RNA-binding protein PCBP2 modulates glioma growth by regulating FHL3
Wei Han, … , Boqin Qiang, Xiaozhong Peng
Wei Han, … , Boqin Qiang, Xiaozhong Peng
Published April 15, 2013
Citation Information: J Clin Invest. 2013;123(5):2103-2118. https://doi.org/10.1172/JCI61820.
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Research Article Oncology

RNA-binding protein PCBP2 modulates glioma growth by regulating FHL3

Abstract

PCBP2 is a member of the poly(C)-binding protein (PCBP) family, which plays an important role in posttranscriptional and translational regulation by interacting with single-stranded poly(C) motifs in target mRNAs. Several PCBP family members have been reported to be involved in human malignancies. Here, we show that PCBP2 is upregulated in human glioma tissues and cell lines. Knockdown of PCBP2 inhibited glioma growth in vitro and in vivo through inhibition of cell-cycle progression and induction of caspase-3–mediated apoptosis. Thirty-five mRNAs were identified as putative PCBP2 targets/interactors using RIP-ChIP protein-RNA interaction arrays in a human glioma cell line, T98G. Four-and-a-half LIM domain 3 (FHL3) mRNA was downregulated in human gliomas and was identified as a PCBP2 target. Knockdown of PCBP2 enhanced the expression of FHL3 by stabilizing its mRNA. Overexpression of FHL3 attenuated cell growth and induced apoptosis. This study establishes a link between PCBP2 and FHL3 proteins and identifies a new pathway for regulating glioma progression.

Authors

Wei Han, Zhongshuai Xin, Zhiqiang Zhao, Wen Bao, Xihua Lin, Bin Yin, Jizong Zhao, Jiangang Yuan, Boqin Qiang, Xiaozhong Peng

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Figure 8

Overexpression of FHL3 inhibits glioma growth in vitro and in vivo.

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Overexpression of FHL3 inhibits glioma growth in vitro and in vivo. (A) ...
(A) Western blot showing overexpression of Flag-FHL3 plasmid in T98G cells using anti-Flag or anti-FHL3 antibody. (B) MTT assay of T98G cells after transfection with the empty vector (—) or flag-FHL3 plasmid. (C) Approximately 48 hours after transfection, T98G cells were analyzed by flow cytometry. The proportions of cells in the G1, G2, and S phases are presented in the right histogram. (D and E) Representative Western blot showing P27, P21, and P16 protein levels (D) and expression levels of differentially phosphorylated pRb (E) in FHL3-overexpressing T98G cells. (F) Nuclear TUNEL staining for apoptotic T98G cells approximately 48 hours after transfection. The percentage of TUNEL-positive cells was calculated (n = 5) and plotted in the histogram. (G) Representative Western blot showing (cleaved) caspase-3 and its substrate (cleaved) PARP in FHL3-overexpressing T98G cells. (H) Anchorage-independent growth of U87MG and U251 cell lines after infection with the adenovirus vector or adenovirus FHL3 as evaluated by the soft agar assay. Scale bars: 50 μm. The number of colonies in each field was scored and the results are expressed as the means ± SD (n = 5; *P < 0.05 by a 2-tailed Student’s t test). (I) Vector U87MG and FHL3-U87MG stable transfectants (5 × 105) were each injected intracranially into 6 nude mice and then imaged after 6 days and 22 days. Tumor sizes were quantified by in vivo bioluminescence imaging. The graph illustrates the distribution of tumor growth factor (22nd day photon flux values versus sixth day photon flux value) between the 2 groups.