Smad4 silencing in pancreatic cancer cell lines using stable RNA interference and gene expression profiles induced by transforming growth factor-β
Smad4 silencing in pancreatic cancer cell lines using stable RNA interference and gene expression profiles induced by transforming growth factor-β
复制标题
DOI：
10.1038/sj.onc.1208102
复制
发表时间：
2005-01-20
期刊：
影响因子：
8
通讯作者：
Omata, M
中科院分区：
文献类型：
--
作者：
Jazag, A;Ijichi, H;Omata, M
转化生长因子- β (tgf - β)-Smad信号通路抑制人上皮细胞的生长，发挥抑瘤作用。Smad4基因在50%的胰腺癌中发生突变或缺失。在本研究中，我们利用稳定RNA干扰（RNAi）方法成功建立了Smad4敲低（S4KD）的胰腺癌细胞系。在S4KD细胞系中，Smad4蛋白表达显著降低，tgf - β - smad信号明显抑制。用tgf - β刺激S4KD细胞和对照细胞，并使用包含3756个基因的cDNA微阵列进行分析，以筛选tgf - β下游的靶分子。微阵列分析显示，对照细胞中的187个S4KD基因和155个基因在tgf - β刺激后立即受到调节。对其中几个基因进行定量RT-PCR分析产生的结果证实了微阵列分析的结果。该阵列鉴定的S4KD和对照细胞中的大多数基因不同，这表明Smad4状态不同，信号通路也不同。在已确定的基因中，246个基因位于tgf - β的下游，此前没有报道过。参与细胞增殖、粘附和运动的基因被发现在S4KD和对照细胞中受到不同的调节。tgf - β诱导的细胞迁移在S4KD细胞中受到抑制，这可能与整合素β 7的不同调控有关。使用稳定的RNAi敲除特定基因似乎是分析内源性基因功能的一种很有前途的工具。
The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using the stable RNA interference (RNAi) method. Smad4 protein expression was reduced dramatically and TGF-beta-Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF-beta and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-beta. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF-beta stimulation. Quantitative RT-PCR analysis on several of these genes produced results that corroborated the outcome of the microarray analysis. Most of the genes in the S4KD and control cells identified by the array differed, which suggests signaling pathways that differ according to Smad4 status. Of the identified genes, 246 have not been reported previously as genes that lie downstream of TGF-beta. Genes that are involved in cell proliferation, adhesion, and motility were found to be regulated differentially with respect to S4KD and control cells. Cell migration induced by TGF-beta was inhibited in the S4KD cells, which might be associated with a different regulation of integrin beta7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.