key: cord-0741206-kpfbduf0
authors: Mao, Jianhui; Sun, Zhaosheng; Cui, Yongjian; Du, Naiyi; Guo, Hong; Wei, Jianhui; Hao, Zhenmin; Zheng, Lei
title: PCBP2 promotes the development of glioma by regulating FHL3/TGF‐β/Smad signaling pathway
date: 2019-11-06
journal: J Cell Physiol
DOI: 10.1002/jcp.29104
sha: 360067c3633b8cac0a2845356bcd755e61bbd02c
doc_id: 741206
cord_uid: kpfbduf0

The purpose of this study was to investigate the role of Poly (C)‐binding protein 2 (PCBP2) and the related signaling pathway in glioma progression. Quantitative real‐time polymerase chain reaction (qRT‐PCR) and immunohistochemistry (IHC) were performed to measure PCBP2 messenger RNA and protein expression in glioma tissues or cells. Cell transfection was completed using Lipofectamine 2000. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay, Transwell assay and flow cytometry assay were used to explore the effects of PCBP2 expression on biological behaviors of glioma cells. Western blot assay was used for the detection of pathway related proteins. Expression of PCBP2 in glioma tissues and cells were higher than that in paracancerous tissues and normal cells (both p < .01). Moreover, the elevated expression of PCBP2 was significantly correlated with tumor size (p = .001) and WHO stage (p = .010). Knockdown of PCBP2 could suppress proliferation, migration and invasion of glioma cells and promote apoptosis. Besides, the expression of transforming growth factor‐β (TGF‐β) pathway related proteins TGF‐β1, p‐Smad2 and p‐Smad7 were decreased following the downregulation of PCBP2. PCBP2 also inhibited FHL3 expression by binding to FHL3‐3′UTR. The inhibition of FHL3 could reverse the antitumor action caused by PCBP2 silencing. In vivo assay, PCBP2 was also found to inhibit the tumor growth of glioma. PCBP2 activates TGF‐β/Smad signaling pathway by inhibiting FHL3 expression, thus promoting the development and progression of glioma.

the molecular mechanisms underlying the aggressive progression of glioma may provide the novel therapeutic targets for the fatal cancer.

Poly(C) binding protein 2 (PCBP2), a RNA-binding protein with 39 kDa, can regulate RNA stabilization, translational silencing and enhancement (Collier, Goobar-Larsson, Sokolowski, & Schwartz, 1998; Wan et al., 2016) . It contains three K homology domains employed as the main RNA recognized regions Leffers, Dejgaard, & Celis, 1995) . Dysregulation of PCBP2 may damage multiple biological processes through RNAbinding pathways. For instance, PCBP2 could regulate the genome circularization and replication of various RNA viruses, like norovirus (Lopez-Manriquez et al., 2013) , severe acute respiratory syndrome coronaviruses (SARS-CoV; Shi et al., 2014) , poliovirus (Walter, Parsley, Ehrenfeld, & Semler, 2002) , and hepatitis C virus (Li, Masaki, Shimakami, & Lemon, 2014) . The upregulation of PCBP2 may contribute to the formation of ribosomal initiation complex, thus enhancing the replication of virus (Asnani, Pestova, & Hellen, 2016) . Additionally, PCBP2 could regulate cell growth, migration and invasion (Chen et al., 2018; Mao et al., 2016) . The abnormalities in PCBP2 expression have been observed in several cancers, such as gastric cancer (Chen et al., 2018) and hepatocellular carcinoma . In glioma, it has been reported that the knockdown of PCBP may inhibit tumor cell proliferation and growth (Tang, Gao, & Chen, 2015) . PCBP2 may act as an oncogene in etiology of glioma.

However, the molecular mechanisms of PCBP2 in glioma progression was poorly known.

In this study, we investigated the expression of PCBP2 in glioma tissues and cells and revealing its functional roles in glioma progression. Furthermore, cell experiments were constructed to explore the molecular mechanisms underlying the oncogenic function of PCBP2 in the pathogenesis of glioma.

The present study was carried out in Harrison international Peace Hospital. A total of 106 adult glioma patients who were diagnosed through pathological examinations were enrolled in our study.

