High expression of adenylate cyclase-associated protein 1 accelerates the proliferation, migration and invasion of neural glioma cells

Pathology – Research and Practice 212 (2016) 264–273

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Original article

High expression of adenylate cyclase-associated protein 1 accelerates the proliferation, migration and invasion of neural glioma cells Zhen Bao a,1 , Xiaojun Qiu b,1 , Donglin Wang c , Na Ban d , Shaochen Fan d , Wenjuan Chen d , Jie Sun d , Weikang Xing a , Yunfeng Wang a , Gang Cui a,∗ a

Department of Neurosurgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, People’s Republic of China Department of Oncology, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, People’s Republic of China c Department of Pathology, Medical College of Nantong University, Nantong, Jiangsu Province, People’s Republic of China d Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College of Nantong University, Nantong, Jiangsu Province, People’s Republic of China b

a r t i c l e

i n f o

Article history: Received 7 May 2015 Received in revised form 26 October 2015 Accepted 31 December 2015 Keywords: Glioma CAP1 Proliferation Migration Invasion

a b s t r a c t Adenylate cyclase-associated protein 1 (CAP1), a conserved member of cyclase-associated proteins was reported to be associated with the proliferation, migration or invasion of the tumors of pancreas, breast and liver, and was involved in astrocyte proliferation after acute Traumatic Brain Injury (TBI). In this study, we sought to investigate the character of CAP1 in the pathological process of human glioma by detecting human glioma specimens and cell lines. 43 of 100 specimens showed high expression of CAP1 via immunohistochemistry. With statistics analysis, we found out the expression level of CAP1 was correlated with the WHO grades of human glioma and was great positively related to Ki-67 (p < 0.01). In vitro, silencing CAP1 in U251 and U87MG, the glioma cell lines with the relatively higher expression of CAP1, induced the proliferation of the cells significantly retarded, migration and invasion as well. Obviously, our results indicated that CAP1 participated in the molecular pathological process of giloma indeed, and in a certain sense, CAP1 might be a potential and promising molecular target for glioma diagnosis and therapies in the future. © 2016 Elsevier GmbH. All rights reserved.

1. Introduction Human malignant glioma, the most lethal brain tumor and the most frequent type of primary adult brain neoplasms, has dismal outcome for patient [3,19]. Patients diagnosed with glioma possess a very low 5-years survival, while patients affected by glioblastoma multiforme (GBM), which is also known as grade IV astrocytoma, survival <1 year despite intensive treatment [14,31]. By now, despite significant advances in neurosurgery and chemoradiotherapy, glioma remains highly resistant to conventional treatments and improvements in patient outcome have been modest [25]. For this reason, it is vital and urgent to investigate thoroughly the molecular pathological mechanism, the origination and progression of glioma, for some more specific biomarkers and potential remedy targets.

∗ Corresponding author. Tel.: +86 15106202811; fax: +86 05137613646. E-mail address: [email protected] (G. Cui). 1 Both authors contribute equally to this study. http://dx.doi.org/10.1016/j.prp.2015.12.017 0344-0338/© 2016 Elsevier GmbH. All rights reserved.

Adenylate cyclase-associated protein 1 (CAP1) is one of cyclaseassociated proteins which are conserved actin monomer-binding proteins present in all eukarcyotes [9]. CAPs were first identified in yeast as a protein that regulates both of the actin cytoskeleton and the Ras/cAMP pathway [28]. Deletion of CAP in yeast resulted in an abnormally large cell size, random budding pattern, and abnormal actin distribution [1]. But the function of Ras is confined to yeast [30]. The previous research demonstrated that CAPs interact with adenylate cyclase complex through their Nterminal domain, whereas the C-terminal half of CAPs directly binds to monomeric actin [6,7]. CAP1 and CAP2 are both localized to the cell membrane and contain one C-CAP/cofactor C-like domain [22]. CAP1 is a highly abundant protein that localized to dynamic actin structures [16]. The biological properties of CAP1 are involved in the regulation of actin filaments by promoting cofilin-induced actin filament depolymerization and are thought to mediate processes such as establishment of cell morphogenesis, polarity, motility, receptor-mediated endocytosis, and messenger RNA (mRNA) location [1]. Actin cytoskeleton reorganization is a key factor in many cellular progression, including cell proliferation [15], migration, and differentiation [11]. According to recent reports,

