Lv Xiaoqun, Zhang Jinguo, Zhang Jun, Guan Wencai, Ren Weifang, Liu Yujuan, Xu Guoxiong
Department of Pharmacy, Jinshan Hospital, Fudan University, Shanghai, People's Republic of China.
Research Center for Clinical Medicine, Jinshan Hospital, Fudan University, Shanghai, People's Republic of China.
Onco Targets Ther. 2021 Jan 8;14:187-198. doi: 10.2147/OTT.S282367. eCollection 2021.
Nicotinamide phosphoribosyltransferase (NAMPT) and the transforming growth factor-β (TGF-β) signaling pathway play important roles in colorectal tumorigenesis and progress. However, the underlying regulatory mechanisms between NAMPT and TGF-β signaling in colorectal cancer (CRC) remain poorly understood.
Public data were extracted from the Oncomine database and the PrognoScan database to investigate the mRNA expression and the prognostic value of NAMPT, respectively, in CRC. Western blot tests were performed to detect Smad2, Smad3, p-Smad2, p-Smad3, Smad4 expression in CRC cells transfected with human NAMPT-siRNA or NAMPT-overexpressing plasmid. TGF-β1 concentrations in culture supernatants were assayed using ELISA kits. The effect of TGF-β1 on NAMPT expression was evaluated by quantitative real-time PCR and Western blot. The dual-luciferase reporter assay was employed to confirm the binding of miR-1-3p to NAMPT 3'-UTR. Subsequently, NAMPT levels in HCT116 cells transfected with the mimics and inhibitors of miR-1-3p were detected by quantitative real-time PCR and Western blot.
NAMPT was overexpressed in human CRC and was correlated with short overall survival. NAMPT increased the protein expression levels of components in the TGF-β signaling pathway including Smad2, Smad3, and Smad4. Moreover, NAMPT promoted TGF-β1 secretion. Intriguingly, the TGF-β1 treatment down-regulated NAMPT expression at mRNA and protein levels in CRC cells which were partly through the up-regulation of miR-1-3p that directly bound to the NAMPT 3'-UTR. These outcomes demonstrated that NAMPT was a downstream target of miR-1-3p and there was a negative association between NAMPT and miR-1-3p in CRC.
There is a negative feedback loop between NAMPT and the TGF-β signaling pathway in CRC cells, providing new insight into the mechanism underlying the regulatory pathways in CRC.
烟酰胺磷酸核糖转移酶（NAMPT）和转化生长因子-β（TGF-β）信号通路在结直肠癌的发生和发展中起重要作用。然而，结直肠癌（CRC）中NAMPT与TGF-β信号之间潜在的调控机制仍知之甚少。
从Oncomine数据库和PrognoScan数据库中提取公共数据，分别研究CRC中NAMPT的mRNA表达及预后价值。进行蛋白质免疫印迹试验，以检测转染人NAMPT-siRNA或NAMPT过表达质粒的CRC细胞中Smad2、Smad3、p-Smad2、p-Smad3、Smad4的表达。使用ELISA试剂盒检测培养上清液中的TGF-β1浓度。通过定量实时PCR和蛋白质免疫印迹评估TGF-β1对NAMPT表达的影响。采用双荧光素酶报告基因检测法确认miR-1-3p与NAMPT 3'-UTR的结合。随后，通过定量实时PCR和蛋白质免疫印迹检测转染miR-1-3p模拟物和抑制剂的HCT116细胞中的NAMPT水平。
NAMPT在人CRC中过表达，且与总生存期短相关。NAMPT增加了TGF-β信号通路中包括Smad2、Smad3和Smad4在内的组分的蛋白质表达水平。此外，NAMPT促进TGF-β1分泌。有趣的是，TGF-β1处理在mRNA和蛋白质水平下调了CRC细胞中NAMPT的表达，这部分是通过直接结合NAMPT 3'-UTR的miR-1-3p的上调实现的。这些结果表明NAMPT是miR-1-3p的下游靶点，且在CRC中NAMPT与miR-1-3p之间存在负相关。
CRC细胞中NAMPT与TGF-β信号通路之间存在负反馈环，为CRC调控通路的潜在机制提供了新的见解。
