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Coiled-Coil Domain Containing 109B Is a Hif1α-Regulated Gene Critical For

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Coiled-Coil Domain Containing 109B Is a Hif1α-Regulated Gene Critical For Xu et al. J Transl Med (2017) 15:165 DOI 10.1186/s12967-017-1266-9 Journal of Translational Medicine RESEARCH Open Access Coiled‑coil domain containing 109B is a HIF1α‑regulated gene critical for progression of human gliomas Ran Xu1, Mingzhi Han1, Yangyang Xu1, Xin Zhang1, Chao Zhang1, Di Zhang1, Jianxiong Ji1, Yuzhen Wei1,3, Shuai Wang1, Bin Huang1, Anjing Chen1, Qing Zhang1, Wenjie Li1, Tao Sun4, Feng Wang4, Xingang Li1* and Jian Wang1,2* Abstract Background: The coiled-coil domain is a structural motif found in proteins that participate in a variety of biologi- cal processes. Aberrant expression of such proteins has been shown to be associated with the malignant behavior of human cancers. In this study, we investigated the role of a specifc family member, coiled-coil domain containing 109B (CCDC109B), in human gliomas. Methods and results: We confrmed that CCDC109B was highly expressed in high grade gliomas (HGG; WHO III–IV) using immunofuorescence, western blot analysis, immunohistochemistry (IHC) and open databases. Through Cox regression analysis of The Cancer Genome Atlas (TCGA) database, we found that the expression levels of CCDC109B were inversely correlated with patient overall survival and it could serve as a prognostic marker. Then, a serious of cell functional assays were performed in human glioma cell lines, U87MG and U251, which indicated that silencing of CCDC109B attenuated glioma proliferation and migration/invasion both in vitro and in vivo. Notably, IHC staining in primary glioma samples interestingly revealed localization of elevated CCDC109B expression in necrotic areas which are typically hypoxic. Moreover, small interfering RNA (siRNA) and specifc inhibiters of HIF1α led to decreased expres- sion of CCDC109B in vitro and in vivo. Transwell assay further showed that CCDC109B is a critical factor in mediating HIF1α-induced glioma cell migration and invasion. Conclusion: Our study elucidated a role for CCDC109B as an oncogene and a prognostic marker in human gliomas. CCDC109B may provide a novel therapeutic target for the treatment of human glioma. Keywords: CCDC109B, HIF1α, Glioma, Proliferation, Invasion Background Difuse infltrative invasion of GBM cells into the adja- Glioblastoma multiforme (GBM) is the most aggres- cent normal brain areas is a major cause of invariable sive malignancy in adults and thus persists as a major recurrence and relapse after resection of primary tumors unsolved clinical challenge [1]. Despite impressive [3]. advances in surgical techniques, radiotherapy and chem- A number of pathological features in GBM provide otherapy, the median survival time of patients with GBM the basis for understanding the functional consequences remains dismally at 14.6 months [2]. of changes in gene expression. For example, hypoxia is a pathological hallmark of GBM. Hypoxia-inducible fac- *Correspondence: [email protected]; [email protected] tor 1 (HIF1), a dimeric transcription factor, is one of the 1 Department of Neurosurgery, Qilu Hospital of Shandong University primary regulators that coordinate cellular responses to and Brain Science Research Institute, Shandong University, #107 Wenhua Xi Road, Jinan 250012, China hypoxia. HIF1 is composed of α and β subunits (HIF1α; 2 Department of Biomedicine, University of Bergen, Jonas Lies vei 91, HIF1β). HIF1α is rapidly degraded under normoxic con- 5009 Bergen, Norway ditions but is often stable under hypoxic conditions. Full list of author information is available at the end of the article © The Author(s) 2017. This 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. Xu et al. J Transl Med (2017) 15:165 Page 2 of 14 However, when HIF1α binds to hypoxia-responsive ele- informed consent was obtained from all patients, and ments (HREs), it activates transcription of downstream approval for experiments was obtained from Ethics genes, which are involved in tumor angiogenesis, inva- Committee of the Qilu Hospital. All surgeries and post- sion, cell survival, and glucose metabolism [4]. Terefore, operative animal care were approved by the Institutional identifying HIF1α-targeted molecules will provide fur- Animal Care and Use Committee (IACUC) of Shandong ther understanding in the development and treatment of University (Shandong, China). Our research complies human glioma. with the commonly-accepted ‘3Rs’: replacement of ani- Coiled coils are among the most ubiquitous fold- mals by