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CAP1 is associated with not only the proliferation of breast cancer cells [27], but also the metastasis in breast carcinoma patients [12]. Besides, CAP1 has been shown to be over-expressed in pancreatic tumors, and probably involves in the aggressive behavior of pancreatic cancers through modulating cell motility [26]. CAP1 is found to be over-expressed in human hepatocellular carcinoma (HCC) and positively associated with HCC cell metastasis [13]. Additionally, it has been reported that CAP1 is significantly up-regulated corresponding to the changed expression of Ki-67, a marker of cell proliferation, in rat cortex astrocytes suffering from traumatic brain injury (TBI) and plays a vital role in the activation of astrocyte proliferation after TBI [29]. Notwithstanding, the researches of CAP1 in many kinds of human cancers have been wider and deeper, the function of the key molecule in human glioma is still uncovered. Epithelial–Mesenchymal Transition (EMT) is involved in cell migration and invasion which are key steps in the progression of human glioma [10]. Once a cancer goes into EMT, it sheds connections with neighboring cells and the extracellular matrix and becomes more aggressive in migration and invasion, leading to enhanced metastasis [24]. The loss of E-cadherin and overexpression of mesenchymal cell markers such as N-cadherin, Vimentin, Snail are hallmarks of EMT. According to previous researches, the over-expression of CAP1 may enhance the progression of human glioma. In this report, we evaluated the expression of CAP1 in 100 glioma specimens and carried out scientific and rigorous research plans on the glioma cell lines, U251 and U87MG, to investigate the possible impact of CAP1 on proliferation, metastasis and invasion, the possible molecular pathological process of human glioma, wishing to discover a conceivable therapy target. 2. Material and methods 2.1. Patients and tissue specimens The human glioma tissues collected from 100 astrocytomas surgical specimens without any therapy in advance and the clinical–pathological data were provided by the Department of Pathology, Affiliated Hospital of Nantong University. Normal brain specimens obtained from 10 patients who received epilepsy surgery were verified for the absence of any tumor organization. Specimens were immediately frozen in liquid nitrogen and stored at −80 ◦ C until use. Some parts of the specimens (including WHO grade II–IV) were fixed in 10% formalin and embedded in paraffin for immunohistochemical analysis. All the tissues were collected and applied in accordance with The Code of Ethics of the World Medical Association. And the study has been approved by the ethics committee of Soochow University. 2.2. Cell lines and cell culture The human glioblastoma cell lines H4, SHG44, U87MG, U251, A172 and U373 were purchased from Shanghai Institute of Cell Biology. All cells were cultured in the DMEM high-glucose medium (Gibco BRL, Grand Island, NY) with 10% heat-inactivated fetal bovine serum at 37 ◦ C with 5% CO2 . 2.3. Antibodies The antibodies applied to immunohistochemistry included: anti-CAP1 (diluted 1:400, Santa Cruz, Biotechnology), antiKi-67 (diluted 1:400, Santa Cruz, Biotechnology). The antibodies applied to Western blot included: anti-CAP1 (diluted 1:2000, Santa Cruz, Biotechnology), anti-proliferating cell nuclear

