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β-catenin pathway
Biochemical and Biophysical Research Communications xxx (2016) 1e9
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Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway Ting Lu a, 1, Zhen Bao b, 1, Yunfeng Wang a, Lixiang Yang a, Bing Lu a, Ke Yan a, Shaozhen Wang a, He Wei a, Zhe Zhang c, Gang Cui a, * a
Department of Neurosurgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, People's Republic of China Department of Neurosurgery, The Affiliated Dushuhu Hospital of Soochow University, Suzhou, Jiangsu Province, People's Republic of China Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College of Nantong University, Nantong, Jiangsu Province, People's Republic of China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 August 2016 Accepted 14 August 2016 Available online xxx
Karyopherinb1 (KPNB1), one of the cytosolic factors involved in the selective protein transport across nucleus, docked at nuclear pore complex and transported through nuclear envelope in an ATPdependent style, assisting proteins to be recognized as import substrates. It has been reported to be bound up with the origination and progress of lung cancer, cervical cancer, head and neck cancer and hepatocellular carcinoma. In current study, we demonstrated for the first time that the role of KPNB1 in human glioma. KPNB1 was over-expressed as the well-known trend of Ki-67(p < 0.01) and tightly closed to poor prognosis, as an independent prognostic factor. In vitro, up-regulation of KPNB1 was accompanied by certain rising levels of proliferation markers, employing U251 and U87MG cells as serumstarve models. Silencing KPNB1 in U251 and U87MG led to G1 phase arrested directly via flow cytometry analysis. In the nucleus of KPNB1-depletion cell models, the decreasing expression of KPNB1 and bcatenin was detected respectively, which indicated that KPNB1 functioned via b-catenin signal. Besides, the interaction between KPNB1 and b-catenin was proved clearly by immunoprecipitation. Taken together, it showed that KPNB1 might enhance human glioma proliferation via Wnt/b-Catenin Pathway. © 2016 Elsevier Inc. All rights reserved.
Keywords: Human glioma KPNB1 Wnt/b-Catenin Proliferation
1. Introduction According to the most authoritative reports, human neural glioma is the most lethal brain tumor. It is also the most common kind of primary adult brain neoplasms with poor outcome for patient [1,2]. The 5-years survival is very low to the glioma patients, when they are diagnosed as glioblastoma multiform [3,4]. Even though there is developmentof neurosurgery and chemo-radiotherapy, glioma still shows high resistance to current treatments, and the improvements in patient's outcome is not significant [5]. As a result, it will make sense to investigate the molecular pathological mechanism thoroughly and discover more specific biomarkers or potential treatment targets.
* Corresponding author. Department of Neurosurgery, The First Affiliated Hospital of Soochow University, NO.188 Shizi Street, Gusu District, Suzhou, Jiangsu Province, People's Republic of China. E-mail address: [email protected] (G. Cui). 1 The authors contributed equally to this work.
Wnts, a kind of secreted glycoproteins, play an important part in cell proliferation, differentiation and oncogenesis [6]. Wnt signal, transduced through the disheveled protein, can regulate glycogen synthase kinase-3b (GSK-3b) negatively, leading to the cytoplasmic b-catenin accumulation [7]. Then, b-catenin is carried into the nucleus and forms a complex with T cell factor/lymphocyte enhancer factors (TCF/LEF) to activate the expression of b-catenin responsive genes, such as c-Jun, cyclin-D1, c-Myc, and peroxisome proliferator-activated receptor-d [8e10]. More study shows that the phosphorylation of GSK-3b is always result from Wnt and other growth stimuli, and it inactivates GSK-3b-mediated phosphorylation at the Ser-33/37/Thr-41 sites and strengthens the stabilization of b-catenin, resulting in the subsequent translocation to the nucleus [11]. Proteins transport across nucleus is a selective, multistep process. Karyopherinb1 (KPNB1) is one of the karyopherin proteins functioning as transporting cargo proteins of cell nucleus [12], and some study has proved they are associated with cell-cycle [13]. According to the classical nuclear transport mechanism, karyopherin-a protein recognizes and binds to the nuclear localization
http://dx.doi.org/10.1016/j.bbrc.2016.08.093 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093
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T. Lu et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e9
signal of the cytoplasmic cargo protein [14]. Karyopherinb1 acts as an adapter, tethering the karyopherina1-substrate complex to docking proteins on nuclear envelope. Then, karyopherinb1 binds to karyopherina. Through the nuclear pore complex, the karyopherinb1-karyopherin-a-cargo complex is actively transported into nucleus [15e17]. In the nucleus, the karyopherinb1karyopherin-a-cargo complex dissociates which is triggered by the binding of the small GTPase Ran to karyopherinb1 [14]. Unbound karyopherinb1 is then recycled directly back to the cytoplasm [18]. It has been demonstrated that KPNB1 is associated with cell proliferation and survival in lung, head and neck cancer [19]. The fact indicates that KPNB1 might play a key role in tumorigenesis. According to previous researches, KPNB1 is able to assist the translocation of b-catenin which accelerates the proliferation of human glioma. In this study, we investigated the expression and interaction of KPNB1 and b-catenin in human glioma tissues and cell lines. The results demonstrated that KPNB1 assisted the translocation of b-catenin to nucleus and this function might be helpful to accelerate cells proliferation as many other pathways. We sought to discovery the molecular pathological mechanisms of human glioma with better understanding and more researches are on the way.
