Chen Sheng, Zhang Min, Xing Lili, Wang Yue, Xiao Yongtao, Wu Yeming
Department of Pediatric Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
Shanghai Institute for Pediatric Research, Shanghai, China.
PLoS One. 2015 Mar 26;10(3):e0121115. doi: 10.1371/journal.pone.0121115. eCollection 2015.
The aim of this study was to investigate the effects of hypoxia-inducible factor-1α (HIF-1α) on the proliferation, migration and invasion of neuroblastoma (NB) cells and the mechanisms involved. We here initially used the real-time polymerase chain reaction (real-time PCR), Western blotting and immunohistochemistry (IHC) to detect the expression of HIF-1α and components of the sonic hedgehog (SHH) signaling pathway in NB cells and human specimens. Subsequently, cell proliferation, migration and invasion were analyzed using the cell counting assay, wound healing assay and Transwell system in two types of human NB cell lines, SH-SY5Y and IMR32. In addition, the role of HIF-1α in NB cells growth was determined in a xenograft nude mouse model. We found that the level of HIF-1α was significantly upregulated during NB progression and was associated with the expression of two components of SHH signaling, SHH and GLI1. We next indicated that the proliferation, migration and invasiveness of SH-SY5Y and IMR32 cells were significantly inhibited by HIF-1α knockdown, which was mediated by small interfering RNAs (siRNAs) targeting against its mRNA. Furthermore, the growth of NB cells in vivo was also suppressed by HIF-1α inhibition. Finally, the pro-migration and proliferative effects of HIF-1α could be reversed by disrupting SHH signaling. In conclusion, our results demonstrated that upregulation of HIF-1α in NB promotes proliferation, migration and invasiveness via SHH signaling.
本研究旨在探讨缺氧诱导因子-1α（HIF-1α）对神经母细胞瘤（NB）细胞增殖、迁移和侵袭的影响及其相关机制。我们首先采用实时聚合酶链反应（real-time PCR）、蛋白质免疫印迹法和免疫组织化学（IHC）检测NB细胞和人体标本中HIF-1α及音猬因子（SHH）信号通路相关成分的表达。随后，运用细胞计数法、伤口愈合实验和Transwell系统对两种人NB细胞系SH-SY5Y和IMR32进行细胞增殖、迁移和侵袭分析。此外，通过异种移植裸鼠模型确定HIF-1α在NB细胞生长中的作用。我们发现，在NB进展过程中HIF-1α水平显著上调，且与SHH信号通路的两个成分SHH和GLI1的表达相关。接下来我们表明，针对HIF-1α mRNA的小干扰RNA（siRNAs）介导的HIF-1α敲低可显著抑制SH-SY5Y和IMR32细胞的增殖、迁移和侵袭能力。此外，HIF-1α抑制也可抑制NB细胞在体内的生长。最后，破坏SHH信号通路可逆转HIF-1α的促迁移和增殖作用。总之，我们的结果表明，NB中HIF-1α的上调通过SHH信号通路促进细胞增殖、迁移和侵袭。