Glioma tissues and corresponding noncancerous tissues were collected from the patients who received surgical procedures and put into liquid nitrogen immediately. Then the specimens were stored at −80℃ for usage in the next step. None of the patients had received any treatments before tissue collection, such as chemotherapy or/and radiotherapy. Clinical characteristics of glioma patients were also obtained from medical records. This study was approved by the Ethics Committee of Harrison international Peace Hospital. All patients had signed the written informed contents. forward: 5′-CATGGCATGAGCACTGCTTCCTG-3′, reverse: 5′-GCTTAGG GCCCTGCCTGGCTACAGC-3′. GAPDH was employed as the internal control, and its primer sequences were as the following, forward: 5′-T GCACCACCAACTGCTTAGC-3′; reverse: 5′-GGCATGGACTGTGGTCA TGAG-3′. The relative expression level of the detected genes was calculated using −ΔΔ 2 Ct method. Each test was repeated three times.

Protein expression of PCBP2 in glioma tissue samples were estimated using IHC analysis staining with streptavidin-perosidase (S-P). IHC kit (Boster, Wuhan, China) was used according to manufacturer's instruction. The used antibody included anti-PCBP2 antibody (1:200, Abcam, UK). Staining results were recorded by two independent investigators, and they did not know the histopathologic features and patients' information. Staining results were scored according to the following standards: 0: for staining < 5%; 1: staining 6-25%; 2: staining 26-50%; 3: staining 51-75%; 4: staining > 75%.

Patients with score <1 were confirmed as negative staining, otherwise, the patients were confirmed as positive staining.

To investigate the role of PCBP2 in glioma progression, siRNA-PCBP2 was designed to silence PCBP2 expression in glioma cells in vitro. si-PCBP2 and corresponding negative control (si-NC) sequences were synthesized by GenePharma (Shanghai, China). The obtained sequences were cloned into pLV-shRNA plasmid, and then transfected into giloma cells using Lipofectamine TM 2000 (Invitrogen, Thermo Fisher Scientific, Inc.). Transfection for 48 hr, qRT-PCR was conducted to measure the relative expression of PCBP2 so as to estimate the transfection effects.

In addition, to explore the molecular mechanisms underlying the function of PCBP2 in the progression of glioma, si-FHL3 and FHL3overexpression (pLV-FHL3) recombinant plasmids and corresponding controls were also constructed.

2.6 | 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay Then the vectors were transfected into U251 cells with or without si-PCBP2 vector. Transfection for 48 hr, the cells were harvested and the luciferase activity was detected using a dual-luciferase reporter assay system (Promega) and normalized to Renilla activity.

Expression of proteins was detected using western blot analysis. 

Twenty Balb/c nude mice (6-8 weeks) were routinely cultured, U251 cells (3 × 10 6 /200 μl) were inoculated on the upper part of the groin, mice were performed the subcutaneous injection. They were equally divided into two groups: experimental group (si-PCBP2) and the control group (si-NC), every group was 10 cases. The major axis (a) and minor axis (b) of the subcutaneous tumor were measured once 3 days, tumor volume: V = 1/2 × a × b 2 . After 4 weeks, nude mice were killed for the measurement of tumor weight.

All the data analysis and figure drawing were performed using SPSS 18.0 software (SPSS Inc., Chicago, IL), and GraphPad Prism version 5.0 (GraphPad, San Diego, CA), respectively. Continuous variables were shown as mean ± standard deviation (SD). Comparison between case and control groups via student's t test.

χ 2 test was used to explore the association of PCBP2 expression with clinical parameters of glioma patients. p < .05 were considered as statistical significance. Figure 1a ,b).Then the protein levels of PCBP2 in glioma tissues were also examined by western blot assay. The data showed that PCBP2 protein level in glioma tissues was significantly increased compared with noncancerous tissues (p < 0.01; Figure 1b) . Additionally, IHC was applied to investigate the expression of PCBP2 protein in glioma tissues. As shown in Figure 1d , PCBP2 exhibited the strong positive expression in glioma tissues, but weak positive expression in noncancerous tissues.

The positive rate was 87.73% (93/106) in glioma tissues, while the positive rate was only 16.98% (18/106) in adjacent normal tissues.