negative-control groups with non-specific immunoglobulin IgG(Sigma Chemical Co. St.Louis,MO,USA). Finally, we rinsed the slides in PBS and incubated in hematoxylin, dehydrated and mounted successively in resin mount. 2.5. Immunohistochemical evaluation In a blinded way, two independent veteran pathologists evaluated the stained sections without any patients' clinical-pathological variables. We selected five random high-power fields(Leica microscope Germany) and counted more than 300 cells in each field to determine the labeling index(LI) as the percentage of immunostained cells relative to the total number of cells [20]. We evaluated intensity comparing to the control and scored as follows: negative staining, 0; weak staining, 1; moderate staining, 2; strong staining, 3. We scored the percentage of tumor cells stained positive as follows: <1% positive tumor cells, 0; 1e10% positive tumor cells, 1; 10e50% positive tumor cells, 2; 50e75% positive tumor cells, 3; >75% positive tumor cells, 4. Then the scores of intensity and percentage were added as 0 and 2e7. Negative: 0; weak stained: 2e3; moderate stained: 4e5; strong stained: 6e7.0e3 meant low expression, while 4e7 meant over-expression [21]. 2.6. Cellular fractionation
2. Materials and methods 2.1. Patients and tissue specimens The work of tissue collection was in accordance with The Code of Ethics of the World Medical Association. We collected the human glioma tissues from 100 astrocytomas surgical specimens without any therapies in advance. The clinical-pathological data derived from the Department of Pathology, Affiliated Hospital of Nantong University. 10 patients with epilepsy surgery provided normal brain which was verified for not any tumor organization. 2.2. Cell lines and cell culture Shanghai Institute of Cell Biology supplied us with the human glioma cell lines H4, SHG44, U87MG, U251, A172 and U373. We cultured the cells with DMEM high-glucose medium (GibcoBRL, GrandIsland,NY) containing 10% fetal bovine serum (heat-inactivated) at 37 C with 5% CO2. 2.3. Antibodies The antibodies for immunohistochemistry included: antiKPNB1(diluted 1:400),anti-Ki-67(diluted 1:400). The antibodies for Western Blot included: anti-KPNB1(diluted 1:2000), anti-bcatenin(diluted 1:2000), anti-proliferating cell nuclear antigen(PCNA, diluted 1:1000), anti-cyclinD1(diluted 1:1000), antip27(diluted 1:1000), anti-a-Tublin(diluted 1:1000), antiLaminB(diluted 1:800), anti-glyceraldehyde-3-phosphate dehydrogenase(GAPDH, diluted 1:1500). 2.4. Immunohistochemistry(IHC) We deparaffinized the glioma tissues sections in xylene, rehydrated in graded ethanol solution and blocked the endogenous peroxidase activity in 0.3% hydrogen peroxide. We heated the sections at 105 C in 0,1 M citrate buffer, pH 6.0 for 10 min. To retrieve the antigen, we incubated them at room temperature for 1 h. To block non-specific protein binding, we incubated with 5% bovine serum in PBS(phosphate buffer saline)(pH 7.2), then incubated with certain antibodies at 4 Covernight. We treated the
We washed cells twice with phosphate-buffered saline(PBS) and resuspended in buffer A(10 mM HEPES, 50mMNaCl, pH 8.0, 1 mM EDTA, 500 mM sucrose, 0.2% Triton X-100, 1 mMNaF, 1 mM PMSF, 0.5 mM Na3VO4, and 0.5 mM 2-mercaptoethanol, 2 mg/ml aprotinin) for 15 min on ice. We used a Dounce homogenizer to homogenizecells with 20 strokes. After centrifugation, we collected the supernantant as a cytoplasmic fraction. The pelleted nuclei were further washed three times with isotonic sucrose buffer (6 mM MgCl2, 250 mM sucrose, 10 mMTris-HCl, pH 7.4) containing 0.5% nonionic detergent Triton X-100 to dissolve cytoplasmic membrane contaminants [22]. For extracting nuclear proteins, we resuspended the isolated nuclei in buffer C (10 mM HEPES, 350mMNaCl, pH 8.0, 0.1 mM EDTA, 25%glycerol, 2 mg/ml aprotinin, 1 mM PMSF and 0.5 mM 2-mercaptoethanol) with gentle rocking for 30 min at 4 C. We collected the supernatant as nuclear fractionafter centrifugation [23]. 2.7. Immunoprecipitation We cleaved cells with lysis buffer(1 mM EDTA, 150mMNaCl, 0.5% NP-40, 20mMTris, 1 mMNaF, 1 mMPMSF, 1 mM Na3VO4and 2 mg/ml aprotinin) and incubated in primary antibodies at 4 C, then incubated with protein A-Sepharose(Roche) or protein G-Sepharose. Then, we washed the immunocomplexes three times with lysis buffer. We eluted the bound proteins by boiling the samples in sodium dodecyl sulfate (SDS) sample buffer containing 2mercaptoethanol for single immunoprecipitation [23]. 2.8. Western blot assay We homogenized glioma tissue samples in lysis buffer(50 mmol/L Tirs,1% Nonidet P-40, pH 7.5, 1% SDS, 5 mmol/ LEDTA, 1% triton X-100,1% sodium deoxycholate, 1 mmol/L PMSF, 1 mg/ml leupeptin, 10 mg/ml aprotinin). We washed cell samples three times with PBS, suspended in 2X lysis buffer(120mMNaCl,50mMTris-HCl, 100 mMNaF,0.5% Nonidet P-40, protease inhibitor mixture, 200 M Na3VO4), then denatured at 100 C for 15min and evaluated with Bio-Rad protein assay(BioRad, Hercules, CA, USA) for total protein concentration, and finally stored at 20 C. We separated the protein samples via SDS-
Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093
T. Lu et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e9
polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride filter(PVDF) membranes(Milipore Bedford MA). Then we soaked the membranes in TBST(20mMTris, 150mMNaCl, 0.05% Tween-20) with 5% evaporated milk for 2 h at room temperature, incubated with antibodies at 4 C for 6e8 h, washed with TBST for 5 min, 3 times, incubated in horseradish peroxidase-linked IgG for 2 h at room temperature, and we detected via infrared imaging system(Odyssey, USA). We quantified the blot band intensity by ImageJ analysis file(Wayne Rasband, National Institutes of Health, US). 2.9. RNA interference of KPNB1 We customized the KPNB1-shRNAs inGeneChemcompany.KPNB1-shRNA#1 target sequence: shRNA#1: 50 GAGATCGAAGACTAACAA-30 , shRNA#2: 50 -CAGTGTAGTTGTTCGAGAT-30 , shRNA#3: 50 -ACGAGAAGTCAAGAACTAT-30 , shRNA#4: 50 GCTGTTAGTGAGCTAAGTA-3’. We transfected cells with 100 nmol/L of shRNAs performed with Lipofectamine 2000(Invitrogen, Carlsbad, CA) as experiment groups and with control shRNA as negativecontrol groups. The mock groups were treated with noting. 2.10. Flow cytometry analysis of cell cycle We fixed cells with 70% methanol in PBSat-20 C for 48 h, incubated with 1 mg/mL RNase A for 20 min at 37 C and stained with 0.5 mg/mL of propidium iodide(PI). DNA contents were analyzed via Becton Dickinsonflow cytometer BD FACScan (San Jose, CA).
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experiment repeats (n) ¼ 3). P < 0.05 was considered statistically significant. 3. Results 3.1. The expression of KPNB1 and Ki67 in human glioma were correlative with the WHO grades of human glioma We detected the expression of KPNB1 by Western Blot analysis in normal brain tissues and three couples of glioma tissues in the rank of WHO grade II, III and IV. We found the significant rising-up tendency of KPNB1 in normal brain tissues, low-grade and highgrade glioma tissues (Fig. 1A.B. p < 0.01). The consistent correlation of the expression of Ki-67 and KPNB1 was shown in 100 specimens of human glioma tissues by immunohistochemical staining (r ¼ 0.556, p < 0.01) (Fig. 1C D). We summarized the clinical-pathological statistics and testified the close relationship of KPNB1 to WHO grades of glioma(Table 1, P ¼ 0.004), as the same as Ki-67 (Table 1, p ¼ 0.001). There was no obvious relevance to other clinical-pathological factors. 3.2. The over-expression of KPNB1 in the specimens was correlative with glioma patients' low 5-years survival ratio and poor prognosis As the Kaplan-Meier survival curves showed, the patients of high expression of KPNB1 suffered lower accumulative 5-years survival ratio (Table 2, p ¼ 0.013), the same as Ki-67(Fig. 1E, F, p < 0.01). The result of univariate survival analysis implied that the over-expression of the two molecules was notably relative to the poor prognosis of the glioma patients (Table 2).
