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HIF-1? contributes to proliferation and invasiveness of neuroblastoma cells via SHH signaling.

ABSTRACT: The aim of this study was to investigate the effects of hypoxia-inducible factor-1? (HIF-1?) 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-1? 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-1? in NB cells growth was determined in a xenograft nude mouse model. We found that the level of HIF-1? 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-1? 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-1? inhibition. Finally, the pro-migration and proliferative effects of HIF-1? could be reversed by disrupting SHH signaling. In conclusion, our results demonstrated that upregulation of HIF-1? in NB promotes proliferation, migration and invasiveness via SHH signaling.

SUBMITTER: Chen S

PROVIDER: S-EPMC4374675 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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HIF-1α contributes to proliferation and invasiveness of neuroblastoma cells via SHH signaling.

Chen Sheng S Zhang Min M Xing Lili L Wang Yue Y Xiao Yongtao Y Wu Yeming Y

PloS one 20150326 3

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 ...[more]

PMID: 25811359

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Project description:Although expression of ribosomal protein L27 (RPL27) is upregulated in clinical colorectal cancer (CRC) tissue, to the best of our knowledge, the oncogenic role of RPL27 has not yet been defined. The present study aimed to investigate whether targeting RPL27 could alter CRC progression and determine whether RPL27 gains an extra‑ribosomal function during CRC development. Human CRC cell lines HCT116 and HT29 were transfected with RPL27‑specific small interfering RNA and proliferation was assessed in vitro and in vivo using proliferation assays, fluorescence‑activated cell sorting (FACS) and a xenograft mouse model. Furthermore, RNA sequencing, bioinformatic analysis and western blotting were conducted to explore the underlying mechanisms responsible for RPL27 silencing‑induced CRC phenotypical changes. Inhibiting RPL27 expression suppressed CRC cell proliferation and cell cycle progression and induced apoptotic cell death. Targeting RPL27 significantly inhibited growth of human CRC xenografts in nude mice. Notably, polo‑like kinase 1 (PLK1), which serves an important role in mitotic cell cycle progression and stemness, was downregulated in both HCT116 and HT29 cells following RPL27 silencing. RPL27 silencing reduced the levels of PLK1 protein and G2/M‑associated regulators such as phosphorylated cell division cycle 25C, CDK1 and cyclin B1. Silencing of RPL27 reduced the migration and invasion abilities and sphere‑forming capacity of the parental CRC cell population. In terms of phenotypical changes in cancer stem cells (CSCs), RPL27 silencing suppressed the sphere‑forming capacity of the isolated CD133+ CSC population, which was accompanied by decreased CD133 and PLK1 levels. Taken together, these findings indicated that RPL27 contributed to the promotion of CRC proliferation and stemness via PLK1 signaling and RPL27 may be a useful target in a next‑generation therapeutic strategy for both primary CRC treatment and metastasis prevention.

Project description:BackgroundAdipose tissue-derived stem cells (ASCs) have been recently isolated from human subcutaneous adipose tissue. ASCs may be useful in regenerative medicine as an alternative to bone marrow-derived stem cells. Changes in the oxygen concentration influence physiological activities, such as stem cell proliferation. However, the effects of the oxygen concentration on ASCs remain unclear. In the present study, the effects of hypoxia on ASC proliferation were examined.MethodsNormal human adipose tissue was collected from the lower abdomen, and ASCs were prepared with collagenase treatment. The ASCs were cultured in hypoxic (1%) or normoxic (20%) conditions. Cell proliferation was investigated in the presence or absence of inhibitors of various potentially important kinases. Hypoxia inducible factor (HIF)-1α expression and MAP kinase phosphorylation in the hypoxic culture were determined with western blotting. In addition, the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 in hypoxic or normoxic conditions were determined with real-time RT-PCR. The effects of these growth factors on ASC proliferation were investigated. Chromatin immunoprecipitation (ChIP) of the HIF-1α-binding hypoxia responsive element in FGF-2 was performed. HIF-1α was knocked down by siRNA, and FGF-2 expression was investigated.ResultsASC proliferation was significantly enhanced in the hypoxic culture and was inhibited by ERK and Akt inhibitors. Hypoxia for 5-15 minutes stimulated the phosphorylation of ERK1/2 among MAP kinases and induced HIF-1α expression. The levels of VEGF and FGF-2 mRNA and protein in the ASCs were significantly enhanced in hypoxia, and FGF-2 increased ASC proliferation. The ChIP assay revealed an 8-fold increase in the binding of HIF-1α to FGF-2 in hypoxia. HIF-1α knockdown by siRNA partially inhibited the FGF-2 expression of ASCs induced by hypoxia.ConclusionASC proliferation was enhanced by hypoxia. HIF-1α activation, FGF-2 production, and the ERK1/2 and Akt pathway were involved in this regulatory mechanism.

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HIF-1? contributes to proliferation and invasiveness of neuroblastoma cells via SHH signaling.

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HIF-1α contributes to proliferation and invasiveness of neuroblastoma cells via SHH signaling.

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