Abstract
DIX domain containing 1 (DIXDC1), the human homolog of coiled-coil-DIX1 (Ccd1), is a positive regulator of Wnt signaling pathway. Recently, it was found to act as a candidate oncogene in colon cancer, non-small-cell lung cancer, and gastric cancer. In this study, we aimed to investigate the clinical significance of DIXDC1 expression in human glioma and its biological function in glioma cells. Western blot and immunohistochemistry analysis showed that DIXDC1 was overexpressed in glioma tissues and glioma cell lines. The expression level of DIXDC1 was evidently linked to glioma pathological grade and Ki-67 expression. Kaplan–Meier curve showed that high expression of DIXDC1 may lead to poor outcome of glioma patients. Serum starvation and refeeding assay indicated that the expression of DIXDC1 was associated with cell cycle. To determine whether DIXDC1 could regulate the proliferation and migration of glioma cells, we transfected glioma cells with interfering RNA-targeting DIXDC1; investigated cell proliferation with Cell Counting Kit (CCK)-8, flow cytometry assays, and colony formation analyses; and investigated cell migration with wound healing assays and transwell assays. According to our data, knockdown of DIXDC1 significantly inhibited proliferation and migration of glioma cells. These data implied that DIXDC1 might participate in the development of glioma, suggesting that DIXDC1 can become a potential therapeutic strategy for glioma.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Brosicke N, Sallouh M, Prior LM, Job A, Weberskirch R, Faissner A (2015) Extracellular matrix glycoprotein-derived synthetic peptides differentially modulate glioma and sarcoma cell migration. Cell Mol Neurobiol 35:741–753. doi:10.1007/s10571-015-0170-1
Denysenko T, Annovazzi L, Cassoni P, Melcarne A, Mellai M, Schiffer D (2016) WNT/beta-catenin signaling pathway and downstream modulators in low- and high-grade glioma. Cancer Genomics Proteomics 13:31–45
Ferguson SD (2011) Malignant gliomas: diagnosis and treatment. Dis Mon 57:558–569. doi:10.1016/j.disamonth.2011.08.020
Hazan RB, Phillips GR, Qiao RF, Norton L, Aaronson SA (2000) Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis. J Cell Biol 148:779–790
Jing XT et al (2009) DIXDC1 promotes retinoic acid-induced neuronal differentiation and inhibits gliogenesis in P19 cells. Cell Mol Neurobiol 29:55–67. doi:10.1007/s10571-008-9295-9
Kleihues P, Burger PC, Scheithauer BW (1993) The new WHO classification of brain tumours. Brain Pathol 3:255–268
Louis DN et al (2007) The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 114:97–109. doi:10.1007/s00401-007-0243-4
Luo W et al (2005) Axin contains three separable domains that confer intramolecular, homodimeric, and heterodimeric interactions involved in distinct functions. J Biol Chem 280:5054–5060. doi:10.1074/jbc.M412340200
Mahaley MS Jr, Mettlin C, Natarajan N, Laws ER Jr, Peace BB (1989) National survey of patterns of care for brain-tumor patients. J Neurosurg 71:826–836. doi:10.3171/jns.1989.71.6.0826
Mrugala MM (2013) Advances and challenges in the treatment of glioblastoma: a clinician’s perspective. Discov Med 15:221–230
Namba T, Kaibuchi K (2010) Switching DISC1 function in neurogenesis: Dixdc1 selects DISC1 binding partners. Dev Cell 19:7–8. doi:10.1016/j.devcel.2010.07.002
Ozerlat I (2012) Neuro-oncology: imaging measurements can predict outcome for glioma after radiotherapy. Nat Rev Neurol 8:238. doi:10.1038/nrneurol.2012.70
Robertson T, Koszyca B, Gonzales M (2011) Overview and recent advances in neuropathology. Part 1: central nervous system tumours. Pathology 43:88–92. doi:10.1097/PAT.0b013e3283426e86
Rolon-Reyes K et al (2015) Microglia activate migration of glioma cells through a Pyk2 intracellular pathway. PLoS ONE 10:e0131059. doi:10.1371/journal.pone.0131059
Shiomi K, Uchida H, Keino-Masu K, Masu M (2003) Ccd1, a novel protein with a DIX domain, is a positive regulator in the Wnt signaling during zebrafish neural patterning. Curr Biol 13:73–77
Shiomi K, Kanemoto M, Keino-Masu K, Yoshida S, Soma K, Masu M (2005) Identification and differential expression of multiple isoforms of mouse coiled-coil-DIX1 (Ccd1), a positive regulator of Wnt signaling. Brain Res Mol Brain Res 135:169–180. doi:10.1016/j.molbrainres.2004.12.002
Sigala I et al (2016) Expression of SRPK1 in gliomas and its role in glioma cell lines viability. Tumour Biol. doi:10.1007/s13277-015-4738-7
Tan C et al (2015) DIXDC1 activates the Wnt signaling pathway and promotes gastric cancer cell invasion and metastasis. Mol Carcinog. doi:10.1002/mc.22290
Tao T, Cheng C, Ji Y, Xu G, Zhang J, Zhang L, Shen A (2012) Numbl inhibits glioma cell migration and invasion by suppressing TRAF5-mediated NF-kappaB activation. Mol Biol Cell 23:2635–2644. doi:10.1091/mbc.E11-09-0805
Uchida F et al (2012) Overexpression of cell cycle regulator CDCA3 promotes oral cancer progression by enhancing cell proliferation with prevention of G1 phase arrest. BMC Cancer 12:321. doi:10.1186/1471-2407-12-321
van Roy F, Berx G (2008) The cell–cell adhesion molecule E-cadherin. Cell Mol Life Sci 65:3756–3788. doi:10.1007/s00018-008-8281-1