2.11. Cell Counting Kit (CCK)-8 We plated stably-transfected cells on a 96-well cell culture cluster(Corning Inc, Corning, NY, USA)at 2 104 cells/well in 100 mL medium. We added Cell Counting Kit-8(Dojindo, Kumamoto, Japan) reagents to a subset of wells with different treatments. Then absorbance was measured at 450 nm wavelength an automated plate reader. The 630 nm wavelength worked as reference group. 2.12. Colony formation assay We selected and plated stably-transfected cells in 6 cm culture plates at 100 cells/well and incubated for 12 days at 37 C. Then we washed cells twice with PBS and stained with Giemsa solution. Under a microscope, we counted the number of colonies containing >50 cells. 2.13. Reporter assay (TOP/FOP assay) We plated 3 104 cells in 24-well plates, incubated for 24 h, then transfected cells with a p-TOP-FLASH-Luc reporter construct. P-RL-SV40, a renilla luciferase expression plasmid, as an internal control for transfection efficiency, was co-transfected at a concentration of 10 ng per well. 48 h later, we cleaved cells with passive lysis buffer and used a Dual-Luciferase Reporter Assay System (Promega) to detect luciferase activity. 2.14. Statistics analysis Statistics analysis was operated via SPSS19.0 statistics analysis software. Chi square test was used to analyze the statistical correlations between KPNB1 and Ki-67 expression and other clinicalpathological features. Employing KaplaneMeier method and logrank test, survival analysis was made. All of numerical values were shown as mean ± SEM (Standard error of the mean,
3.3. The expression of KPNB1 in glioma cell lines and the function in cell proliferation For verifying the function of KPNB1 in the development of glioma, two glioma cell lines, U251 and U87MG, were selected for the following experiments. The expression of KPNB1 was highest in them than others (Fig. 1G, H, p < 0.05). In U251 and U87MG cell lines, we conducted the serumstarvation and re-feeding experiment. The flow cytometry analysis told that, when the cells were held in the serum-deprivation environment for 72 h, the proportion of the cells arrested in G1 phase increased to 55.86% and 54.02%. After re-feeding, the proportion of G1 phase decreased and the S phase proportion grew up gradually (Fig. 2A,B,E,F, p < 0.01). Moreover, the levels of the expression of KPNB1 and some other reported proteins concerning cell proliferation, such as PCNA, cyclinD1, p27, were detected. (Fig. 2C,D,G,H, p < 0.01). 3.4. Down-regulated KPNB1 inhibitedglioma proliferation in vitro The silencing efficiency of all KPNB1-shRNAs was compared by western blot from 1 to 4 respectively (Fig. 3A,B, p < 0.01). Obviously, KPNB1-shRNA#3 achieved better knock-down efficiency than others in U251 cell line, as KPNB1-shRNA#1 to U87MG. We operated the flow cytometry analyses to detect the proportion of the transfected cells in G1 and S phases (Fig. 3C,D p < 0.01). Significantly, the cells of G1 phase increased from 60.59% to 70.47% in U251, 51.47%e61.63% in U87MG, and the cells of S phase reduced from 23.48% to 10.73% in U251, 28.48%e16.91% in U87MG. In an addition, we detected the expression level of the PCNA, cyclinD1, p27 (Fig. 3E,F, p < 0.01). CCK-8 assay was made to compare the proliferation of the transfected cells to control-shRNA-transfected groups (Fig. 4A,B, p < 0.01). The relative absorbance of experiment groups was less than the negative-control groups. Employing
Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093
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T. Lu et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e9
Fig. 1. The over-expression of KPNB1 and Ki-67 was detected in glioma tissues, and it was corresponding to low accumulative survival. (A-B)Expression of KPNB1 in 3 couples of glioma tissues and 1 couple of normal tissues were detected via western blot for showing the significant tendency. GAPDH was used as cross reference. Data were presented as mean ± SEM (n ¼ 3, *, P < 0.01). (C)Immunohistochemical staining of KPNB1 and Ki-67 were carried out in 100 glioma specimens. The immunoreactivity rose up as the WHO grades of glioma. Scale bar ¼ 100 mm. (D)The correlation tests showed the strong consistency between KPNB1 and Ki-67(p < 0.01). (EeF) High expression of KPNB1 was close to low accumulative survival, as well as Ki-67(p < 0.01). (GeH) KPNB1 was expressed highest in U251 than others, followed by U87MG (p < 0.05).
Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093
T. Lu et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e9 Table 1 The correlation between the expression of KPNB1 and Ki-67 and clinicopathological parameters in 100 glioma specimens. Variable
Total
Age <40 17 40 83 Gender Female 37 Male 63 Tumor location Frontal 26 Parietal 25 Occipital 20 Temporal 23 Unknown 6 Surgery Biopsy 17 Partial 41 resection Gross total 42 resection Vessel density Normal 23 Increase 77 Tumor diameter <4 cm 41 4 cm 59 Necrosis Absence 40 Presence 60 WHO grade II 36 III 35 IV 29
KPNB1 expression Low
High
12 46
5 37
22 36
P Value
Ki-67 expression
P Value
Low
High
0.291
12 44
5 39
0.283
15 27
0.837
24 32
13 31
0.212
17 9 11 11 5
9 16 9 12 1
0.570
18 12 9 11 1
8 13 11 12 5
0.246
11 23
6 18
0.824
11 22
6 19
0.726
24
18
23
19
16 42
7 35
0.235
15 41
8 36
0.347
23 35
18 24
0.838
23 33
18 26
0.575
27 31
13 29
0.149
27 29
13 31
0.101
27 21 10
9 14 19
0.004*
28 19 9
8 16 20
0.001*
Statistical analyses were performed by the Pearson c2 test. *P < 0.05 was considered significant.
colony formation assay, we observed the same difference (Fig. 4C,D,E, p < 0.01). Furthermore, the results of reporter assay showed weaker relative luciferase activity of experiment groups than the negative groups (Fig. 4G,H, p < 0.01). 3.5. Down-regulated KPNB1 weakened b-catenin transport into nuclear and the direct interaction between them was detected We observed the great declined b-catenin expression in the nucleus of KPNB1-depletion cells, and the expression level in cytoplasm was higher than the control groups (Fig. 4I, J). It meant knock-down KPNB1 actually cut down on b-catenin transport across the nuclear envelope. In other words, KPNB1 indeed played a part in Wnt/b-catenin pathway. According to the evidence above, we desired to identify the molecule mechanism between them. The immunoprecipitation presented us an interesting result that direct interaction exited (Fig. 4K). 4. Discussion As we all know, in human central nervoussystem(CNS), human glioma is the most common type of malignancy [24]. It is still a typical deadly cancer among the neurological tumors. Significantly, glioblastoma is the most aggressive and lethal cancer with an average 15 months survival time after diagnosis [25]. The current standard therapy is made up with maximal safe resection, radiotherapy and temozolomide [25]. The key stages in cancer progression of malignant glioma cell include proliferation and invasion [26]. Therefore, investigating the underlying molecular
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Table 2 Contribution of various potential prognostic factors to survival by univariate analysis in 100 glioma specimens. Characteristics
Age <40 40 Gender Female Male Tumor Location Frontal Parietal Occipital Temporal Unknown Surgery Biopsy Partial resection Gross Total resection Vessel Density Normal Increase Tumor Diameter <4 cm 4 cm Necrosis Absence Presence WHO Grade II III IV Kpnb1expression Low High Ki-67 expression Low High
Total
Survival status
P Value
<5years
5years
17 83
9 46
8 37
0.529
37 63
14 41
23 22
0.054
26 25 20 23 6
13 14 14 11 3
13 11 6 12 3
0.622
17 41 42
10 23 22
7 18 20
0.888
23 77
13 42
10 35
0.530
41 59
21 34
20 25
0.547
40 60
19 36
21 24
0.228
36 35 29
8 22 25
28 13 4
0.002*
58 42
26 29
32 13
0.013*
56 44
21 34
35 10
0.000*
Statistical analyses were performed by the Pearson c2 test. *P < 0.05 was considered significant.
