Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jan 21, 2015; 21(3): 868-877
Published online Jan 21, 2015. doi: 10.3748/wjg.v21.i3.868
NOB1 is essential for the survival of RKO colorectal cancer cells
Xiao-Wen He, Tao Feng, Qiao-Ling Yin, Yuan-Wei Jian, Ting Liu
Xiao-Wen He, Tao Feng, Qiao-Ling Yin, Department of General Surgery, Shaoxing People’s Hospital, Shaoxing Hospital of Zheijiang University, Shaoxing 312000, Zhejiang Province, China
Yuan-Wei Jian, Ting Liu, Department of Gastroenterology, Xiangya Hospital of Central South University, Changsha 410008, Hunan Province, China
Author contributions: He XW and Feng T developed the concept and design of the research; Jian YW and Liu T analyzed the data; and Yin QL wrote the manuscript.
Supported by National Natural Science Foundation of China, No. 81272735; Class A Medical and Health Technology Program Project from Zhejiang Province, No. 2010KY178; and the Science and Technology Department of Hunan Province, No. 2010Ck3013.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Ting Liu, PhD, Department of Gastroenterology, Xiangya Hospital of Central South University, 87 Xiangya Road, Changsha 410008, Hunan Province, China. liuting818@126.com
Telephone: +86-731-89753023 Fax: +86-731-89753723
Received: March 1, 2014
Peer-review started: March 5, 2014
First decision: March 27, 2014
Revised: June 13, 2014
Accepted: July 22, 2014
Article in press: July 22, 2014
Published online: January 21, 2015
Processing time: 325 Days and 1.9 Hours
Abstract

AIM: To determine the role of NOB1, a regulator of cell survival in yeast, in human colorectal cancer cells.

METHODS: 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.

RESULTS: 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.

CONCLUSION: NOB1 gene silencing by lentivirus-mediated RNA interference can inhibit tumor growth by inducing apoptosis of cancerous human colorectal cells.

Keywords: NOB1; Small RNA interference; Apoptosis; Colorectal cancer; BAX; WNT

Core tip: NOB1, a critically important regulator in yeast, is also required for regulation of cell growth and survival in RKO human colorectal cancer cells. NOB1 knockdown promotes cell apoptosis in both in vitro and in vivo model systems. The gene expression profile suggests the importance of the WNT pathway, cell proliferation, apoptosis, the fibroblast growth factor, and angiogenesis signaling pathways in the function of NOB1.