NOB1 is essential for the survival of RKO colorectal cancer cells
NOB1 is essential for the survival of RKO colorectal cancer cells
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NOB1 对于 RKO 结直肠癌细胞的存活至关重要
DOI：
10.3748/wjg.v21.i3.868
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发表时间：
2015-01-21
影响因子：
4.3
通讯作者：
Liu, Ting
中科院分区：
文献类型：
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作者：
He, Xiao-Wen;Feng, Tao;Liu, Ting
目的：为了确定NOB1，酵母细胞存活的调节剂，在人结直肠癌cells. METHODS：慢病毒介导的小干扰RNA（siRNA）被用来抑制NOB1在RKO人结直肠癌细胞在体外和在体内的小鼠异种移植模型的表达。使用蛋白质印迹和定量逆转录聚合酶链反应（qRT-PCR）测定体外和体内敲低功效。qRT-PCR也用于分析NOB1敲低后的下游信号。使用细胞生长和集落形成测定来确定NOB1抑制对RKO增殖及其形成集落的能力的影响。通过末端脱氧转移酶介导的dUTP缺口末端标记（TUNEL）和膜联蛋白V染色评价的核酸内切酶活性用于确定NOB1抑制前后是否存在凋亡性细胞死亡。细胞周期分析用于确定NOB 1抑制对RKO细胞周期的影响。结果：病毒介导的siRNA抑制NOB 1后，RKO细胞中NOB 1的mRNA和蛋白表达均下调，在体内外实验系统中NOB 1的表达均受到抑制，在体内外实验系统中NOB 1的表达均受到抑制，在体内外实验系统中NOB 1的表达均受到抑制。（3）通过显著诱导细胞凋亡抑制细胞生长，而不改变细胞周期分布;和（4）与对照NOB1-siRNA组相比，NOB1-siRNA组中异种移植肿瘤的平均重量和体积显著降低（P = 0.001，P <0.05）。在NOB1-siRNA组的肿瘤内检测到的凋亡显著多于对照组。微阵列分析检测到2336个可能受NOB1调控的基因。这些基因中的大多数与WNT、细胞增殖、细胞凋亡、成纤维细胞生长因子和血管生成信号通路相关，其中BAX和WNT通过qRT-PCR验证。其中，代表963个独特基因的1451个探针上调，代表1373个独特基因的2308个探针下调。结论：慢病毒介导的RNA干扰沉默NOB 1基因可以通过诱导人结直肠癌细胞凋亡来抑制肿瘤生长。
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.