pathological mechanism of human glioma is very urgent. In current study, we presented that KPNB1 could be involved in the molecule mechanisms of human glioma proliferation, at least partially, via the Wnt/b-catenin pathway. Karyopherinb1 (KPNB1) is one of the karyopherin proteins, which functions as the transporting cargo proteins into and out of the cell nucleus [12], a selective, multistep process. It was elucidated that cell cycle could be arrested by KPNB1 inhibition which was associated with NF-kB pathway in hepatocellular carcinoma [27]. Based on the World Health Organization(WHO) pathological criteria, there are four grades of human glioma as follows: WHO grade I, pilocytic astrocytoma; WHO grade II, diffuse low grades glioma; WHO grade III, anaplastic gliomas; WHO grade IV, glioblastoma(GBM) [28]. According to the clinical-pathological statistics, we confirmed that the over-expression of KPNB1 was great correlative with the WHO grades of human glioma, which meant a low 5-years survival ratio. Moreover, we observed that the expression of KPNB1 was correlative with certain proliferation markers, cyclin D1, PCNA and p27, a cell cycle suppressor gene. It demonstrated the function of KPNB1 in proliferation. However, KNPB1 mediates the nucleolar import of some other proteins as well, such as NF kappa b, XPO7, and IPO8. The potential molecule mechanism of KPNB1 was still unclear. In some reports, the suppression of Wnt/b-catenin pathway was able to inhibit the phenotype and proliferation rate in human colon carcinoma cells [29]. Since KPNB1 was an important across-nuclear envelope transport protein, we could make this fearless hypothesis
Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093
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that KPNB1 promotes proliferation by inducing cell cycle via Wnt/ b-catenin pathway. Human Wnts consist of 19 secreted proteins which work in proliferation, migration, differentiation, survival, and lineage decisions in many kinds of cells during development and adult life [30]. If the Wnt signals are absent, a cytoplasmic
protein complex, the b-catenin destruction complex, will target free b-catenin [29]. When the Wnts and membrane heterodimeric receptors are bound up together, the destruction complexes and subsequent unphosphorylatedb-catenin molecules accumulation in the cytoplasm will be inhibited. At the same time, a part of the b-
Fig. 2. The expression of KPNB1 and the different phases of cell cycle in starve and re-feeding models. (A-B, E-F)Flow cytometry showed the changes in cell cycles of U251 and U87MG cells after re-feeding from serum-starve for 72 h (C-D, G-H)Several cell cycle and proliferation makers, PCNA, cyclin D1 were in the increasing trend with KPNB1 at each time point, p27 was in opposite. GAPDH was used as control protein load. Data were presented as mean ± SEM (n ¼ 3, *, P < 0.01, compared with control-0h).
Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093
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Fig. 3. Depletion of KPNB1 weakened the ability of proliferation in U251 and U87MG cells. (AeB) shRNA#3 showed the highest level of effectiveness to U251 and shRNA#1 was the same to U87MG cells. (CeD) 70.47% and 61.63% of the KPNB1-depletion cells were arrested in G1 phase. (E-F, G-H)CyclinD1, p27, PCNA were detected via western blot showing the variation trend as the inhibited proliferation. Data were presented as mean ± SEM (n ¼ 3, *, P < 0.01, compared to the Ctrl-groups).
Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093
Fig. 4. The weakened proliferation of KPNB1-depletion cells in CCK8 and colonies formation. (AeB) Cells transfected with shRNA#1 and #3 were cultivated in the 96-wells plates for detect the relative absorbance on 450 nm at each time point of 0 h, 24 h, 48 h, 72 h, 96 h. The obvious difference was observed at 72 h and 96 h. Data were presented as mean ± SEM (n ¼ 3, *, P < 0.01). (CeF) The selected and stably-transfected cells were cultivated into the 6 cm culture plates and incubated for 12 days at 37 C with 5% CO2. Stained with Giemsa solution, we observed the diversity in the KPNB1-depletion cells. (G-H)Reporter assay was carried out in the KPNB1-depletion U251 and U87MG cells Data was presented as mean ± SEM (n ¼ 3, *, P<0.01). (I-J)Via cells fractionation, b-catenin was detected to be decreased in the nucleus of KPNB1-depletion U251 and U87MG cells. (K)A kind of direct interaction exists between b-catenin and KPNB1. With immunoprecipitation and western-blot, the result showed the direct interaction between b-catenin and KPNB1.
Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093
T. Lu et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e9
cateninmolecules enter the nucleus and work as a co-activator of LEF/TCF transcription factors [31]. The association withb-catenin transiently converts TCF into a transcriptional activatorto its target genes, with modulation of TCF comingfrom phosphorylation [32]. Neverthless, the details are unclear that howb-catenin shuttles between the cytoplasmand the nucleus, although recent evidence suggestsa role for microtubules and active transport [33]. Here, we find that KPNB1 works as a transport-protein in nuclear membrane as known. It might assist b-catenin transcription across nuclear membrane with Wnt signal. Besides, b-catenin/LEF/TCF target genes encode proteins involved in many cellular processes, such as proliferation, cell cycle regulation, metabolism, migration, lineage commitment, and differentiation [34]. In current study, we demonstrated the function of KPNB1 in the Wnt/b-catenin pathway as medium. With more b-catenin transported into nucleus, the TCF/ LEF complex was necessarily activated. However, there is an outstanding question turning up. As we all know, KNPB1 also mediates the nucleolar import of some other proteins, such as NF-kb, XPO7, and IPO8 and so on which may be also involved in cell proliferation more or less. Though we have demonstrated the cofunction of KNPB1 and Wnt/b-cateninto cell proliferation, it is unwise to neglect the crosstalk of other signal pathways. Moreover, to identify our hypothesis fully and particularly, further study to uncover the mechanism is currently in progress. Conflict of interest No competing financial interests exist. Acknowledgements This work was supported by grants from National Natural Science Foundation of China (No.81372687). References [1] S. Deorah, C.F. Lynch, Z.A. Sibenaller, T.C. Ryken, Trends in brain cancer incidence and survival in the United States: surveillance, epidemiology, and end results program, 1973 to 2001, Neurosurg. focus 20 (2006) E1. [2] D.A. Reardon, J.E. Herndon 2nd, K.B. Peters, et al., Bevacizumab continuation beyond initial bevacizumab progression among recurrent glioblastoma patients, Br. J. cancer 107 (2012) 1481e1487. [3] E.A. Maher, F.B. Furnari, R.M. Bachoo, et al., Malignant glioma: genetics and biology of a grave matter, Genes & Dev. 15 (2001) 1311e1333. [4] W. Zhuang, Z. Qin, Z. Liang, The role of autophagy in sensitizing malignant glioma cells to radiation therapy, Acta biochimica biophysica Sinica 41 (2009) 341e351. [5] P.Y. Wen, S. Kesari, Malignant gliomas in adults, N. Engl. J. Med. 359 (2008) 492e507. [6] P. Polakis, Wnt signaling and cancer, Genes & Dev. 14 (2000) 1837e1851. [7] I.R. Cho, S.S. Koh, H.J. Min, et al., Pancreatic adenocarcinoma up-regulated factor (PAUF) enhances the expression of beta-catenin, leading to a rapid proliferation of pancreatic cells, Exp. Mol. Med. 43 (2011) 82e90. [8] T.C. He, A.B. Sparks, C. Rago, et al., Identification of c-MYC as a target of the APC pathway, Science 281 (1998) 1509e1512. [9] T.C. He, T.A. Chan, B. Vogelstein, K.W. Kinzler, PPARdelta is an APC-regulated
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Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway Ting Lu a, 1, Zhen Bao b, 1, Yunfeng Wang a, Lixiang Yang a, Bing Lu a, Ke Yan a, Shaozhen Wang a, He Wei a, Zhe Zhang c, Gang Cui a, * a
Department of Neurosurgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, People's Republic of China Department of Neurosurgery, The Affiliated Dushuhu Hospital of Soochow University, Suzhou, Jiangsu Province, People's Republic of China Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College of Nantong University, Nantong, Jiangsu Province, People's Republic of China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 August 2016 Accepted 14 August 2016 Available online xxx
Karyopherinb1 (KPNB1), one of the cytosolic factors involved in the selective protein transport across nucleus, docked at nuclear pore complex and transported through nuclear envelope in an ATPdependent style, assisting proteins to be recognized as import substrates. It has been reported to be bound up with the origination and progress of lung cancer, cervical cancer, head and neck cancer and hepatocellular carcinoma. In current study, we demonstrated for the first time that the role of KPNB1 in human glioma. KPNB1 was over-expressed as the well-known trend of Ki-67(p < 0.01) and tightly closed to poor prognosis, as an independent prognostic factor. In vitro, up-regulation of KPNB1 was accompanied by certain rising levels of proliferation markers, employing U251 and U87MG cells as serumstarve models. Silencing KPNB1 in U251 and U87MG led to G1 phase arrested directly via flow cytometry analysis. In the nucleus of KPNB1-depletion cell models, the decreasing expression of KPNB1 and bcatenin was detected respectively, which indicated that KPNB1 functioned via b-catenin signal. Besides, the interaction between KPNB1 and b-catenin was proved clearly by immunoprecipitation. Taken together, it showed that KPNB1 might enhance human glioma proliferation via Wnt/b-Catenin Pathway. © 2016 Elsevier Inc. All rights reserved.