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antigen (PCNA, diluted 1:1000, Santa Cruz, Biotechnology), anti-cyclin A (diluted 1:1000, Santa Cruz, Biotechnology), anticyclin dependent kinase 2 (CDK2, diluted 1:1000, Santa Cruz, Biotechnology), anti-P21 (diluted 1:1000, Santa Cruz, Biotechnology), anti-E-cadherin (diluted 1:1000, Santa Cruz, Biotechnology), anti-N-cadherin (diluted 1:1000, Santa Cruz, Biotechnology), antiVimentin (diluted 1:1000, Santa Cruz, Biotechnology), anti-Snail (diluted 1:1000, Santa Cruz, Biotechnology), anti-glyceraldehyde3-phosphate dehydrogenase (GAPDH, diluted 1:1500, Santa Cruz, Biotechnology). 2.4. Immunohistochemistry (IHC) The glioma tissues sections were deparaffinized in xylene, rehydrated in graded ethanol solutions, and then we block the endogenous peroxidase activity in 0.3% hydrogen peroxide. Then the sections were heated at 105 ◦ C in 0.1 M citrate buffer, pH 6.0 for 10 min and incubated at room temperature for 1 h to retrieve the antigen. Afterwards, the section were incubated with 5% bovine serum in PBS (phosphate buffer saline) (pH 7.2) for blocking nonspecific protein binding, followed by overnight incubation with certain antibodies at 4 ◦ C. Negative-control groups were treated with non-specific immunoglobulin IgG (Sigma Chemical Co. St. Louis, MO, USA) as the first antibody. Then we incubated the sections. According to the manufacturer’s instructions, the slides were rinsed in PBS and incubated with hematoxylin, dehydrated and mounted successively in resin mount. 2.5. Immunohistochemical evaluation All of the stained sections were evaluated in a blinded way by two independent veteran pathologists without any of the clinical–pathological variables of the patients. Five high-power fields (Leica microscope Germany) were selected randomly and more than 300 cells in each field were counted to determine the labeling index (LI), which means the percentage of immunostained cells relative to the total number of cells [8]. Intensity was evaluated in comparison with the control and scored as follows: 0, negative staining; 1, weak staining; 2, moderate staining; 3, strong staining. The percentage of tumor cells stained positive were scored as follows: 0, <1% positive tumor cells; 1, 1–10% positive tumor cells; 2, 10–50% positive tumor cells; 3, 50–75% positive tumor cells; 4, >75% positive tumor cells. Then we added the scores of intensity and percentage as 0 and 2–7. 0: negative; 2–3: weak stained; 4–5: moderate stained; 6–7: strong stained. For statistical analysis, 0–3 were counted as low expression, while 4–7 were counted as over-expression [4]. 2.6. Western blot assay Glioma tissue samples were homogenized in lysis buffer (1% Nonidet P-40, 50 mmol/L Tirs, pH 7.5, 5 mmol/L EDTA, 1% SDS, 1% sodium deoxycholate, 1% triton X-100, 1 mmol/L PMSF, 10 mg/mL aprotinin, and 1 mg/mL leupeptin). Cell samples were washed three times with PBS, suspended in 2X lysis buffer (50 mM Tris–HCl, 120 mM NaCl, 0.5% Nonidet P-40, 100 mM NaF, 200 M Na3 VO4 , protease inhibitor mixture). Then, samples were denatured at 100 ◦ C for 15 min and evaluated with Bio-Rad protein assay (BioRad, Hercules, CA, USA) for the total protein concentration, and then stored at −20 ◦ C. The protein samples were separated via SDSpolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride filter (PVDF) membranes (Milipore Bedford MA). The membranes were blocked in TBST (20 mM Tris, 150 mM NaCl, 0.05% Tween-20) with 5% evaporated milk for 2 h at room temperature and then incubated in certain antibodies at 4 ◦ C for 6–8 h. The membranes were washed with TBST for three times,

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Fig. 1. The over-expression of CAP1 and Ki-67 is detected in glioma tissues, and it is consistent with low accumulative survival. (A and B) Expression of CAP1 in 6 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 = 3, *, p < 0.01). (C) Immunohistochemical staining of CAP1 and Ki-67 were carried out in 100 glioma specimens. The immunoreactivity rose up as the WHO grades of glioma. Scale bar = 100 ␮m above. Scale bar = 50 ␮m below. (D) The correlation test showed the strong consistency between CAP1 and Ki-67 (p < 0.01). (E and F) High expression of CAP1 was close to low accumulative survival, as well as Ki-67 (p < 0.01).

5 min/time and incubated with horseradish peroxidase-linked IgG for 2 h at room temperature, and then detected by infrared imaging system (Odyssey, USA). The blot band intensity was quantified by ImageJ analysis file (Wayne Rasband, National Institutes of Health, US).

the negative-control shRNA sequence: 5 -TTCTCCGAACGTGTCACGT-3 . U251 cell line were transfected with 100 nmol/L of shRNA#1 performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and U87MG cell line with shRNA#2. The cells were identified as negative-control groups treated with control shRNA and mock groups treated with noting.

