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Jci.Org Volume 123 Number 5 May 2013 2103 Research Article Research article RNA-binding protein PCBP2 modulates glioma growth by regulating FHL3 Wei Han,1 Zhongshuai Xin,1,2 Zhiqiang Zhao,1 Wen Bao,1 Xihua Lin,1 Bin Yin,1 Jizong Zhao,3 Jiangang Yuan,1 Boqin Qiang,1 and Xiaozhong Peng1 1The State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. 2Division of Biochemical and Gene Engineering Medicines, National Institute for Food and Drug Control, Beijing, China. 3Department of Neurosurgery, Beijing Tiantan Hospital, Beijing, China. PCBP2 is a member of the poly(C)-binding protein (PCBP) family, which plays an important role in posttran- scriptional 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 apop- tosis. 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. Introduction expression in oral cancer (14), PCBP2 is one of the least studied Poly(C)-binding proteins (PCBPs) are characterized by their proteins in human cancers among PCBPs. Most of the reports on high-affinity and sequence-specific interactions with polycytosine PCBP2 have focused on its posttranscriptional and translational (poly(C)). In mammalian cells, these PCBPs belong to 1 of 2 sub- controls in RNA viruses. For example, PCBP2 has been reported to sets: hnRNP K/J, or the α-complex proteins (e.g., PCBP1-4) (1, 2). participate in the replication and translation of many RNA viruses, hnRNP K, PCBP1, and PCBP2 have been studied in the greatest including poliovirus (15), coxsackievirus (16), and rhinovirus (17). detail. The latter 2 proteins are also known as αCP1 and αCP2, PCBP2 can also bind to the 5′ UTR (18) and 3′ UTR (19) of the or hnRNPE1 and hnRNPE2 (3). Recently, 2 other members of the HCV gene. PCBP2 was induced after viral infection and interacted αCP family were discovered: PCBP3 (αCP3) and PCBP4 (αCP4) (4). with MAVS, which showed that PCBP2 was a negative regulator of PCBPs regulate gene expression at various levels, including MAVS-mediated antiviral signaling (20). transcription, mRNA processing, mRNA stabilization, and trans- Recent discoveries have revealed that PCBP2 is a regulator of lation. It has been suggested that PCBPs, specifically hnRNPK tumor development. Molinaro et al. found that 2′,5′-oligoadeny- and PCBP1, play a critical role in carcinogenesis. Transcriptional late synthetase (OAS) activation may occur in prostate cancer cells activation of the oncogenes c-SRC and c-Myc was increased by in vivo when stimulated by Raf kinase inhibitor protein (RKIP) and hnRNP K (5, 6), suggesting that hnRNPK may cooperate with PCBP2 (21). In leukemic blasts, PCBP2 expression was induced by additional oncoproteins to overexpress genes that promote can- BCR/ABL through constitutive activation of MAPKERK1/2 (22) and cerous growth. PCBP1 has been reported to be downregulated its regulation of CEBPA mRNA was influenced by the decoy activ- in metastatic cervical cancer (7) and metastatic breast cancer ity of miR-328 (23). Given that more target mRNAs of PCBP2 were cells (8). Knockdown of PCBP1 in the prostate cancer cell line found by RIP-ChIP and biotin pull-down, we believe that the bind- LNCaP increased androgen receptor (AR) protein targeting ing of PCBP2 to its target mRNAs may play important functions to the 3′ untranslated region (UTR) of AR transcripts (9). The in human cancers. recent finding that transforming growth factor-β–mediated Here, we showed the upregulation of PCBP2 in human glioma (TGFβ-mediated) phosphorylation of PCBP1 induced epithe- tissues and cell lines. Knockdown of PCBP2 inhibits glioma cell lial-mesenchymal transdifferentiation (EMT) (10), and the evi- growth both in vitro and in nude mice. To elucidate how PCBP2 dence that PCBP1 can downregulate metastasis-associated PRL-3 functions as an RNA-binding protein with its target mRNAs in phosphatase translation (11) indicates that PCBP1 could be a gliomas, we identified 35 mRNAs that preferentially bind PCBP2 tumor suppressor. by RIP-ChIP analysis. We also showed that four- and-a-half LIM PCBP2 mapped to 12q13.12-q13.13 and PCBP1 mapped to domain 3 (FHL3) is downregulated in gliomas in which mRNA is 2p12-p13 (12) are highly homologous proteins with 82% amino a novel binding target of PCBP2. acid identity. It has been suggested that PCBP2 also plays an important role in human malignancies. However, in addition Results to its overexpression during leukomegenesis (13) and its under- To examine the expression pattern of PCBP2 in gliomas, total RNA and proteins were extracted from 26 primary glioma sam- Conflict of interest: The authors have declared that no conflict of interest exists. ples, which contain 9 grade II, 9 grade III, and 8 grade IV glioma Citation for this article: J Clin Invest. 