Keywords: Human glioma KPNB1 Wnt/b-Catenin Proliferation
1. Introduction According to the most authoritative reports, human neural glioma is the most lethal brain tumor. It is also the most common kind of primary adult brain neoplasms with poor outcome for patient [1,2]. The 5-years survival is very low to the glioma patients, when they are diagnosed as glioblastoma multiform [3,4]. Even though there is developmentof neurosurgery and chemo-radiotherapy, glioma still shows high resistance to current treatments, and the improvements in patient's outcome is not significant [5]. As a result, it will make sense to investigate the molecular pathological mechanism thoroughly and discover more specific biomarkers or potential treatment targets.
* Corresponding author. Department of Neurosurgery, The First Affiliated Hospital of Soochow University, NO.188 Shizi Street, Gusu District, Suzhou, Jiangsu Province, People's Republic of China. E-mail address: [email protected] (G. Cui). 1 The authors contributed equally to this work.
Wnts, a kind of secreted glycoproteins, play an important part in cell proliferation, differentiation and oncogenesis [6]. Wnt signal, transduced through the disheveled protein, can regulate glycogen synthase kinase-3b (GSK-3b) negatively, leading to the cytoplasmic b-catenin accumulation [7]. Then, b-catenin is carried into the nucleus and forms a complex with T cell factor/lymphocyte enhancer factors (TCF/LEF) to activate the expression of b-catenin responsive genes, such as c-Jun, cyclin-D1, c-Myc, and peroxisome proliferator-activated receptor-d [8e10]. More study shows that the phosphorylation of GSK-3b is always result from Wnt and other growth stimuli, and it inactivates GSK-3b-mediated phosphorylation at the Ser-33/37/Thr-41 sites and strengthens the stabilization of b-catenin, resulting in the subsequent translocation to the nucleus [11]. Proteins transport across nucleus is a selective, multistep process. Karyopherinb1 (KPNB1) is one of the karyopherin proteins functioning as transporting cargo proteins of cell nucleus [12], and some study has proved they are associated with cell-cycle [13]. According to the classical nuclear transport mechanism, karyopherin-a protein recognizes and binds to the nuclear localization
http://dx.doi.org/10.1016/j.bbrc.2016.08.093 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093
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T. Lu et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e9
signal of the cytoplasmic cargo protein [14]. Karyopherinb1 acts as an adapter, tethering the karyopherina1-substrate complex to docking proteins on nuclear envelope. Then, karyopherinb1 binds to karyopherina. Through the nuclear pore complex, the karyopherinb1-karyopherin-a-cargo complex is actively transported into nucleus [15e17]. In the nucleus, the karyopherinb1karyopherin-a-cargo complex dissociates which is triggered by the binding of the small GTPase Ran to karyopherinb1 [14]. Unbound karyopherinb1 is then recycled directly back to the cytoplasm [18]. It has been demonstrated that KPNB1 is associated with cell proliferation and survival in lung, head and neck cancer [19]. The fact indicates that KPNB1 might play a key role in tumorigenesis. According to previous researches, KPNB1 is able to assist the translocation of b-catenin which accelerates the proliferation of human glioma. In this study, we investigated the expression and interaction of KPNB1 and b-catenin in human glioma tissues and cell lines. The results demonstrated that KPNB1 assisted the translocation of b-catenin to nucleus and this function might be helpful to accelerate cells proliferation as many other pathways. We sought to discovery the molecular pathological mechanisms of human glioma with better understanding and more researches are on the way.