2.7. RNA interference of CAP1 2.8. Flow cytometry analysis of cell cycle The CAP1-shRNAs were synthesized by GeneChem. CAP1shRNA#1 target sequence: 5 -AGTTCAAGACCCTATGGAA-3 , CAP1-shRNA#2 target sequence: 5 -AGGCTTACATTAAGGAGTT-3 , CAP1-shRNA#3 target sequence: 5 -TGGCCCTTATGTGAAAGAA-3 , CAP1-shRNA#4 target sequence: 5 -CATCTCAGAGCAGATCAAA-3 ,

Cells with certain treatment before were fixed with 70% methanol in PBS at −20 ◦ C for 48 h, and then with 1 mg/mL RNase A for 20 min at 37 ◦ C. Afterwards, the cells were stained with 0.5 mg/mL of propidium iodide (PI). The DNA contents were

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Table 1 The correlation between CAP1, Ki-67 expression and clinicopathological parameters in 100 glioma specimens. Variable

Total

CAP1 expression

p Value

Low

High

Ki-67 expression

p Value

Low

High

Age <40 ≥40

17 83

11 46

6 37

0.481

11 44

6 39

0.432

Gender Female Male

37 63

21 36

16 27

0.567

23 32

14 31

0.270

Tumor location Frontal Parietal Occipital Temporal Unknown

26 25 20 23 6

16 13 11 12 5

10 12 9 11 1

0.659

17 12 9 12 5

9 13 11 11 1

0.348

Surgery Biopsy Partial resection

17 41

10 23

7 18

0.982

10 22

7 19

0.937

Gross total resection

42

24

18

23

19

Vessel density Normal Increase

23 77

15 42

8 35

0.364

14 41

9 36

0.519

Tumor diameter <4 cm ≥4 cm

41 59

22 35

19 24

0.360

22 33

19 26

0.822

Necrosis Absence Presence

40 60

26 31

14 29

0.133

26 29

14 31

0.101

WHO grade II III IV

42 35 23

37 20 0

5 15 23

0.000*

35 18 2

7 17 21

0.000*

Statistical analyses were performed by the Pearson 2 test. * p < 0.05 was considered significant.

analyzed by a Becton Dickinson flow cytometer BD FACScan (San Jose, CA). 2.9. Cell counting kit (CCK)-8 assay The cells transfected by shRNAs with the untreated groups were seeded on a 96-well cell culture cluster (Corning Inc, Corning, NY, USA) at 2 × 104 cells/well in 100 ␮L medium and incubated overnight. Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) reagents were added to a subset of wells with different treatments, after which absorbance was measured at a test wavelength of 450 nm on an automated plate reader with the 630 nm wavelength as a reference group. 2.10. Colony formation assay The selected and stably-transfected cells were plated in 6 cm culture plates at 100 cells/well. After incubation for 12 days at 37 ◦ C, the cells were washed twice with PBS and stained with Giemsa solution. The number of colonies containing >50 cells was counted under a microscope. 2.11. Wound healing assay Cells were seeded on 6-well plates at a density of 5 × 105 cells/well. 48 h after transfection, the cells were scratched on the monolayer with a 10 ␮L, pipette tip, and washed twice with

PBS. Cells were then cultured in serum-free DMEM medium for an additional 48 h. Photographs were taken by an inverted Leica phase-contrast microscope (Leica DFC 300 FX) at the time points of 0, 4, 8, 12, 24, 48 h. 2.12. Migration and invasion assay Cells were suspended with serum-free DMEM medium. And 1 × 105 cells were added to the top chambers of 24-well transwell plates (Corning, 8 ␮m pore size), and DMEM with 10% FBS was added to the bottom chambers. After overnight incubation, the bottom (migrated) cells were fixed and stained with crystal violet to visualize nuclei. The number of migrating cells in 5 fields was counted under 200× magnification, and the means for each chamber were determined. In all experiments, data represent the average number of cells from triplicate chambers. 2.13. Statistics analysis Statistics analysis was performed by the SPSS17.0 statistics analysis software. The statistical correlations between CAP1 and Ki-67 expression and the clinical–pathological features were analyzed using the Chi square test. Survival analysis was carried out using the Kaplan–Meier method, and curves were compared using the logrank test. All of values were expressed as the mean ± SEM (Standard error of the mean), and p < 0.05 was considered statistically significant.

