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Depletion of B cell CLL/Lymphoma 11B Gene Expression Represses Glioma Cell Growth

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Abstract

B cell CLL/lymphoma 11B (Bcl11b), a C2H2 zinc finger transcription factor, not only serves as a critical regulator in development but also plays the controversial role in T cell acute lymphoblastic leukemia (T-ALL). We previously found that the enriched expression of Bcl11b was detected in high tumorigenic C6 glioma cells. However, the role of Bcl11b in glioma malignancy and its mechanisms remains to be uncovered. In this study, using the lentivirus-mediated knockdown (KD) approach, we found that Bcl11b KD in tumorigenic C6 cells reduced the cell proliferation, colony formation, and migratory ability. The results were further verified using two human malignant glioma cell lines, U87 and U251 cells. A cyclin-dependent kinase inhibitor p21, a known Bcl11b target, was significantly upregulated in tumorigenic C6, U87, and U251 cells after Bcl11b KD. Cellular senescence was observed by examination of the β-galactosidase activity in U87 and U251 cells with Bcl11b KD. Reduced expression of stemness gene Sox-2 and its downstream effector Bmi-1 was also observed in U87 and U251 cells with Bcl11b KD. These results suggest that the ablation of Bcl11b gene expression induced glioma cell senescence. Propidium iodide (PI) staining combined with flow cytometry analysis also showed that Bcl11b KD led to the cell cycle arrest of U87 and U251 cells at the G0/G1 or at the S phase, indicating that Bcl11b is required for glioma cell cycle progression. Together, this is the first study to show that the inhibition of Bcl11b suppresses glioma cell growth by regulating the expression of the cell cycle regulator p21 and stemness-associated genes (Sox-2/Bmi-1).

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Abbreviations

Bcl11b:

B cell CLL/lymphoma 11B

CKI:

Cyclin-dependent kinase inhibitor

EtOH:

Ethanol

HCl:

Hydrogen chloride

HDAC:

Histone deacetylase

KD:

Knockdown

PI:

Propidium iodide

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Acknowledgments

The work was supported by the National Science Council, Taiwan (NSC99-2628-B-006-030-MY3, NSC 102-2314-B-006-058, and NSC 102-2811-B-006-017).

Ethical Statement

The study did not involve any human participants and/or animals.

Conflict of Interest

The authors declare that they have no competing interests.

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    Authors and Affiliations

    1. Department of Life Sciences, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan

      Chih-Kai Liao, Kuan-Min Fang, Kitman Chai, Chin-Hsien Wu, Chia-Hsin Ho & Shun-Fen Tzeng

    2. Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Zhunan, Miaoli County, Taiwan

      Chung-Shi Yang

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    1. Chih-Kai Liao
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    Correspondence to Shun-Fen Tzeng.

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    Chih-Kai Liao and Kuan-Min Fang contributed equally to this work.

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    Supplementary Material Fig. S1

    Bcl11b knockdown induces cell senescence of U87 and C6 cells. U87 (A) and C6-1 (B) cells infected by lenti-sh-ctrl (mock), lenti-sh-hBcl11b (KD1) and lenti-sh-rno-1682 (KD2) were reseeded in a 96-well plate. Following incubation for 24 h, the cells were lysed and then subjected to 96-well cellular senescence assay kit (Cell Biolabs, Inc.). Absorbance was measured at 360 nm (Excitation)/465 nm (Emission) using an ELISA-plated reader. The quantitative data represent as relative fluorescence units (RFU) of 3 independent experiments.* p < 0.05 versus mock. (DOCX 67 kb)

    Supplementary Material Table 1

    The measurement of in vivo tumor size in C6 glioma with Bcl11b kncokdown. 1×106 cells of C6-1 cells were infected by lentivirus containing sh-ctrl (mock), by sh-hBcl11b-916 (KD1), or by sh-rnoBcl11b-1682 (KD2). These cells were then implanted into rat cerebral cortex. At 14 dpi, the isolated rat brains were sectioned and then were subjected into H&E staining. UTHSCSA Image tool for Windows was used for the measurement of the tumor size. The tumor size (mm3) in each glioma-bearing brain slice was analyzed by measurement of tumor area (mm2) multiplied by slice thickness (20 μm). The total tumor volume (mm3) in one animal was estimated by summing up the tumor sizes (mm3). (DOCX 14 kb)

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    Liao, CK., Fang, KM., Chai, K. et al. Depletion of B cell CLL/Lymphoma 11B Gene Expression Represses Glioma Cell Growth. Mol Neurobiol 53, 3528–3539 (2016). https://doi.org/10.1007/s12035-015-9231-1

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