Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells. MM is one of the most common cancers among hematological malignancies. It is characterized by the overproduction and accumulation of non-functional immunoglobulins or immunoglobulin chains in the plasma cells. The product of the TMED9 gene is the carrier protein expressed on the endoplasmic reticulum (ER) membrane. It plays a role in the transport of proteins. As we previously found the high RPKM value of TMED9 gene in MM patients with the transcriptome study, we hypothesized TMED9 may have a role in the pathogenesis of MM. Therefore, in this study, we aimed to investigate the function of the TMED9 gene in MM cell lines.

TMED9 gene expression was suppressed by siRNA with the electroporation method in U266 and RPMI8226 cell lines. TMED9 gene was overexpressed by using expression vector in both cell lines. After gene suppression and overexpression, the expression levels were determined by qPCR. The change in the protein level of TMED9 was shown by western blot analysis. Cells were stained with three fluorescent dyes which are Apopxin green,7-AAD, and CytoCalcein Violet 450. Cells were visualized under confocal microscopy to determine apoptosis in the control and treated groups. The number of apoptotic and viable cells were counted with the ImageJ program. The cell proliferation was determined with the MTT assay.

In siRNA-treated groups, the suppression of TMED9 was calculated by ΔΔCT method at 24 and 48 hours compared to control groups. Likewise, the overexpression level in the vector-treated cells was calculated by ΔΔCT method compared to control groups. Suppression and overexpression of TMED9 were demonstrated at the protein level by Western Blot. As a result of the suppression of siRNA-mediated gene expression, it was shown, the rate of apoptotic cells increased compared to the control group. MTT results revealed that cell proliferation decreased siRNA-treated group compared to the control group. On the other hand, TMED9 overexpression showed increased cell viability and proliferation compared to the control group.

In this study, we found that suppression of TMED9 gene expression decreased the viability of MM cells and triggered apoptosis. Our results are promising in MM, as TMED9 has been shown to be associated with poor prognosis in a different types of cancers. It is also the first functional study with the TMED9 gene in U266 and RPMI8266 cells.