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3. Results 3.1. The correlation of the expression of CAP1 and Ki-67 in human glioma tissues with the WHO grades of human glioma For investigating the possible role of CAP1 in the development of glioma, we detected and compared the expression of CAP1 in normal brain tissues and three couples of glioma tissues in the rank of WHO grade II–IV by Western blot analysis. According to the statistics analysis, we observed the significant rising-up tendency of CAP1 in normal brain tissues, low-grade and high-grade glioma tissues (Fig. 1A and B). As an important reported proliferation marker, Ki-67 was examined in 100 specimens of human glioma tissues with CAP1 by immunohistochemical staining. It was just like what we had expected that the consistency correlation of the expression of Ki-67 and CAP1 (Fig. 1D), which did prove that CAP1 was relative to the proliferation of tumor cells. To testify the possibility of the correlation, we summarized the clinical–pathological statistics and found out that the close relationship of CAP1 to WHO grades of glioma (p < 0.01), which was just the same as Ki-67 as cross reference (Table 1). There was no obvious relevance to other clinical–pathological factors. In this way, we guessed that CAP1 might take a relatively great part in the progression of glioma, the over-expression of which might lead to poor prognosis.

3.2. The relationship between the expression of CAP1 in the specimens and the patients’ 5-years survival ratio and the prognosis of human glioma To verify the possibility of the presumption, we carried out statistics analysis via SPSS. According to the Kaplan–Meier survival curves, the patients with high expression of CAP1 suffered lower accumulative 5-years survival ratio (p < 0.01). It is just the same to the impact of over-expression of Ki-67 (Fig. 1E and F). Subsequently, we operated the univariate survival analysis to compare the impact of other clinical–pathological factors on the 5-years survive to that of CAP1 and Ki-67, and we made the conclusion that the over-expression of the two molecular was vitally relative to the poor prognosis of the patients of glioma (Tables 2 and 3).

Table 2 Contribution of various potential prognostic factors to survival by univariate analysis in 100 glioma specimens. Characteristics

Total

Survival status

p Value

<5 years

≥5 years

Age <40 ≥40

17 83

10 46

7 37

0.797

Gender Female Male

37 63

15 41

22 22

0.057

Tumor location Frontal Parietal Occipital Temporal Unknown

26 25 20 23 6

14 14 14 11 3

12 11 6 12 3

0.671

Surgery Biopsy Partial resection

17 41

11 23

6 18

0.689

Gross total resection

42

22

20

Vessel density Normal Increase

23 77

14 42

9 35

0.592

Tumor diameter <4 cm ≥4 cm

41 59

22 34

19 25

0.694

Necrosis Absence Presence

40 60

20 36

20 24

0.324

WHO grade II III IV

42 35 23

15 23 18

27 12 5

0.002*

CAP1 expression Low High

57 43

26 30

31 13

0.016*

Ki-67 expression Low High

55 45

21 35

34 10

0.000*

Statistical analyses were performed by the Pearson 2 test. * p < 0.05 was considered significant.

3.3. The expression of CAP1 in glioma cell lines and the relationship to cell proliferation To identify the function of CAP1 in pathological procession of glioma, we selected only one glioma cell line, U251, for the experiments followed, for the reason that CAP1 was expressed highest in it, followed by U87MG (Fig. 2A and B). In the view of the positive relationship between CAP1 and Ki-67, the over-expression of CAP1 was likely to accelerate cell proliferation. To make it clear, we carried out a serum starvation and re-feeding experiment in U251 cell line. As the flow cytometry analysis told that when the cells were controlled in the serum deprivation environment for 72 h, the proportion of the cells arrested in the G1 phase increased to 63.63%. After re-feeding, the proportion of the cells in the G1 phase reduced and that in S phase grew up gradually (Fig. 2C and D). At the same time, we detected the levels of the expression of CAP1 and some other reported proteins related to cell proliferation, such as PCNA, cyclin A, CDK2, P21. With all of the proofs provided above (Fig. 2E and F), we could just make a tentative conclusion that CAP1 might be related to the proliferation of glioma cells and accelerate the pathological process of glioma.

Table 3 Cox regression analysis in 100 glioma specimens.