alternatives wherever possible, reduction in the ing motifs identifed in proteins and have not only been number of animals used, and refnement of experimental found in structural proteins but also play a necessary role conditions and procedures to minimize harm to animals. in various intracellular regulation processes [5]. Coiled coils are involved in signal-transducing events and act Cell culture and hypoxic treatment as a molecular recognition system. Furthermore, they Human glioma cell lines, U87MG, U251 and T98 were provide mechanical stability to cells and are involved in obtained from the Culture Collection of the Chinese movement processes [6]. Increasing evidence suggests Academy of Sciences (Shanghai, China). Te normal that aberrant expression of coiled-coil domain containing human astrocytes (NHA) cell line was a kind gift from proteins infuences the migration, invasion and prolifera- the Department of Biomedicine at the University of Ber- tion of various human cancers, including bladder cancer gen (Bergen, Norway). Cells were cultured in Dulbecco’s [7], pancreatic cancer [8], gastric cancer [9], papillary modifed Eagle’s medium (DMEM; Termo Fisher Sci- thyroid carcinoma [10], leukemia [11], prostate cancer entifc, Waltham, MA, USA) supplemented with 10% [12], breast cancer [13]. fetal bovine serum (FBS; Termo Fisher Scientifc) and CCDC109B, also known as mitochondrial calcium uni- maintained at 37 °C in a humidifed chamber contain- porter b (MCUb), is an MCU isogene [14]. CCDC109B ing 5% CO 2. For hypoxic treatment, cells were placed in is an evolutionarily conserved protein, which pos- a modulator incubator (HERAcell 150i, Termo Fisher sesses two coiled-coil domains and two transmembrane Scientifc) in 94% N2, 5% CO2, and 1% O2. For sta- domains [15]. Functionally, MCUb acts as a negative ble CCDC109B-knockdown, U87MG and U251 cells subunit of the MCU channel, and the MCU/MCUb ratio were infected with lentivirus expressing short hairpin seems to vary in diferent tissues, providing a molecular RNA (shRNA) (sh-CCDC109B-1). After 48 h, U87MG mechanism to mediate the efciency of mitochondrial or U251 cells were exposed to 0.5 or 2 µg/mL puromy- calcium (Ca2+) intake [16]. Te failure of mitochondria cin (A1113802, Termo Fisher Scientifc), respectively, to intake calcium leads to the abnormal activation of in complete DMEM for an additional 2 weeks. Cells cytosolic Ca2+-dependent enzymes, including calpain were subsequently treated with PX478 (S7612, Selleck proteases [17] and calmodulin-dependent kinases [18] Chemicals; Shanghai, China) and HIF1α siRNA to inhibit and ultimately leads to changes in cellular signaling cas- HIF1α expression and harvested after 48 h. Sequences cades which directly regulate cell growth [19], tumor of synthesized shRNAs (Genepharma; Shanghai, China) cell invasion [20]. However, the biological signifcance of were the following: sh-Negative Control (sh-NC) CCDC109B in human glioma remains unclear. 5′-TTCTCCGAACGTGTCACGTtt-3′; sh-CCDC109B-1 Here, we investigated expression of CCDC109B in 5′-CAGTCACACCATTATAGTAtt-3′; sh-CCDC109B-2 human glioma tissues and cell lines by analyzing our own 5′-CTCGACAGGATTATACTTAtt-3′; sh-CCDC109B-3 cohort and publicly available molecular databases. Ten, 5′-GCAAGTAGAAGAACTCAATtt-3′. Sequences of functional experiments were performed with model synthesized siRNAs (Genepharma) were the following: systems in vitro and in vivo. We uncovered a potential si-NC 5′-TTCTCCGAAGGTGTCACGG-3′; si-HIF1α-1 oncogenic role for CCDC109B in glioma progression and 5′TACGTTGTGAGTGGTATTATT-3′; si-HIF1α-2 5′-CT identifed HIF1α as a possible transcriptional regulator. GATGACCAGCAACTTGA-3′. Tese results, support CCDC109B as a new therapeutic target for the treatment of human glioma. IHC Samples were fxed in 4% formalin, parafn-embedded, Methods and sectioned (4 µm). After de-waxing and rehydration, Ethics statement the sections were incubated with 0.01 M citrate bufer for Human brain tumor (n = 68; WHO grade II–IV) and 20 min at 95 °C for antigen retrieval. Endogenous per- non-neoplastic tissue (n = 4) samples were obtained oxidase activity and non-specifc antigens were blocked from surgeries performed at the Department of Neu- with 3% hydrogen peroxide (ZSGB-Bio; Beijing, China) rosurgery at Qilu Hospital (Shandong, China). Written and 10% normal goat serum (ZSGB-Bio) respectively, Xu et al. J Transl Med (2017) 15:165 Page 3 of 14 followed by incubation with primary antibody at 4 °C numbers were determined using Kodak MI software. overnight. Sections were rinsed with phosphate bufered Each experiment was repeated three times in
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