Wang X et al (2006) DIXDC1 isoform, l-DIXDC1, is a novel filamentous actin-binding protein. Biochem Biophys Res Commun 347:22–30. doi:10.1016/j.bbrc.2006.06.050
Wang L, Cao XX, Chen Q, Zhu TF, Zhu HG, Zheng L (2009) DIXDC1 targets p21 and cyclin D1 via PI3K pathway activation to promote colon cancer cell proliferation. Cancer Sci 100:1801–1808. doi:10.1111/j.1349-7006.2009.01246.x
Wang Y et al (2013) Interaction with cyclin H/cyclin-dependent kinase 7 (CCNH/CDK7) stabilizes C-terminal binding protein 2 (CtBP2) and promotes cancer cell migration. J Biol Chem 288:9028–9034. doi:10.1074/jbc.M112.432005
Wang Y et al (2014) Knockdown of CRM1 inhibits the nuclear export of p27 (Kip1) phosphorylated at serine 10 and plays a role in the pathogenesis of epithelial ovarian cancer. Cancer Lett 343:6–13. doi:10.1016/j.canlet.2013.09.002
Wong CK, Luo W, Deng Y, Zou H, Ye Z, Lin SC (2004) The DIX domain protein coiled-coil-DIX1 inhibits c-Jun N-terminal kinase activation by Axin and dishevelled through distinct mechanisms. J Biol Chem 279:39366–39373. doi:10.1074/jbc.M404598200
Wu Y, Jing X, Ma X, Wu Y, Ding X, Fan W, Fan M (2009) DIXDC1 co-localizes and interacts with gamma-tubulin in HEK293 cells. Cell Biol Int 33:697–701. doi:10.1016/j.cellbi.2009.04.001
Xu Z, Liu D, Fan C, Luan L, Zhang X, Wang E (2014) DIXDC1 increases the invasion and migration ability of non-small-cell lung cancer cells via the PI3K-AKT/AP-1 pathway. Mol Carcinog 53:917–925. doi:10.1002/mc.22059
Yamashita Y et al (2010) CDC25A mRNA levels significantly correlate with Ki-67 expression in human glioma samples. J Neurooncol 100:43–49. doi:10.1007/s11060-010-0147-3
Acknowledgments
This work was supported by the Natural Science Foundation of Jiangsu Province (BK20130386), Chinese Projects for Postdoctoral Science Funds (No. 2015M571792), Jiangsu Planned Projects for Postdoctoral Research Funds (No. 1402200C), and the Scientific Research and Innovation Project of Nantong University (YKC15090).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflicts of interest
All authors declare no conflict of interest.
Additional information
Jianguo Chen and Chaoyan Shen have contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
10571_2016_433_MOESM1_ESM.tif
Supplementary Fig. S1 Knockdown of DIXDC1 decreases U251MG cells proliferation. a and b Western blot analysis showing the effect of decreased DIXDC1 on the protein expression of PCNA, cyclin D1 in U251MG cells. The bar chart demonstrates the ratio of DIXDC1, PCNA and cyclin D1 protein to GAPDH by densitometry. Mean ± SEM of three independent experiments. (*, #, ^ P < 0.05, compared with the control) c Cell vitality of U251MG cells transfected with the shDIXDC1-2 or control shRNA were examined by CCK-8 assay at the indicated time. Mean ± SEM (*P < 0.05, compared with the control) d Flow cytometric analysis of cell cycle distribution 48 h later following control shRNA and shDIXDC1-2 transfection. e and f Knocking down of DIXDC1 suppressed U251MG cells growth as determined by colony formation assays. The colonies (> 50 cells/colony) were counted. Colony-formation ability was ratio of the number of colony to the number of cell plated. Mean ± SEM of three independent experiments. (*P < 0.05, compared with the control group) Comparisons between groups were undertaken by Student’s t-test. Supplementary material 1 (TIFF 754 kb)
10571_2016_433_MOESM2_ESM.tif
Supplementary Fig. S2 Knockdown of DIXDC1 will inhibit the migration of U251MG cells. a and c Wound healing assays with control-shRNA and shDIXDC1-2 transfected U251MG cells. Migration of the cells to the wound was visualized at 0, 24, and 48 h with an inverted Leica phase-contrast microscope (200 × magnification). Each time point is derived from three independent experiments. (*P < 0.05, compared with the control-sh) b and d Crystal violet staining of glioma cells that crossed the polycarbonate membrane of the trans-well chamber to detect the effect of DIXDC1 on migration of glioma cells. Number of cells that migrated through the member was counted by an inverted Leica phase-contrast microscope (400 × magnification) in 5 fields. Columns, mean of triplicate experiments (*P < 0.05, compared with the control-sh). e and f Western blot analysis of DIXDC1, E-cadherin, N-cadherin, and GAPDH in control-shRNA and shDIXDC1-2 cell lines. The bar graph demonstrated the relative expression of DIXDC1, N-cadherin, and E-cadherin versus GAPDH in U251MG cells transfected with control-shRNA or shDIXDC1-2. Mean ± SEM of three independent experiments. (*, #, ^ P < 0.05 compared with the control group).Comparisons between groups were undertaken by Student’s t-test. Supplementary material 2 (TIFF 920 kb)
Rights and permissions
About this article
Cite this article
Chen, J., Shen, C., Shi, J. et al. Knockdown of DIXDC1 Inhibits the Proliferation and Migration of Human Glioma Cells. Cell Mol Neurobiol 37, 1009–1019 (2017). https://doi.org/10.1007/s10571-016-0433-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10571-016-0433-5