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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE78195 | GEO | 2017/02/23

SECONDARY ACCESSION(S): PRJNA312903

REPOSITORIES: GEO

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Project description:CD24 is a potential oncogene reported to be overexpressed in a large variety of human malignancies. We have shown that CD24 is overexpressed in 90% of colorectal tumors at a fairly early stage in the multistep process of carcinogenesis. Anti-CD24 monoclonal antibodies (mAb) induce a significant growth inhibition in colorectal and pancreatic cancer cell lines that express the protein. This study is designed to investigate further the effects of CD24 down-regulation using mAb or small interfering RNA in vitro and in vivo. Western blot analysis showed that anti-CD24 mAb induced CD24 protein down-regulation through lysosomal degradation. mAb augmented growth inhibition in combination with five classic chemotherapies. Xenograft models in vivo showed that tumor growth was significantly reduced in mAb-treated mice. Similarly, stable growth inhibition of cancer cell lines was achieved by down-regulation of CD24 expression using short hairpin RNA (shRNA). The produced clones proliferated more slowly, reached lower saturation densities, and showed impaired motility. Most importantly, down-regulation of CD24 retarded tumorigenicity of human cancer cell lines in nude mice. Microarray analysis revealed a similar pattern of gene expression alterations when cells were subjected to anti-CD24 mAb or shRNA. Genes in the Ras pathway, mitogenactivated protein kinase, or BCL-2 family and others of oncogenic association were frequently down-regulated. As a putative new oncogene that is overexpressed in gastrointestinal malignancies early in the carcinogenesis process, CD24 is a potential target for early intervention in the prevention and treatment of cancer. The study compared gene expression profiles between human CRC cells HT29 before and after expression of 1 and 2 shRNA vectors directed at the human CD24 gene, GFP control gene, HT29 cells and Colo357, human pancreatic cancer cells, before and after the inhibition of the CD24 molecule using 72h treatment with anti-CD24 monoclonal antibodies.

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

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