negative-control groups with non-specific immunoglobulin IgG(Sigma Chemical Co. St.Louis,MO,USA). Finally, we rinsed the slides in PBS and incubated in hematoxylin, dehydrated and mounted successively in resin mount. 2.5. Immunohistochemical evaluation In a blinded way, two independent veteran pathologists evaluated the stained sections without any patients' clinical-pathological variables. We selected five random high-power fields(Leica microscope Germany) and counted more than 300 cells in each field to determine the labeling index(LI) as the percentage of immunostained cells relative to the total number of cells [20]. We evaluated intensity comparing to the control and scored as follows: negative staining, 0; weak staining, 1; moderate staining, 2; strong staining, 3. We scored the percentage of tumor cells stained positive as follows: <1% positive tumor cells, 0; 1e10% positive tumor cells, 1; 10e50% positive tumor cells, 2; 50e75% positive tumor cells, 3; >75% positive tumor cells, 4. Then the scores of intensity and percentage were added as 0 and 2e7. Negative: 0; weak stained: 2e3; moderate stained: 4e5; strong stained: 6e7.0e3 meant low expression, while 4e7 meant over-expression [21]. 2.6. Cellular fractionation
2. Materials and methods 2.1. Patients and tissue specimens The work of tissue collection was in accordance with The Code of Ethics of the World Medical Association. We collected the human glioma tissues from 100 astrocytomas surgical specimens without any therapies in advance. The clinical-pathological data derived from the Department of Pathology, Affiliated Hospital of Nantong University. 10 patients with epilepsy surgery provided normal brain which was verified for not any tumor organization. 2.2. Cell lines and cell culture Shanghai Institute of Cell Biology supplied us with the human glioma cell lines H4, SHG44, U87MG, U251, A172 and U373. We cultured the cells with DMEM high-glucose medium (GibcoBRL, GrandIsland,NY) containing 10% fetal bovine serum (heat-inactivated) at 37 C with 5% CO2. 2.3. Antibodies The antibodies for immunohistochemistry included: antiKPNB1(diluted 1:400),anti-Ki-67(diluted 1:400). The antibodies for Western Blot included: anti-KPNB1(diluted 1:2000), anti-bcatenin(diluted 1:2000), anti-proliferating cell nuclear antigen(PCNA, diluted 1:1000), anti-cyclinD1(diluted 1:1000), antip27(diluted 1:1000), anti-a-Tublin(diluted 1:1000), antiLaminB(diluted 1:800), anti-glyceraldehyde-3-phosphate dehydrogenase(GAPDH, diluted 1:1500). 2.4. Immunohistochemistry(IHC) We deparaffinized the glioma tissues sections in xylene, rehydrated in graded ethanol solution and blocked the endogenous peroxidase activity in 0.3% hydrogen peroxide. We heated the sections at 105 C in 0,1 M citrate buffer, pH 6.0 for 10 min. To retrieve the antigen, we incubated them at room temperature for 1 h. To block non-specific protein binding, we incubated with 5% bovine serum in PBS(phosphate buffer saline)(pH 7.2), then incubated with certain antibodies at 4 Covernight. We treated the
We washed cells twice with phosphate-buffered saline(PBS) and resuspended in buffer A(10 mM HEPES, 50mMNaCl, pH 8.0, 1 mM EDTA, 500 mM sucrose, 0.2% Triton X-100, 1 mMNaF, 1 mM PMSF, 0.5 mM Na3VO4, and 0.5 mM 2-mercaptoethanol, 2 mg/ml aprotinin) for 15 min on ice. We used a Dounce homogenizer to homogenizecells with 20 strokes. After centrifugation, we collected the supernantant as a cytoplasmic fraction. The pelleted nuclei were further washed three times with isotonic sucrose buffer (6 mM MgCl2, 250 mM sucrose, 10 mMTris-HCl, pH 7.4) containing 0.5% nonionic detergent Triton X-100 to dissolve cytoplasmic membrane contaminants [22]. For extracting nuclear proteins, we resuspended the isolated nuclei in buffer C (10 mM HEPES, 350mMNaCl, pH 8.0, 0.1 mM EDTA, 25%glycerol, 2 mg/ml aprotinin, 1 mM PMSF and 0.5 mM 2-mercaptoethanol) with gentle rocking for 30 min at 4 C. We collected the supernatant as nuclear fractionafter centrifugation [23]. 2.7. Immunoprecipitation We cleaved cells with lysis buffer(1 mM EDTA, 150mMNaCl, 0.5% NP-40, 20mMTris, 1 mMNaF, 1 mMPMSF, 1 mM Na3VO4and 2 mg/ml aprotinin) and incubated in primary antibodies at 4 C, then incubated with protein A-Sepharose(Roche) or protein G-Sepharose. Then, we washed the immunocomplexes three times with lysis buffer. We eluted the bound proteins by boiling the samples in sodium dodecyl sulfate (SDS) sample buffer containing 2mercaptoethanol for single immunoprecipitation [23]. 2.8. Western blot assay We homogenized glioma tissue samples in lysis buffer(50 mmol/L Tirs,1% Nonidet P-40, pH 7.5, 1% SDS, 5 mmol/ LEDTA, 1% triton X-100,1% sodium deoxycholate, 1 mmol/L PMSF, 1 mg/ml leupeptin, 10 mg/ml aprotinin). We washed cell samples three times with PBS, suspended in 2X lysis buffer(120mMNaCl,50mMTris-HCl, 100 mMNaF,0.5% Nonidet P-40, protease inhibitor mixture, 200 M Na3VO4), then denatured at 100 C for 15min and evaluated with Bio-Rad protein assay(BioRad, Hercules, CA, USA) for total protein concentration, and finally stored at 20 C. We separated the protein samples via SDS-
Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093
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polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride filter(PVDF) membranes(Milipore Bedford MA). Then we soaked the membranes in TBST(20mMTris, 150mMNaCl, 0.05% Tween-20) with 5% evaporated milk for 2 h at room temperature, incubated with antibodies at 4 C for 6e8 h, washed with TBST for 5 min, 3 times, incubated in horseradish peroxidase-linked IgG for 2 h at room temperature, and we detected via infrared imaging system(Odyssey, USA). We quantified the blot band intensity by ImageJ analysis file(Wayne Rasband, National Institutes of Health, US). 2.9. RNA interference of KPNB1 We customized the KPNB1-shRNAs inGeneChemcompany.KPNB1-shRNA#1 target sequence: shRNA#1: 50 GAGATCGAAGACTAACAA-30 , shRNA#2: 50 -CAGTGTAGTTGTTCGAGAT-30 , shRNA#3: 50 -ACGAGAAGTCAAGAACTAT-30 , shRNA#4: 50 GCTGTTAGTGAGCTAAGTA-3’. We transfected cells with 100 nmol/L of shRNAs performed with Lipofectamine 2000(Invitrogen, Carlsbad, CA) as experiment groups and with control shRNA as negativecontrol groups. The mock groups were treated with noting. 2.10. Flow cytometry analysis of cell cycle We fixed cells with 70% methanol in PBSat-20 C for 48 h, incubated with 1 mg/mL RNase A for 20 min at 37 C and stained with 0.5 mg/mL of propidium iodide(PI). DNA contents were analyzed via Becton Dickinsonflow cytometer BD FACScan (San Jose, CA).
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experiment repeats (n) ¼ 3). P < 0.05 was considered statistically significant. 3. Results 3.1. The expression of KPNB1 and Ki67 in human glioma were correlative with the WHO grades of human glioma We detected the expression of KPNB1 by Western Blot analysis in normal brain tissues and three couples of glioma tissues in the rank of WHO grade II, III and IV. We found the significant rising-up tendency of KPNB1 in normal brain tissues, low-grade and highgrade glioma tissues (Fig. 1A.B. p < 0.01). The consistent correlation of the expression of Ki-67 and KPNB1 was shown in 100 specimens of human glioma tissues by immunohistochemical staining (r ¼ 0.556, p < 0.01) (Fig. 1C D). We summarized the clinical-pathological statistics and testified the close relationship of KPNB1 to WHO grades of glioma(Table 1, P ¼ 0.004), as the same as Ki-67 (Table 1, p ¼ 0.001). There was no obvious relevance to other clinical-pathological factors. 3.2. The over-expression of KPNB1 in the specimens was correlative with glioma patients' low 5-years survival ratio and poor prognosis As the Kaplan-Meier survival curves showed, the patients of high expression of KPNB1 suffered lower accumulative 5-years survival ratio (Table 2, p ¼ 0.013), the same as Ki-67(Fig. 1E, F, p < 0.01). The result of univariate survival analysis implied that the over-expression of the two molecules was notably relative to the poor prognosis of the glioma patients (Table 2).
