- Open Access
HOXC8: a predictive glioma biomarker that induces epithelia-mesenchymal transition
- Tingyu Liang†2,
- Xiaoxuan Wang†3,
- Peiliang Li2,
- Yang Cao2,
- Enshan Feng2Email author and
- Gan You1, 4Email author
© The Author(s) 2018
- Received: 6 March 2018
- Accepted: 7 August 2018
- Published: 28 August 2018
The transcription factor Homeobox C8 (HOXC8) is overexpressed and regulates many important genes involved in the proliferation and invasion of many malignant tumors. However, the function of HOXC8 in gliomas remains unclear.
Based on the Chinese Glioma Genome Atlas (CGGA) set, HOXC8 expression is negatively correlated with overall survival (OS). Small interfering RNA (si-HOXC8) was used to downregulate the mRNA and protein expression levels of HOXC8 to assess glioma cell proliferation, migration and invasion.
Patients with higher HOXC8 levels showed poorer prognosis. DAVID analysis results indicated that HOXC8 was related to cell cycle, cell adhesion and immune response. In U251 and LN229 glioma cells treated with small interfering RNA for HOXC8 (si-HOXC8) for gene knockdown, significantly lower cell capacity of growth, migration and invasion was observed. Moreover, HOXC8 knockdown could reduce the protein expression of classical epithelial mesenchymal transition (EMT) related markers.
HOXC8 may play an important role in glioma proliferation, migration and invasion. These findings indicated that HOXC8 may constitute a novel target for glioma treatment.
- Cell cycle
Gliomas account for most of malignant tumors of the central nervous system (CNS). Despite the recent progress in surgical resection, radiotherapy and chemotherapy, prognosis for glioma remains poor [1, 2]. Gliomas possessed strong proliferation, migration and invasion abilities, causing high mortality. Therefore, multiple studies have identified key genes that could help achieve a breakthrough in gliomas treatment [3, 4]. In this study, Homeobox C8 (HOXC8), a transcript factor, was selected for in-depth assessment.
HOXC8, located in a cluster on chromosome 12, belongs to the 39-member HOX family of proteins . Under normal circumstances, HOXC8 plays an indispensable role in embryonic morphogenesis and system morphogenesis [6, 7]. However, in various tumors, including ovarian cancer, hepatocellular carcinoma, and breast cancer, HOXC8 induces cell proliferation, migration and invasion, and is inversely correlated with overall survival (OS) through different mechanisms [8-10]. Although HOXC8 is important in tumorigenesis, its mechanism in glioma remains unclear.
Epithelial mesenchymal transition (EMT), a cellular alteration that confers a more invasive and drug-resistant phenotype, was initially observed in embryonic development [11, 12]. EMT was a complex process reflected by epithelial cells acquiring the mesenchymal phenotype and motility through a cascade of biological events . In gliomas, the mechanism by which HOXC8 affected EMT is unclear.
In summary, the aim of our study was to assess the oncogenic function of HOXC8. HOXC8 knockdown could inhibit glioma cell proliferation, migration and invasion. Therefore, HOXC8 could provide novel insights for glioma treatment.
Dataset and glioma samples
We obtained mRNA samples and follow-up information from the Chinese Glioma Genome Atlas (CGGA). OS was defined as the time from surgery to death. Glioma samples were collected from surgical resection, snap-frozen in liquid nitrogen and stored at − 80 °C until RNA extraction . This study was approved by the Ethics Committee of Beijing Tiantan Hospital, and written informed consent was obtained from all the patients.
Cell culture and siRNA transfection
Two glioma cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin and 100 μg/mL streptomycin), in a humidified atmosphere with 5% CO2 at 37 °C . The siRNA segmentand a non-specific control siRNA sequence (NC) were purchased from Genepharma (Shanghai, China), and transfected into LN229 and U251 cells using Lipofectamine™ 2000 (Invitrogen, Shanghai, China), according to the manufacturer’s instructions. After 48 h of incubation, samples were collected for RT-PCR and Western blot assay .
Real-time polymerase chain reaction (RT-PCR) and western blot (WB)
RT-PCR and Western blot were performed as previously described [4, 17].The following primers were used for RT-PCR: HOXC8, Forward 5’-ACCGGCCTATTACGACTGC-3′ and Reverse 5′-TGCTGGTAGCCTGAGTTGGA-3′; GAPDH (Forward 5′- GGAGCGAGATCCCTCCAAAAT -3′ and Reverse 5′- GGCTGTTGTCATACTTCTCATGG-3′ was used as an internal control, and fold changes were calculated by the 2 − ΔΔCt method. For Western blotting, densities of specific protein bands were quantified after normalization to Tubulin or Actin level in the same sample.
