NOB1 is essential for the survival of RKO colorectal cancer cells.

Abstract

To determine the role of NOB1, a regulator of cell survival in yeast, in human colorectal cancer cells. Lentivirus-mediated small interfering RNA (siRNA) was used to inhibit NOB1 expression in RKO human colorectal cancer cells in vitro and in vivo in a mouse xenograft model. The in vitro and in vivo knockdown efficacy was determined using both Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR was also used to analyze the downstream signals following NOB1 knockdown. Cell growth and colony formation assays were used to determine the effect of NOB1 inhibition on RKO proliferation and their ability to form colonies. Endonuclease activity, as evaluated by terminal deoxytransferase-mediated dUTP nick end labeling (TUNEL), and annexin V staining were used to determine the presence of apoptotic cell death prior to and following NOB1 inhibition. Cell cycle analysis was used to determine the effect of NOB1 inhibition on RKO cell cycle. A cDNA microarray was used to determine global differential gene expression following NOB1 knockdown. Virus-mediated siRNA inhibition of NOB1 resulted in (1) the down-regulation of NOB1 expression in RKO cells for both the mRNA and protein; (2) inhibition of NOB1 expression both in vitro and in vivo experimental systems; (3) cell growth inhibition via significant induction of cell apoptosis, without alteration of the cell cycle distribution; and (4) a significant decrease in the average weight and volume of xenograft tumors in the NOB1-siRNA group compared to the control scr-siRNA group (P = 0.001, P < 0.05). Significantly more apoptosis was detected within tumors in the NOB1-siRNA group than in the control group. Microarray analysis detected 2336 genes potentially regulated by NOB1. Most of these genes are associated with the WNT, cell proliferation, apoptosis, fibroblast growth factor, and angiogenesis signaling pathways, of which BAX and WNT were validated by qRT-PCR. Among them, 1451 probes, representing 963 unique genes, were upregulated; however, 2308 probes, representing 1373 unique genes, were downregulated. NOB1 gene silencing by lentivirus-mediated RNA interference can inhibit tumor growth by inducing apoptosis of cancerous human colorectal cells.