Expression of PCBP2 protein was obviously higher in glioma tissues than that in adjacent normal tissues.

In this study, the relationship between PCBP2 expression and clinical parameters of glioma patients was evaluated. The However, the expression of PCBP2 was not correlated with age, gender, family history, and KPS score of glioma patients (p > .05 for all; Table 1 ).

To si-PCBP2 transfection, compare with si-NC group (p < .05, Figure 3b ,c). Furthermore, the apoptotic rate of si-PCBP2 transfected cells was obviously increased, compared with si-NC group (p < .001 and Figure 3d ). Therefore, the knockdown of PCBP2 could obviously inhibit the progression of glioma cells.

In our study, qRT-PCR and western blot were carried out to investigate the expression of FHL3 in si-PCBP2 transfected cells.

F I G U R E 1 Poly (C)-binding protein 2 (PCBP2) was upregulated in glioma tissues. The relative expression of PCBP2 mRNA in glioma tissue specimens and noncancerous tissues were examined by quantitative real-time polymerase chain reaction (qRT-PCR) assay (a). The levels of PCBP2 in HEB cells and U251 cells were examined by qRT-PCR assay (b). PCBP2 protein expression in noncancerous tissues and glioma tissues were examined by western blot analysis (c). The representative immunohistochemical (IHC) staining images showing the expression patterns of PCBP2 protein in noncancerous tissues and glioma tissues (×200) (d). Data are presented as mean ± SD (n = 3). **p < .01. SD, standard deviation F I G U R E 2 The efficiency of si-PCBP2 was confirmed by quantitative real-time polymerase chain reaction (a) and Western blot analysis (b). Data are presented as mean ± SD (n = 3). ***p < .001

F I G U R E 3 Effects of poly (C)-binding protein 2 (PCBP2) expression on biological behaviors of glioma cells in vitro. The knockdown of PCBP2 expression could significantly inhibit proliferation (a), migration (b) and invasion (c) of glioma cells. Furthermore, inhibition of PCBP2 expression could promote cell apoptosis (d). Data are presented as mean ± SD (n = 3). *p < .05, **p < .01, and ***p < .001. SD, standard deviation

The results suggested that the expression of FHL3 was obviously increased in glioma cells with the knockdown of PCBP2 (p < .05 and Figure 4a ,b). PCBP2 could regulate the expression of FHL3 in glioma.

Moreover, western blot method was used to investigate the expressions of TGF-β/Smad signaling pathway proteins in glioma cells transfected by si-PCBP2 and the results showed that the expression of TGF-β1, p-Smad2, and p-Smad7 proteins were downregulated in glioma cells after PCBP2 knockdown. However, the expression of Smad2 and Smad7 did not show obvious changes (Figure 4c ).

Luciferase reporter assay was used to investigate the link between PCBP2 and FHL3. The glioma cell U251 was cotransfected by wt or mt and si-PCBP2 or si-NC plasmids. The relative luciferase activity was significantly reduced after PCBP2 knockdown in cells transfected by FHL3-wt (p < .01, Figure 5a ). However, the luciferase activity in cells transfected by FHL3-mt did not show obvious changes after PCBP2 silencing (NS: nonsignificant). All the data revealed the target relationship between PCBP2 and FHL3.

Nucleotide sequence of FHL3 target binding site and the core recognition sequence are red (Figure 5a and Han et al., 2013) .

in glioma cells. The glioma cell line U251 was transfected by pLV-FHL3 and corresponding control (pLV-NC) in vitro. qRT-PCR method was used to investigate the relative expression of PCBP2

and FHL3 in transfected cells. Expression of FHL3 mRNA was significantly increased after pLV-FHL3 transfection (p < .01, Figure 5b ), while PCBP2 expression did not show obvious changes (p > .05, Figure 5d ). The results of western blot analysis was also showed after pLV-FHL3 transfection, the expression of FHL3

protein was increased significantly (p < 0.01 and Figure 5c ), while the expression of PCBP2 did not change significantly (p > .05 and Figure 5e ). Therefore, PCBP2 could inhibit FHL3 expression, while FHL3 did not influence PCBP2 expression. The data indicated that FHL3 was located on the downstream of PCBP2. FHL3 might be a potential target gene of PCBP2 in glioma.

