Xue Yuyang, Chen Cong, Xu Wei, Xu Hao, Zheng Junnian, Gu Yuming
Department of Interventional Radiology, The No. 1 Hospital of Xuzhou, Xuzhou, Jiangsu 221002, P.R. China.
State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, P.R. China.
Oncol Lett. 2018 May;15(5):7693-7701. doi: 10.3892/ol.2018.8292. Epub 2018 Mar 19.
The aim of the present study was to investigate the functional role of Frizzled-7 (FZD7) in the apoptosis of hepatoma cells. HepG2 and Huh-7 hepatocellular carcinoma (HCC) cell lines with FZD7 expression were selected for use in the present study. The small hairpin RNA (shRNA) eukaryotic expression vector specific to FZD7 was constructed using gene recombination, and was then transfected into HepG2 and Huh-7 hepatoma cell lines using Lipofectamine 2000 to assess whether the downregulation of FZD7 could affect the proliferative ability of these cells. The results demonstrated that the downregulation of FZD7 expression significantly inhibited the proliferative ability of both cell types through the induction of cell apoptosis, as evidenced using Cell Counting kit-8 assays and flow cytometry. Furthermore, the western blotting results demonstrated that silencing of FZD7 increased the activities of caspase-3 and caspase-9. These increases were also associated with the downregulation of the inhibitor of the apoptosis protein family. Additionally, it was revealed that silencing of FZD7 expression caused the downregulation of apoptosis regulator Bcl-2 and Bcl-XL in HepG2, and Huh-7 cells, as determined through western blot analysis and reverse transcription-quantitative polymerase chain reaction. In the following work, ELISA and western blot analysis revealed that the knockdown of FZD7 inhibited the expression and activities of nuclear factor-κB (NF-κB) p65. Furthermore, it was demonstrated that the expression levels of phosphylated-Smad2/3 were markedly upregulated in sh-FZD7-transfected HepG2 and Huh-7 cells. Then, shRNA eukaryotic expression vector specific to transforming growth factor (TGF)-β receptor II was transfected into both cell lines to investigate the association between the TGF-β/Smad signaling pathway and NF-κB p65. Notably, when the TGF-β/Smad signaling pathway was inhibited, no significant differences in the cell apoptosis rate and NF-κB expression levels were identified in HCC cells. Overall, the results of the present study suggest that the shRNA-mediated knockdown of FZD7 induces apoptosis of hepatoma cell lines through the inhibition of NF-κB. In addition, the TGF-β/Smad signaling pathway appeared to partially participate in the underlying molecular mechanism of FZD7 in HCC.
本研究的目的是探讨卷曲蛋白7（FZD7）在肝癌细胞凋亡中的功能作用。本研究选用了具有FZD7表达的HepG2和Huh-7肝癌（HCC）细胞系。利用基因重组构建了针对FZD7的小发夹RNA（shRNA）真核表达载体，然后使用Lipofectamine 2000将其转染到HepG2和Huh-7肝癌细胞系中，以评估FZD7的下调是否会影响这些细胞的增殖能力。结果表明，FZD7表达的下调通过诱导细胞凋亡显著抑制了两种细胞类型的增殖能力，这通过细胞计数试剂盒-8检测和流式细胞术得到证实。此外，蛋白质印迹结果表明，FZD7的沉默增加了caspase-3和caspase-9的活性。这些增加也与凋亡蛋白家族抑制剂的下调有关。此外，通过蛋白质印迹分析和逆转录-定量聚合酶链反应确定，FZD7表达的沉默导致HepG2和Huh-7细胞中凋亡调节因子Bcl-2和Bcl-XL的下调。在接下来的工作中，酶联免疫吸附测定和蛋白质印迹分析表明，FZD7的敲低抑制了核因子-κB（NF-κB）p65的表达和活性。此外，结果表明，在转染了sh-FZD7的HepG2和Huh-7细胞中，磷酸化-Smad2/3的表达水平明显上调。然后，将针对转化生长因子（TGF）-β受体II的shRNA真核表达载体转染到这两种细胞系中，以研究TGF-β/Smad信号通路与NF-κB p65之间的关联。值得注意的是，当TGF-β/Smad信号通路受到抑制时，在肝癌细胞中未发现细胞凋亡率和NF-κB表达水平有显著差异。总体而言，本研究结果表明shRNA介导的FZD7敲低通过抑制NF-κB诱导肝癌细胞系凋亡。此外，TGF-β/Smad信号通路似乎部分参与了FZD7在肝癌中的潜在分子机制。
