Notch1 is overexpressed in human intrahepatic cholangiocarcinoma and is associated with its proliferation, invasiveness and sensitivity to 5-fluorouracil in vitro.
Notch1 is overexpressed in human intrahepatic cholangiocarcinoma and is associated with its proliferation, invasiveness and sensitivity to 5-fluorouracil in vitro.
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DOI：
10.3892/or.2014.3123
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发表时间：
2014-06
期刊：
影响因子：
4.2
通讯作者：
Wenrui Wu;Rui Zhang;Xiang-De Shi;Man-Sheng Zhu;Lei-bo Xu;H. Zeng;Chao Liu
中科院分区：
文献类型：
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作者：
Wenrui Wu;Rui Zhang;Xiang-De Shi;Man-Sheng Zhu;Lei-bo Xu;H. Zeng;Chao Liu
据报道，Notch信号通路在抑制肝细胞分化和允许肝内胆管形成中起关键作用。然而，对其在肝内胆管癌（ICC）中的意义知之甚少。本研究的目的是探讨Notch1表达在ICC组织和细胞中的影响。免疫组织化学检测ICC石蜡包埋切片（n=44） Notch1的表达。在培养的ICC细胞（RBE和hcc -9810）中，Notch1被RNA干扰（RNAi）敲低。分别采用细胞计数试剂盒-8 （CCK-8）、菌落形成试验、Transwell试验和流式细胞术检测细胞的增殖、侵袭性和对5-氟尿嘧啶（5-FU）的敏感性。采用实时荧光定量聚合酶链式反应（qRT-PCR）和免疫印迹法检测多药耐药（MDR）相关基因mdr1 - p -糖蛋白（ABCB - 1）、乳腺癌耐药蛋白（ABCG - 2）和多药耐药蛋白亚型1 （MRP - 1）的表达水平。与邻近肝组织相比，Notch1在ICC细胞膜和细胞质中过表达（35/44,79.5%），且在肿瘤大小≥5 cm （p=0.021）和HBs-Ag阳性（p=0.018）的病例中更为常见。通过沉默Notch1，可以抑制ICC细胞的增殖和侵袭性，显著提高5-FU的抑制率。5-FU在RBE细胞中的IC50值从148.74±0.72降至5.37±0.28µg/ml，在hcc -9810细胞中的IC50值分别从326.92±0.87降至42.60±0.35µg/ml。此外，与对照组相比，Notch1沉默明显增加了5-FU处理的凋亡细胞的百分比。Notch1敲低导致ABCB‑1和MRP‑1表达水平降低。因此，Notch可能在ICC的发展中起着重要的作用。在体外实验中，沉默Notch1可抑制ICC细胞的增殖和侵袭性，提高ICC细胞对5-FU的敏感性。
The Notch signaling pathway has been reported to play crucial roles in inhibiting hepatocyte differentiation and allowing formation of intrahepatic bile ducts. However, little is known about its significance in intrahepatic cholangiocarcinoma (ICC). The aim of the present study was to investigate the effects of Notch1 expression in ICC tissues and cells. The expression of Notch1 was examined in paraffin-embedded sections of ICC (n=44) by immunohistochemistry. Notch1 was knocked down by RNA interference (RNAi) in cultured ICC cells (RBE and HCCC-9810). The proliferation, invasiveness and sensitivity to 5-fluorouracil (5-FU) were detected by Cell Counting Kit-8 (CCK-8), colony formation assays, Transwell assays and flow cytometry, respectively. The expression levels of several multidrug resistance (MDR)-related genes, MDR1-P-glycoprotein (ABCB‑1), breast cancer resistance protein (ABCG‑2) and the multidrug resistance protein isoform 1 (MRP‑1), were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Notch1 was overexpressed in cell membranes and cytoplasm of ICC compared with the adjacent liver tissue (35/44, 79.5%) and this was more common in cases with tumor size≥5 cm (p=0.021) and HBs-Ag positive (p=0.018). By silencing Notch1, the proliferation and invasiveness of ICC cells were inhibited and the inhibition rate of 5-FU was markedly increased. In addition, IC50 values of 5-FU in RBE cells were decreased from 148.74±0.72 to 5.37±0.28 µg/ml and the corresponding values for HCCC-9810 cells were 326.92±0.87 to 42.60±0.35 µg/ml, respectively. Furthermore, Notch1 silencing clearly increased the percentage of apoptotic cells treated by 5-FU compared with the control. Notch1 knockdown led to diminished expression levels of ABCB‑1 and MRP‑1. Therefore, Notch may play important roles in the development of ICC. Silencing Notch1 can inhibit the proliferation and invasiveness of ICC cells and increase their sensitivity to 5-FU in vitro.