2.11. Cell Counting Kit (CCK)-8 We plated stably-transfected cells on a 96-well cell culture cluster(Corning Inc, Corning, NY, USA)at 2 104 cells/well in 100 mL medium. We added Cell Counting Kit-8(Dojindo, Kumamoto, Japan) reagents to a subset of wells with different treatments. Then absorbance was measured at 450 nm wavelength an automated plate reader. The 630 nm wavelength worked as reference group. 2.12. Colony formation assay We selected and plated stably-transfected cells in 6 cm culture plates at 100 cells/well and incubated for 12 days at 37 C. Then we washed cells twice with PBS and stained with Giemsa solution. Under a microscope, we counted the number of colonies containing >50 cells. 2.13. Reporter assay (TOP/FOP assay) We plated 3 104 cells in 24-well plates, incubated for 24 h, then transfected cells with a p-TOP-FLASH-Luc reporter construct. P-RL-SV40, a renilla luciferase expression plasmid, as an internal control for transfection efficiency, was co-transfected at a concentration of 10 ng per well. 48 h later, we cleaved cells with passive lysis buffer and used a Dual-Luciferase Reporter Assay System (Promega) to detect luciferase activity. 2.14. Statistics analysis Statistics analysis was operated via SPSS19.0 statistics analysis software. Chi square test was used to analyze the statistical correlations between KPNB1 and Ki-67 expression and other clinicalpathological features. Employing KaplaneMeier method and logrank test, survival analysis was made. All of numerical values were shown as mean ± SEM (Standard error of the mean,
3.3. The expression of KPNB1 in glioma cell lines and the function in cell proliferation For verifying the function of KPNB1 in the development of glioma, two glioma cell lines, U251 and U87MG, were selected for the following experiments. The expression of KPNB1 was highest in them than others (Fig. 1G, H, p < 0.05). In U251 and U87MG cell lines, we conducted the serumstarvation and re-feeding experiment. The flow cytometry analysis told that, when the cells were held in the serum-deprivation environment for 72 h, the proportion of the cells arrested in G1 phase increased to 55.86% and 54.02%. After re-feeding, the proportion of G1 phase decreased and the S phase proportion grew up gradually (Fig. 2A,B,E,F, p < 0.01). Moreover, the levels of the expression of KPNB1 and some other reported proteins concerning cell proliferation, such as PCNA, cyclinD1, p27, were detected. (Fig. 2C,D,G,H, p < 0.01). 3.4. Down-regulated KPNB1 inhibitedglioma proliferation in vitro The silencing efficiency of all KPNB1-shRNAs was compared by western blot from 1 to 4 respectively (Fig. 3A,B, p < 0.01). Obviously, KPNB1-shRNA#3 achieved better knock-down efficiency than others in U251 cell line, as KPNB1-shRNA#1 to U87MG. We operated the flow cytometry analyses to detect the proportion of the transfected cells in G1 and S phases (Fig. 3C,D p < 0.01). Significantly, the cells of G1 phase increased from 60.59% to 70.47% in U251, 51.47%e61.63% in U87MG, and the cells of S phase reduced from 23.48% to 10.73% in U251, 28.48%e16.91% in U87MG. In an addition, we detected the expression level of the PCNA, cyclinD1, p27 (Fig. 3E,F, p < 0.01). CCK-8 assay was made to compare the proliferation of the transfected cells to control-shRNA-transfected groups (Fig. 4A,B, p < 0.01). The relative absorbance of experiment groups was less than the negative-control groups. Employing
Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093
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T. Lu et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e9
Fig. 1. The over-expression of KPNB1 and Ki-67 was detected in glioma tissues, and it was corresponding to low accumulative survival. (A-B)Expression of KPNB1 in 3 couples of glioma tissues and 1 couple of normal tissues were detected via western blot for showing the significant tendency. GAPDH was used as cross reference. Data were presented as mean ± SEM (n ¼ 3, *, P < 0.01). (C)Immunohistochemical staining of KPNB1 and Ki-67 were carried out in 100 glioma specimens. The immunoreactivity rose up as the WHO grades of glioma. Scale bar ¼ 100 mm. (D)The correlation tests showed the strong consistency between KPNB1 and Ki-67(p < 0.01). (EeF) High expression of KPNB1 was close to low accumulative survival, as well as Ki-67(p < 0.01). (GeH) KPNB1 was expressed highest in U251 than others, followed by U87MG (p < 0.05).
Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093
T. Lu et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e9 Table 1 The correlation between the expression of KPNB1 and Ki-67 and clinicopathological parameters in 100 glioma specimens. Variable
Total
Age <40 17 40 83 Gender Female 37 Male 63 Tumor location Frontal 26 Parietal 25 Occipital 20 Temporal 23 Unknown 6 Surgery Biopsy 17 Partial 41 resection Gross total 42 resection Vessel density Normal 23 Increase 77 Tumor diameter <4 cm 41 4 cm 59 Necrosis Absence 40 Presence 60 WHO grade II 36 III 35 IV 29
KPNB1 expression Low
High
12 46
5 37
22 36
P Value
Ki-67 expression
P Value
Low
High
0.291
12 44
5 39
0.283
15 27
0.837
24 32
13 31
0.212
17 9 11 11 5
9 16 9 12 1
0.570
18 12 9 11 1
8 13 11 12 5
0.246
11 23
6 18
0.824
11 22
6 19
0.726
24
18
23
19
16 42
7 35
0.235
15 41
8 36
0.347
23 35
18 24
0.838
23 33
18 26
0.575
27 31
13 29
0.149
27 29
13 31
0.101
27 21 10
9 14 19
0.004*
28 19 9
8 16 20
0.001*
Statistical analyses were performed by the Pearson c2 test. *P < 0.05 was considered significant.
colony formation assay, we observed the same difference (Fig. 4C,D,E, p < 0.01). Furthermore, the results of reporter assay showed weaker relative luciferase activity of experiment groups than the negative groups (Fig. 4G,H, p < 0.01). 3.5. Down-regulated KPNB1 weakened b-catenin transport into nuclear and the direct interaction between them was detected We observed the great declined b-catenin expression in the nucleus of KPNB1-depletion cells, and the expression level in cytoplasm was higher than the control groups (Fig. 4I, J). It meant knock-down KPNB1 actually cut down on b-catenin transport across the nuclear envelope. In other words, KPNB1 indeed played a part in Wnt/b-catenin pathway. According to the evidence above, we desired to identify the molecule mechanism between them. The immunoprecipitation presented us an interesting result that direct interaction exited (Fig. 4K). 4. Discussion As we all know, in human central nervoussystem(CNS), human glioma is the most common type of malignancy [24]. It is still a typical deadly cancer among the neurological tumors. Significantly, glioblastoma is the most aggressive and lethal cancer with an average 15 months survival time after diagnosis [25]. The current standard therapy is made up with maximal safe resection, radiotherapy and temozolomide [25]. The key stages in cancer progression of malignant glioma cell include proliferation and invasion [26]. Therefore, investigating the underlying molecular
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Table 2 Contribution of various potential prognostic factors to survival by univariate analysis in 100 glioma specimens. Characteristics
Age <40 40 Gender Female Male Tumor Location Frontal Parietal Occipital Temporal Unknown Surgery Biopsy Partial resection Gross Total resection Vessel Density Normal Increase Tumor Diameter <4 cm 4 cm Necrosis Absence Presence WHO Grade II III IV Kpnb1expression Low High Ki-67 expression Low High
Total
Survival status
P Value
<5years
5years
17 83
9 46
8 37
0.529
37 63
14 41
23 22
0.054
26 25 20 23 6
13 14 14 11 3
13 11 6 12 3
0.622
17 41 42
10 23 22
7 18 20
0.888
23 77
13 42
10 35
0.530
41 59
21 34
20 25
0.547
40 60
19 36
21 24
0.228
36 35 29
8 22 25
28 13 4
0.002*
58 42
26 29
32 13
0.013*
56 44
21 34
35 10
0.000*
Statistical analyses were performed by the Pearson c2 test. *P < 0.05 was considered significant.