Gender WHO grade CAP1 expression Ki-67 expression

Hazard ratio

95% confidence interval

p Value

1.915 2.062 2.609 4.490

1.058–3.463 1.480–2.873 1.534–4.437 2.581–7.811

0.132 0.017* 0.005* 0.003*

Statistical analyses were performed by the Cox regression analysis. * p < 0.05 was considered significant.

3.4. Down-regulated of CAP1 inhibits the proliferation of U251 cell line in vitro Since we had discovered that CAP1 was up-regulated in the starve-released cells, it is necessary to verify its character in the way of silencing CAP1. We transfected the U251 cells with control-shRNA, CAP1-shRNA#1, CAP1-shRNA#2, CAP1-shRNA#3 and CAP1-shRNA#4 respectively. The efficiency of all the shRNAs was compared by Western blot (Fig. 3A and B). As it was shown, all the expression of CAP1 with the CAP1-siRNAs was reduced contract

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Fig. 2. The expression of CAP1 in several glioma cell lines and the different phases of cell cycle in starve and re-feeding models. (A and B) The highest expression level was showed up in U251 cell lines, followed by U87MG. Data were presented as mean ± SEM (n = 3, *, p < 0.05). (C and D) Flow cytometry showed 63.63% of the cells were arrested in G1 phase and the obvious cell cycle progression in U251 cells in the process of re-feeding after serum-starve for 72 h. (E and F) Several cell cycle and proliferation makers, PCNA, cyclin A, CDK2, P21, were in the increasing trend with CAP1 at each time point. GAPDH was used as control protein load. Data were presented as mean ± SEM (n = 3, *, ˆ, #, $, &, p < 0.01, compared with control-0 h).

to the negative-control group. Moreover, CAP1-shRNA#1 achieved the best knock-down efficiency in U251 cell line, which provided a straightforward direction for the following experiments. We made a flow cytometry analyses to show the proportion of the transfected cells in G1 and S phase (Fig. 3C and D, p < 0.05). Obviously, on the one hand the cells of G1 phase increased from 60.59% to 69.47%, on the other hand the cells of S phase decreased from 23.48% to 11.73% in U251 cell line, which indicated that down-expression of CAP1 delayed the cell cycle. At the same time, we detected the expression of the PCNA, cyclin A, CDK2, Ki-67, p21 (Fig. 3E and F). It revealed the explicit tendency with the expression of knocked-down CAP1, which meant the CAP1 did play an important role in the cell cycle and the cell proliferation in the level of molecular pathology. To fulfill the evidence for our conclusion, we made a CCK-8 assay to

compare the proliferation of the CAP1-shRNA#1-transfected U251 to control-shRNA-transfected U251 (Fig. 3G p < 0.05). The relative absorbance was less than that of the negative-control group. In the colony formation assay, the same difference was observed (Fig. 3H). 3.5. Knocking-down CAP1 suppresses the migration and invasion of U87MG and U251 cell lines in vitro We detected the changes of certain relevant molecules expression in U251 with the treatment of transfection by shRNA#1 (Fig. 4A and B). Interestingly, we found out the differences were notable in a certain extent, the decline trend of N-cadherin, Snail and Vimentin and the increasing trend of E-cadherin were conspicuous between

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Fig. 3. Depletion of CAP1 weakened the ability of proliferation in U251 cells. (A and B) The highest level of effectiveness is shRNA#1 to U251 cells, #3 following. Data were presented as mean ± SEM (n = 3, *, ˆ, p < 0.01, compared with the mock groups). (C and D) 69.47% of the CAP1-depletion cells were arrested in G1 phase. Data were presented as mean ± SEM (n = 3, *, ˆ, p < 0.01, compared to the Ctrl-groups). (E and F) Cyclin A, CDK2, P21 were detected via Western blot showing the certain change as the decline proliferation. Data were presented as mean ± SEM (n = 3, *, ˆ, #, $, p < 0.01, compared to the Ctrl-groups). (G) Cells transfected with shRNA#1 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 = 3, *, ˆ, p < 0.01). (H) 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 depletion-CAP1 cells.

the mock, control and shRNA groups in U251 cells (p < 0.01), which meant silencing CAP1 might weaken the pathological process of migration and invasion. U87MG cell line was also chosen for the study via the transfection of shRNA#2, the highest effectiveness to U87MG (Fig. 4C and D). We got the similar result as well (Fig. 4E and F). To explore the interesting phenomenon once again, we implemented wound healing (Fig. 5A–D) and transwell assay (Fig. 5E–H) in the two representative cell lines. The ability of wound healing and invasion was conspicuously decreased with the transfection of CAP1-shRNAs comparing to the control-shRNAs in both of U251 and U87MG cell lines (p < 0.05).