F I G U R E 4 Poly (C)-binding protein 2 (PCBP2) knockdown could promote FHL3 expression and suppress TGF-β/Smad signaling pathway. The knockdown of PCBP2 could significantly enhance the expression of FHL3 in glioma cells (a and b). Knockdown of PCBP2 might inhibit the activity of TGF-β/Smad signaling pathway (c). Data are presented as mean ± SD (n = 3). *p < .05, **p < .01, ***p < .001. SD, standard deviation 3.7 | Oncogenic function of PCBP2 in glioma was mediated by FHL3

To explore the molecular mechanisms of PCBP2 in the progression of glioma, U251 was cotransfected by si-PCBP2 and si-FHL3. Western blot analysis suggested that compared with glioma cells only transfected by si-PCBP2, the cotransfection of si-PCBP2 and si-FHL3 could significantly enhance the expression of TGF-β1, p-Smad2, and p-Smad7 proteins, suggesting the activation of TGF-β/Smad signaling pathway (Figure 6 ).

F I G U R E 5 FHL3 was a potential target of poly (C)-binding protein 2 (PCBP2) in glioma.The nucleotide sequence of FHL3 target binding site and the core recognition sequence are red (a). The loss of PCBP2 expression could reduce the luciferase activity of cell transfected by FHL3-wt, but it did not influence the cells transfected by FHL3-mt (b). The transfection of pLV-FHL3 vector could distinctly enhance the expression of FHL3 (c and d), but had no significant influence on PCBP2 expression (e and f). Data are presented as mean ± SD (n = 3). **p < .01, (NS, nonsignificant, p > .05)

In addition, the proliferation (p < .05, Figure 7a ), migration (p < .01

and Figure 7b ) and invasion (p < .01 and Figure 7c ) abilities of cotransfected cells were distinctly enhanced, and the cell apoptosis (p < .001 Figure 7d ) was decreased. All the data revealed that si-FHL3 transfection could reverse the antitumor action caused by the knockdown of PCBP2 in glioma.

In this study, we also carried out the animal assay to verify the role of 

Glioma is a deadly malignancy, accounting for about 80% primary brain cancer (Omuro & DeAngelis, 2013) . To improve the outcome of glioma, it is crucial to explore the molecular mechanisms underlying the tumor progression of glioma. In the current study, we found that PCBP2 silencing could inhibit glioma cell proliferation, migration and invasion, and promote cell apoptosis in vitro. PCBP2 might participate in glioma progression through regulating FHL3/TGF-β/Smad signaling pathway.

PCBP2 is a RNA-binding protein that can regulate RNA processing (Collier et al., 1998; Wan et al., 2016) . Abnormal expression of PCBP2 may lead to genetic alterations, thus leading to human diseases, like cancer. In our study, we found that the expression of PCBP2 was significantly upregulated in glioma tissues and cells, compared with noncancerous tissues and normal cells. Furthermore, the elevated expression of PCBP2 showed the positive association with tumor size and WHO stages of glioma.

The conclusion was consistent with the previous studies. Luo et al. reported that the high expression of PCBP2 was closely correlated with advanced tumor stage and poor prognosis of GBM patients and it might be a potential prognostic biomarker for GBM patients (Luo & Zhuang, 2017) . Increased expression of PCBP2 may be a risk hallmark for glioma patients, predicting aggressive cancer progression.

Given their function in RNA processing process, PCBP2 is involved in several cellular processes, such as cell proliferation, migration and invasion, as well as apoptosis. In the present study, in vitro experiments were performed to investigate the effects of In addition, we also investigated the molecular mechanisms of PCBP2 in glioma progression. We found that the knockdown of PCBP2 might lead to high expression of FHL3 and low activity of TGF- tumor suppressor p21 and repression of oncogene c-myc (Ding et al., 2009 ). In the current study, we also found that knockdown of FHL3 could reverse the tumor-suppressive effects caused by PCBP2

silencing expression in glioma. Therefore, PCBP2 may activate TGF-β/Smad signaling pathway through suppressing FHL3, thus contributing to the progression of glioma.

