RNA-binding protein PCBP2 modulates glioma growth by regulating FHL3
Wei Han, Zhongshuai Xin, Zhiqiang Zhao, Wen Bao, Xihua Lin, Bin Yin, Jizong Zhao, Jiangang Yuan, Boqin Qiang, Xiaozhong Peng
Wei Han, Zhongshuai Xin, Zhiqiang Zhao, Wen Bao, Xihua Lin, Bin Yin, Jizong Zhao, Jiangang Yuan, Boqin Qiang, Xiaozhong Peng
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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 9

Co-knockdown of PCBP2 and FHL3 rescues the phenotype induced by PCBP2 knockdown alone.

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Co-knockdown of PCBP2 and FHL3 rescues the phenotype induced by PCBP2 kn...
(A) Western blot of FHL3 expression in 3 glioma cell lines transiently transfected with FHL3 siRNA. (B) Western blot showing PCBP2 and FHL3 protein levels in glioma cell lines transiently transfected with the PCBP2 siRNA alone, or PCBP siRNA plus FHL3 siRNA. (C) MTT assay of 3 glioma cell lines after transfection. *P < 0.05 compared with the control transfection and cotransfection by a 2-tailed Student’s t test). (D) The transfected cells above were analyzed by flow cytometry. The proportions of cells in the G1, G2, and S phases are depicted in the histograms. *P < 0.05 compared with the control. (E) Nuclear TUNEL staining for apoptosis in the glioma cell lines. The percentage of TUNEL-positive cells was calculated (n = 5) and plotted on the histogram. *P < 0.05 compared with the control transfection and cotransfection. (F) Western blot showing the levels of (cleaved) caspase-3 and (cleaved) PARP in the transfected glioma cell lines. (G) Anchorage-independent cell growth of U87MG and U251 cells after infection with Ad control shRNA, Ad shPCBP2, or Ad shPCBP2 plus Ad shFHL3 were evaluated by a soft-agar assay. Scale bars: 50 μm. The number of colonies in each field was expressed as the means ± SD (n = 5; *P < 0.05). (H) Subcutaneous tumors isolated from nude mice 4 weeks after adenovirus injection (top). Growth curves of tumors in the nude mice (n = 3 per group) are shown (bottom). Each intratumoral injection of adenovirus is indicated by an arrow.