cells by RQ-PCR. Sko-007sh MAGE1/CT7 had an 80% decrease in MAGEC1/CT7 protein expression when compared with Sko-007wt, Sko-007ev and Sko-007sh MAGEC1/CT7 Δ by Western Blot. Our preliminary functional assays did not show any change in cell proliferation and DNA synthesis when Sko-007sh MAGEC1/CT7 was compared with Sko-007wt, Sko-007ev and Sko-007sh MAGEC1/CT7 Δ. Indeed, Sko-007 cell line has invasive potential, but no change was observed in Sko-007sh MAGEC1/CT7 or other cells (Sko-007wt, Sko-007ev and Sko-007sh MAGEC1/CT7 Δ). Conclusions Expression of MAGEC1/CT7 mRNA could be inhibited by the shRNA strategy, but the concomitant reduction of its protein level did not seem to have impact in proliferation, DNA synthesis or in invasive potential of Sko-007 MM cell line (Supported CNPq and LICR Sao Paulo Branch). Disclosures No relevant conflicts of interest to declare.