4. Discussion Human malignant glioma is the most common type of central nervous system (CNS) malignancy [5]. The current standard therapy includes maximal safe resection followed by radiotherapy in combination with temozolomide [21]. It is still a typical deadly cancer among the neurological oncology. Especially, glioblastoma is one of the most aggressive and lethal forms of cancer with an average survival time of 15 months after diagnosis [21]. Malignant glioma cell proliferation and invasion are key stages in cancer progression that affect patient mortality [17]. As a result, it is needed and

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Fig. 4. Conspicuous difference of the expression of some migration and invasion makers was detected in depletion-CAP1 U251 and U87MG cells respectively. (A and B) The decline trend of N-cadherin, Snail and Vimentin and the increasing trend of E-cadherin was conspicuous between the mock, control and shRNA groups in U251 cells. Data were presented as mean ± SEM (n = 3, *, ˆ, #, $, p < 0.05). (C and D) The highest level of transfection effectiveness was shRNA#2 to U87MG cell lines. Data were presented as mean ± SEM (n = 3, *, p < 0.01). (E and F) The same result could be identified in the treatment of U87MG cells. Data were presented as mean ± SEM (n = 3, *, ˆ, #, $, p < 0.05).

necessary to explore the underlying molecular pathological mechanism of human glioma. In this study, we demonstrated adenylate cyclase-associated protein 1 (CAP1) promoted the pathological procession, proliferation, migration and invasion, of human glioma in vitro. According to the World Health Organization (WHO), human glioma is classified into four grades as follows: pilocytic astrocytoma, WHO grade I; diffuse “low grades” glioma, WHO grade II; anaplastic gliomas, WHO grade III; glioblastoma (GBM), WHO grade IV [18]. Via the systemic analysis of the clinical–pathological statistics from experienced doctors of pathology department in the affiliated hospital of Nantong university, we identified the great correlation between the over-expression of CAP1 and the WHO grades of human glioma, which meant a poor prognosis of a low 5years survival ratio. Additionally, the key molecular was expressed

in the glioma cell lines obviously. So, it was extremely possible that CAP1 participated in the pathological procession, at least partially, of human glioma. With the immunohistochemical stain, we discovered the great positive relationship of CAP1 and Ki-67, a reported proliferation marker. On the basis of the research above, the over-expression of CAP1 was connected with the progression and development of human glioma in a macroscopic view. Then, we strongly wanted to uncover the roles of CAP1 in the pathological process in detail and the changes of behavior of glioma cell. The proliferation of cancer and carcinogenesis could never be documented without referring to altered regulation of the cell cycle [20]. Progression of the cell cycle is governed by a family of cyclin-dependent kinases (Cdks), of which activity is regulated

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Fig. 5. In wound healing and transwell experiments, the ability of migration and invasion was identified to be decreased in depletion-CAP1 U251 and U87MG cells respectively with notable difference. (A–D) In wound healing models, the distance of the wound was detected at the time point of 24 h and 48 h after the transfection in U251 and U87MG cells. Data were presented as mean ± SEM (n = 3, *, ˆ, p < 0.05). (E–H) The similar result was observed in the transwell models. Data were presented as mean ± SEM (n = 3, #, $, p < 0.05).