2013;123(5):2103–2118. doi:10.1172/JCI61820. tissues and 2 normal brain tissues. Real-time PCR and Western The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 5 May 2013 2103 research article Figure 1 PCBP2 is upregulated in glioma tissues and cell lines compared with normal brain tissues and normal human astrocytes. (A) Real-time PCR analysis of PCBP2 in 2 normal brain tissues (N1, N2) and 26 glioma tissues (T1–T9 grade II, T10–T18 grade III, and T19–T26 grade IV). Col- umns represent means and bars represent SD. (B) Representative Western blot showing PCBP2 protein levels in 2 normal brain tissues and 27 glioma tissues. β-Actin was used as a loading control. (C) Immunohistochemical staining of PCBP2 in glioma (Grade II, Grade III, Grade IV) and pericarcinous tissues (Normal) using anti-human PCBP2 antibodies. There was no staining with rabbit IgG. Original magnification,× 400. (D) Real-time PCR analysis of PCBP2 in 2 normal human astrocyte cell lines (NHA and HA) and 5 glioma cell lines (T98G, A172, U251, U87MG, and CCF-STTG1). (E) Representative Western blot showing PCBP2 protein levels in 2 NHA cell lines and the 5 indicated glioma cell lines. blot were then performed to analyze the gene expression pro- We also found significantly elevated levels of PCBP2 in T98G, files. We also determined mRNA and protein levels of PCBP2 U87MG, and U251 glioma cell lines (Figure 1, D and E). Therefore, in 5 different human glioma cell lines (T98G, U87MG, U251, these 3 cell lines were used as models for further study of PCBP2 func- A172, and CCF-STTG1) and 2 normal human astrocyte cell tion in gliomas. To investigate the role of PCBP2 in glioma develop- lines (NHA and HA). The results showed that the mRNA and ment, we synthesized 2 siRNAs (PCBP2 siRNA and PCBP2 siRNA*) protein expression of PCBP2 was upregulated in both glioma that specifically target PCBP2, as previously reported (24, 25). After tissues and cell lines compared with normal brain tissues (Fig- transfection for 48 to 72 hours in T98G, U87MG, and U251 glioma ure 1). A strong positive signal of PCBP2 was found in almost cell lines, siRNA-mediated knockdown of PCBP2 reduced its protein all glioma tissues, whereas an obvious loss of PCBP2 in nor- level by at least 80% compared with the control samples (Figure 2A mal brain tissue was observed (Figure 1C). A gradually stronger and Supplemental Figure 1; supplemental material available online PCBP2 expression was found from grade II samples to grade IV with this article; doi:10.1172/JCI61820DS1). Glioma cells trans- samples (Figure 1, A–C). fected with PCBP2 siRNA had a reduced number of metabolically 2104 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 5 May 2013 research article Figure 2 Inhibition of glioma cell growth in vitro by knockdown of PCBP2. (A) Western blot of PCBP2 expression in 3 glioma cell lines (T98G, U87MG, and U251) transiently transfected with the control siRNA or PCBP2 siRNA for 48 to 72 hours using Lipofectamine 2000. β-Actin was used as a loading control. (B) MTT assay on the same 3 glioma cell lines after transfection with the control siRNA or PCBP2 siRNA as above. Data are presented as the mean ± SD and are representative of 3 wells. *P < 0.05 compared with the control siRNA by a 2-tailed Student’s t test. (C) Approximately 72 hours after transfection, the 3 glioma cell lines were analyzed by flow cytometry. The proportions of cells in the G1, G2, and S phases of the cell cycle are depicted in the histograms. *P < 0.05 compared with the control siRNA by a 2-tailed Student’s t test. (D and E) Representative Western blot showing P27, P21, and P16 protein levels (D) and expression levels of differentially phosphorylated pRb (E) in the transfected glioma cell lines. P16 was deleted in U87MG and U251 cells. (F) Nuclear TUNEL staining for apoptotic cells in the glioma cell lines after approximately 72 hours of transfection. The ratio of TUNEL-positive cells was calculated (n = 5) and plotted on the histogram.
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