Next Generation Sequencing Facilitates Quantitative Analysis of colorectal cancer cells transfected with NC siRNA or RPL9 siRNA. Homo sapiens

Ribosomal protein L9 (RPL9), a component of the 60S subunit, is up-regulated in human colorectal cancer (CRC). We thus hypothesized if targeting of RPL9 with small interfering (si) RNA could inhibit CRC progression. We also investigated molecular mechanism to mediate CRC cell death caused by RPL9 silencing. HCT116 and HT29 human CRC cells were transfected with RPL9 siRNA and tested for growth inhibition and apoptotic induction using MTS, FACS and microscopic analysis. To obtain insights into the molecular changes in response to RPL9 knockdown, global changes in gene expression were examined using RNA sequencing. As a result, RPL9 silencing caused inhibition of CRC cell growth through induction of apoptotic cell death. RNA sequencing revealed that RPL9-specific knockdown led to dysregulation of 918 genes in HCT116 and 3178 genes in HT29 cells. Among those, 296 genes showed the same directional regulation (128 up- and 168 down-regulated genes), including up-regulation of tumor suppressors such as KLF6 and ATF3, and were considered as a common RPL9 knockdown signature. Western blotting proved that down-regulation of RPL9 was accompanied with the decrease in the levels of PARP-1 and pro-caspase 3, accelerating apoptotic signaling. Of importance, targeting RPL9 significantly inhibited CRC growth in a murine xenograft model. In conclusion, these results suggest that inhibition of RPL9 expression could be an attractive option for molecular targeted therapy of colorectal cancer. Overall design: Examination of 2 different siRNA transfected cells in 2 cell lines.