by phosphorylation, activated by cyclin binding, and inhibited by Cdk inhibitors [2]. We designed a serum starvation and re-feeding model in U251 cell lines for the relative highest level of expression. It showed up with the rising-up tendency of the expression of CAP1 and corresponding trend of other proliferation markers, PCNA, cyclin A, CDK2, p21. At the same time, the flow cytometry told that the proportion of cells in G1 phase decreased and it grew up in S phase. Furthermore, we operated Western blot, flow cytometry, CCK-8 and colony formation assay with the U251 cells transfected, which provided more evidence for the conclusion that over-expression of CAP1 enhanced the proliferation of human glioma in vitro. Migration and invasion are usually recognized as the main reason for the high recurrence and death rates of glioma and migration

of tumor cells plays an important role in the invasion of glioma and restricts the efficacy of surgery and other therapies [23]. Amount of studies reported CAP1 had impact on the migration and invasion in many other kinds of cancers, breast cancer [12], hepatocellular carcinoma [13], pancreatic cancer [26], precisely. CAP1-knockdown HeLa cells had significantly enhanced cell spreading and adhesion. To expand the function of CAP1, we investigated the character of CAP1 in the pathological process of migration and invasion in human glioma for the first time. The loss of E-cadherin and over-expression of mesenchymal cell markers such as N-cadherin, Vimentin, Snail are hallmarks of EMT. With the Western blot assay, we detected the identified tendency of E-cadherin, N-cadherin, Vimentin, Snail expression in the transfected U251 and U87MG cells to uncover that CAP1

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might influence the cell behavior. Significantly, the tendency was explicit. The decline trend of N-cadherin, Snail and Vimentin and the increasing trend of E-cadherin was conspicuous between the mock, control and shRNA groups in U251 cells (p < 0.05). To fulfill the evidence, the wound healing assay and transwell assay with the U251 and U87MG were constructed for more persuading statistics showed in this report. It happened to be just like expected (p < 0.05). An interesting result turned up that it was just the same obvious outcome as which of depletion-CAP1 in other cancers. In this study, we demonstrated CAP1 was tightly closed to human glioma, and that the over-expression of CAP1 accelerated the molecular–pathological process, proliferation, migration and invasion, in different levels. However, further researches are needed to enrich the molecular mechanism about CAP1 in detail, for the possibility of becoming a potential and promising molecular target for glioma diagnosis and therapies in the future. 5. Compliance with ethical standards 1. Disclosure of potential conflicts of interest. 2. Research involving Human Participants. 3. Informed consent. 6. Conflict of interest statement No competing financial interests exist. Acknowledgements This work was supported by National Natural Science Foundation of China (81372687), National Natural Science Foundation of China (81272789). References [1] E. Bertling, P. Hotulainen, P.K. Mattila, T. Matilainen, M. Salminen, P. Lappalainen, Cyclase-associated protein 1 (CAP1) promotes cofilin-induced actin dynamics in mammalian nonmuscle cells, Mol. Biol. Cell 15 (2004) 2324–2334. [2] M.S. Chang, C.L. Chang, C.J. Huang, Y.C. Yang, p29, a novel GCIP-interacting protein, localizes in the nucleus, Biochem. Biophys. Res. Commun. 279 (2000) 732–737. [3] 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. [4] Z. Ding, X. Liu, Y. Liu, J. Zhang, X. Huang, X. Yang, L. Yao, G. Cui, D. Wang, Expression of far upstream element (FUSE) binding protein 1 in human glioma is correlated with c-Myc and cell proliferation, Mol. Carcinog. 54 (2015) 405–415. [5] S.D. Ferguson, Malignant gliomas: diagnosis and treatment, Dis. Mon.: DM 57 (2011) 558–569. [6] N.L. Freeman, T. Lila, K.A. Mintzer, Z. Chen, A.J. Pahk, R. Ren, D.G. Drubin, J. Field, A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization, Mol. Cell. Biol. 16 (1996) 548–556. [7] J.E. Gerst, K. Ferguson, A. Vojtek, M. Wigler, J. Field, CAP is a bifunctional component of the Saccharomyces cerevisiae adenylyl cyclase complex, Mol. Cell. Biol. 11 (1991) 1248–1257. [8] X. Huang, F. Liu, C. Zhu, J. Cai, H. Wang, X. Wang, S. He, C. Liu, L. Yao, Z. Ding, Y. Zhang, T. Zhang, Suppression of KIF3B expression inhibits human hepatocellular carcinoma proliferation, Dig. Dis. Sci. 59 (2014) 795–806. [9] A.V. Hubberstey, E.P. Mottillo, Cyclase-associated proteins: CAPacity for linking signal transduction and actin polymerization, FASEB J. 16 (2002) 487–499, official publication of the Federation of American Societies for Experimental Biology.

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