pathological mechanism of human glioma is very urgent. In current study, we presented that KPNB1 could be involved in the molecule mechanisms of human glioma proliferation, at least partially, via the Wnt/b-catenin pathway. Karyopherinb1 (KPNB1) is one of the karyopherin proteins, which functions as the transporting cargo proteins into and out of the cell nucleus [12], a selective, multistep process. It was elucidated that cell cycle could be arrested by KPNB1 inhibition which was associated with NF-kB pathway in hepatocellular carcinoma [27]. Based on the World Health Organization(WHO) pathological criteria, there are four grades of human glioma as follows: WHO grade I, pilocytic astrocytoma; WHO grade II, diffuse low grades glioma; WHO grade III, anaplastic gliomas; WHO grade IV, glioblastoma(GBM) [28]. According to the clinical-pathological statistics, we confirmed that the over-expression of KPNB1 was great correlative with the WHO grades of human glioma, which meant a low 5-years survival ratio. Moreover, we observed that the expression of KPNB1 was correlative with certain proliferation markers, cyclin D1, PCNA and p27, a cell cycle suppressor gene. It demonstrated the function of KPNB1 in proliferation. However, KNPB1 mediates the nucleolar import of some other proteins as well, such as NF kappa b, XPO7, and IPO8. The potential molecule mechanism of KPNB1 was still unclear. In some reports, the suppression of Wnt/b-catenin pathway was able to inhibit the phenotype and proliferation rate in human colon carcinoma cells [29]. Since KPNB1 was an important across-nuclear envelope transport protein, we could make this fearless hypothesis
Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093
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T. Lu et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e9
that KPNB1 promotes proliferation by inducing cell cycle via Wnt/ b-catenin pathway. Human Wnts consist of 19 secreted proteins which work in proliferation, migration, differentiation, survival, and lineage decisions in many kinds of cells during development and adult life [30]. If the Wnt signals are absent, a cytoplasmic
protein complex, the b-catenin destruction complex, will target free b-catenin [29]. When the Wnts and membrane heterodimeric receptors are bound up together, the destruction complexes and subsequent unphosphorylatedb-catenin molecules accumulation in the cytoplasm will be inhibited. At the same time, a part of the b-
Fig. 2. The expression of KPNB1 and the different phases of cell cycle in starve and re-feeding models. (A-B, E-F)Flow cytometry showed the changes in cell cycles of U251 and U87MG cells after re-feeding from serum-starve for 72 h (C-D, G-H)Several cell cycle and proliferation makers, PCNA, cyclin D1 were in the increasing trend with KPNB1 at each time point, p27 was in opposite. GAPDH was used as control protein load. Data were presented as mean ± SEM (n ¼ 3, *, P < 0.01, compared with control-0h).
Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093
T. Lu et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e9
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Fig. 3. Depletion of KPNB1 weakened the ability of proliferation in U251 and U87MG cells. (AeB) shRNA#3 showed the highest level of effectiveness to U251 and shRNA#1 was the same to U87MG cells. (CeD) 70.47% and 61.63% of the KPNB1-depletion cells were arrested in G1 phase. (E-F, G-H)CyclinD1, p27, PCNA were detected via western blot showing the variation trend as the inhibited proliferation. Data were presented as mean ± SEM (n ¼ 3, *, P < 0.01, compared to the Ctrl-groups).
Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093
Fig. 4. The weakened proliferation of KPNB1-depletion cells in CCK8 and colonies formation. (AeB) Cells transfected with shRNA#1 and #3 were cultivated in the 96-wells plates for detect the relative absorbance on 450 nm at each time point of 0 h, 24 h, 48 h, 72 h, 96 h. The obvious difference was observed at 72 h and 96 h. Data were presented as mean ± SEM (n ¼ 3, *, P < 0.01). (CeF) The selected and stably-transfected cells were cultivated into the 6 cm culture plates and incubated for 12 days at 37 C with 5% CO2. Stained with Giemsa solution, we observed the diversity in the KPNB1-depletion cells. (G-H)Reporter assay was carried out in the KPNB1-depletion U251 and U87MG cells Data was presented as mean ± SEM (n ¼ 3, *, P<0.01). (I-J)Via cells fractionation, b-catenin was detected to be decreased in the nucleus of KPNB1-depletion U251 and U87MG cells. (K)A kind of direct interaction exists between b-catenin and KPNB1. With immunoprecipitation and western-blot, the result showed the direct interaction between b-catenin and KPNB1.
Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093
T. Lu et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e9
cateninmolecules enter the nucleus and work as a co-activator of LEF/TCF transcription factors [31]. The association withb-catenin transiently converts TCF into a transcriptional activatorto its target genes, with modulation of TCF comingfrom phosphorylation [32]. Neverthless, the details are unclear that howb-catenin shuttles between the cytoplasmand the nucleus, although recent evidence suggestsa role for microtubules and active transport [33]. Here, we find that KPNB1 works as a transport-protein in nuclear membrane as known. It might assist b-catenin transcription across nuclear membrane with Wnt signal. Besides, b-catenin/LEF/TCF target genes encode proteins involved in many cellular processes, such as proliferation, cell cycle regulation, metabolism, migration, lineage commitment, and differentiation [34]. In current study, we demonstrated the function of KPNB1 in the Wnt/b-catenin pathway as medium. With more b-catenin transported into nucleus, the TCF/ LEF complex was necessarily activated. However, there is an outstanding question turning up. As we all know, KNPB1 also mediates the nucleolar import of some other proteins, such as NF-kb, XPO7, and IPO8 and so on which may be also involved in cell proliferation more or less. Though we have demonstrated the cofunction of KNPB1 and Wnt/b-cateninto cell proliferation, it is unwise to neglect the crosstalk of other signal pathways. Moreover, to identify our hypothesis fully and particularly, further study to uncover the mechanism is currently in progress. Conflict of interest No competing financial interests exist. Acknowledgements This work was supported by grants from National Natural Science Foundation of China (No.81372687). References [1] S. Deorah, C.F. Lynch, Z.A. Sibenaller, T.C. Ryken, Trends in brain cancer incidence and survival in the United States: surveillance, epidemiology, and end results program, 1973 to 2001, Neurosurg. focus 20 (2006) E1. [2] D.A. Reardon, J.E. Herndon 2nd, K.B. Peters, et al., Bevacizumab continuation beyond initial bevacizumab progression among recurrent glioblastoma patients, Br. J. cancer 107 (2012) 1481e1487. [3] E.A. Maher, F.B. Furnari, R.M. Bachoo, et al., Malignant glioma: genetics and biology of a grave matter, Genes & Dev. 15 (2001) 1311e1333. [4] W. Zhuang, Z. Qin, Z. Liang, The role of autophagy in sensitizing malignant glioma cells to radiation therapy, Acta biochimica biophysica Sinica 41 (2009) 341e351. [5] P.Y. Wen, S. Kesari, Malignant gliomas in adults, N. Engl. J. Med. 359 (2008) 492e507. [6] P. Polakis, Wnt signaling and cancer, Genes & Dev. 14 (2000) 1837e1851. [7] I.R. Cho, S.S. Koh, H.J. Min, et al., Pancreatic adenocarcinoma up-regulated factor (PAUF) enhances the expression of beta-catenin, leading to a rapid proliferation of pancreatic cells, Exp. Mol. Med. 43 (2011) 82e90. [8] T.C. He, A.B. Sparks, C. Rago, et al., Identification of c-MYC as a target of the APC pathway, Science 281 (1998) 1509e1512. [9] T.C. He, T.A. Chan, B. Vogelstein, K.W. Kinzler, PPARdelta is an APC-regulated
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Please cite this article in press as: T. Lu, et al., Karyopherinb1 regulates proliferation of human glioma cells via Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.093