Clone formation assay
A total of 1000 cells were seeded per well in a 6-well plate in DMEM and 10% FBS, and cultured for 2 weeks before Crystal violet stain. Then, clone numbers were recorded in siRNA and control groups, respectively.
Transwell migration assay
Transwell plates were used to study cell migration and invasion. A total of 1 × 105 LN229 or U251 cells were seeded in each well in DMEM only. Control medium was added to the lower chamber of the transwell plates. After 36 h (invasion assay) or 12 h (migration assay) of incubation, the cells in upper chambers were removed carefully before Crystal violet staining. Analysis was carried out by microscopy, counting cells in siRNA and control groups, respectively .
We used Kaplan-Meier analysis (log-rank test) to evaluate predictive value of HOXC8 expression for OS in different groups. Univariate and multivariate Cox regression analyses were used to determine that HOXC8 was an independent factor to predict OS. Pearson relation analysis was performed in CGGA array set using R language package. All values were considered statistically significant at p < 0.05.
HOXC8 was associated with overall survival in gliomas
Univariate and multivariate Cox analysis in CGGA glioma samples
Age at diagnosis
IDH1 status (mutation)
High HOXC8 expression
Grade (high grade)
TCGA subtype (mesenchymal type)
In conclusion, we hypothesized that HOXC8 could serve as a novel biomarker for predicting glioma OS.
Gene functional analysis
LN229 and U251 glioma cell lines were used to investigate biological processes
HOXC8 could significantly promote glioma proliferation
Downregulation of HOXC8 inhibits cell migration and invasion by blocking the EMT pathway
Massive evidence supports that HOXC8 plays an important role in malignant tumor formation and progression [21, 22]. As a transcription factor, HOXC8 is able to regulate and coordinate multiplevital genes (e.g. Mgl1, Embigin, Meis1, and Fyn) involved in cancer development and progression [8, 22, 23]. Previous findings demonstrated that HOXC8 depletion by small interfering RNA suppresses epithelial ovarian cancer proliferation and migration, and induces apoptosis by increasing ZAC1 expression . HOXC8, overexpressed in hepatocellular carcinoma (HCC) compared with adjacent non-tumor tissues, is associated with poor prognosis. Si-HOXC8 blocks G1-S phase transition, inhibits cell proliferation and renders cells more sensitive to oxaliplatin . Therefore, HOXC8 as a potential oncogenic driver, plays an important role in cancer cell proliferation, migration and invasion, by overregulating many oncogenes and downregulating tumor suppressor genes. In this study, we demonstrated that si-HOXC8 blunted glioma cell proliferation, migration and invasion by reversing EMT.
EMT is known to be implicated in cancer progression, metastasis and drug resistance . EMT reversibly enables polarized epithelial cells to lose their epithelial characteristics and to acquire mesenchymal properties . The availability of antibodies targeting epithelial (E-cadherin, β-catenin, and Claudin-1) and mesenchymal (N-cadherin, Slug, Vimentin and MMP9) markers makes it convenient to assess EMT . Multiple genes and pathways were reported to be involved in EMT [26-28]. However, the mechanisms by which HOXC8 affects EMT remains unclear. This study found that the HOXC8 was involved in EMT. Moreover, si-HOXC8 decreased the expression of classical mesenchymal markers (N-cadherin, Slug, Vimentin and MMP9). Hence, the present findings provide additional insights regarding the implication of HOXC8 and target genes in EMT as well as malignant tumor progression.
In summary, this study demonstrated that higher HOXC8 expression resulted in shorter OS. In agreement, HOXC8 knockdown inhibited glioma cell proliferation. Meanwhile, reduction of HOXC8 could also affected migration and invasion through blocking EMT pathway. The present findings suggested that HOXC8 should be considered a novel biomarker and target for glioma treatment.
Thanks for Chinese Glioma Genome Atlas dataset and The Cancer Genome Atlas dataset for data share.
This study was funded by National Natural Science Fund (Grant No. 81301112) and Yu Miao Plan of Beijing Ditan Hospital (Grant No. DTYM201603) and Beijing Medical Management Bureau Cultivation Plan (Grant No. PX2018079).
Conception and design: ESF and GY; Performed the experiments: TYL and XXW; Data collection and analysis: TYL; Order reagents/materials: TYL and PLL; Wrote the paper: YC. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by the Ethics Committee of Beijing Tiantan Hospital, and written informed consent was obtained from all the patients.
Consent for publication
The authors declare that they have no competing interest.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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