Certainly, several limitations in the current study should be stated. First, the sample size was relatively small that might reduce the statistical power of the current study. Second, the interaction between PCBP2 and other genes, as well as relative signaling pathways which might play potential roles in cancer development were not investigated in our study. The progression F I G U R E 7 Inhibition of FHL3 expression could reverse the antitumor actions induced by the knockdown of poly (C)-binding protein 2 (PCBP2) in glioma cells in vitro. Compared to the control groups, the cells proliferation (a), migration (b) and invasion (c) were significantly enhanced, while cell apoptosis (d) was downregulated in + si-FHL3 group. The cells in control group were transfected by si-PCBP2 vector only, while the cells in + si-FHL3 group were cotransfected by si-PCBP2 and si-FHL3 vectors. Data are presented as mean ± SD (n = 3). *p < .05, **p < .01, and ***p < .001. SD, standard deviation MAO ET AL. 

Data available on request from the authors.

With the approval of Central Laboratory, Harrison International Peace Hospital Ethics Committee, written informed consent was obtaining from every subject.

http://orcid.org/0000-0002-4216-0702

PCBP2 enables the cadicivirus IRES to exploit the function of a conserved GRNA tetraloop to enhance ribosomal initiation complex formation

Poly(rC) binding protein 2 (PCBP2) promotes the viability of human gastric cancer cells by regulating CDK2

Spatiotemporal expression of poly(rC)-binding protein PCBP2 modulates Schwann cell proliferation after sciatic nerve injury

Translational inhibition in vitro of human papillomavirus type 16 L2 mRNA mediated through interaction with heterogenous ribonucleoprotein K and poly(rC)-binding proteins 1 and 2

The worldwide incidence and prevalence of primary brain tumors: A systematic review and metaanalysis

The molecular and phenotypic basis of the glioma invasive perivascular niche

Human four-and-a-half LIM family members suppress tumor cell growth

Tumor volume was significantly decreased in nude mice with PCBP2 knockdown (a and b); Tumor weight was obviously lower in si-PCBP2 group than that in si-NC group (c); The expression level of FHL3 was also upregulated in si-PCBP2 group (d). *p < .05, **p < .01, ***p < .001 through a TGF-beta-like signaling pathway

Ribotrap analysis of proteins associated with FHL3 3′untranslated region in glioma cells

RNAbinding protein PCBP2 modulates glioma growth by regulating FHL3

Characterisation of two major cellular poly(rC)-binding human proteins, each containing three K-homologous (KH) domains

hnRNP L and NF90 interact with hepatitis C virus 5′-terminal untranslated RNA and promote efficient replication

Interplay between PCBP2 and miRNA modulates ARHGDIA expression and function in glioma migration and invasion

Norovirus genome circularization and efficient replication are facilitated by binding of PCBP2 and hnRNP A1

The 2016 World Health Organization Classification of tumors of the central nervous system: aa summary

High expression of PCBP2 is associated with progression and poor prognosis in patients with glioblastoma

PCBP2 modulates neural apoptosis and astrocyte proliferation after spinal cord injury

Molecular and genetic determinants of glioma cell invasion

Glioblastoma and other malignant gliomas: a clinical review

The epidemiology of glioma in adults: A "state of the science

SARS-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the MAVS/TRAF3/TRAF6 signalosome

MicroRNA-214 targets PCBP2 to suppress the proliferation and growth of glioma cells

Advances in genetic and epigenetic analyses of gliomas: A neuropathological perspective

Distinct poly(rC) binding protein KH domain determinants for poliovirus translation initiation and viral RNA replication

beta2-adrenergic receptor signaling promotes pancreatic ductal adenocarcinoma (PDAC) progression through facilitating PCBP2-dependent c-myc expression

PCBP2 enhances the antiviral activity of IFN-alpha against HCV by stabilizing the mRNA of STAT1 and STAT2

Overexpression of PCBP2 contributes to poor prognosis and enhanced cell growth in human hepatocellular carcinoma

PCBP2 promotes the development of glioma by regulating FHL3/ TGF-β/Smad signaling pathway