Mol Cell Biochem DOI 10.1007/s11010-015-2533-9
Overexpression of SYF2 correlates with enhanced cell growth and poor prognosis in human hepatocellular carcinoma Shusen Zhang1 • Weidong Shi2 • Yuyan Chen3 • Zhiwei Xu4 • Jia Zhu4 • Tingting Zhang1 • Wei Huang1 • Runzhou Ni1 • Cuihua Lu1 • Xiubing Zhang2
Received: 11 May 2015 / Accepted: 6 August 2015 Ó Springer Science+Business Media New York 2015
Abstract SYF2, also known as p29/NTC31/CBPIN, encodes a nuclear protein that interacts with Cyclin D-type binding-protein 1. SYF2 has been reported to be involved in pre-mRNA splicing and cell cycle regulation. In the present study, we observed that SYF2 was obviously upregulated in HCC tumor tissues and cell lines, and its level was positively correlated with the tumor grade and Ki-67 expression, as well as poor prognosis of HCC. In vitro, using serum starvation–refeeding experiment, our results suggested that SYF2 was upregulated in proliferating HCC cells, and was positive correlated with the expression of PCNA and Cyclin D1. In addition, depletion
of SYF2 decreased PCNA and Cyclin D1 levels. Accordingly, interference of SYF2 resulted in cells cycle arrest at G1/S phase in Huh7 HCC cells. Furthermore, we found that SYF2 might interact with Cyclin D1 and could confer doxorubicin resistance in HCC cells. These findings revealed that SYF2 might play a regulatory role in the proliferation of HCC cells. In summary, SYF2 may be a novel prognostic marker and serve as a potential therapeutic target in HCC.
Shusen Zhang and Weidong Shi have contributed equally to this work.
Introduction
Electronic supplementary material The online version of this article (doi:10.1007/s11010-015-2533-9) contains supplementary material, which is available to authorized users. & Cuihua Lu
[email protected] & Xiubing Zhang
[email protected] 1
Department of Digestion, Affiliated Hospital of Nantong University, Nantong University, Nantong 226001, Jiangsu, People’s Republic of China
2
Department of Oncology, The Second People’s Hospital of Nantong, Nantong University, Nantong 226001, Jiangsu, People’s Republic of China
3
Class 5 Grade 13, Clinical Medicine, Medical College, Nantong University, Nantong 226001, Jiangsu, People’s Republic of China
4
Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong 226001, Jiangsu, People’s Republic of China
Keywords SYF2 Hepatocellular carcinoma Cyclin D1 Prognosis Cell proliferation
Hepatocellular carcinoma (HCC) ranks the fifth leading cause of cancer mortality worldwide with a 5-year postoperative survival rate of 30–40 %. The dismal prognosis of HCC mainly results from delayed diagnosis and high prevalence of HCC invasion and metastasis [1, 2]. Although basic researchers and clinical practitioners have clarified the major risk factors for HCC, namely HBV and HCV infections, dietary aflatoxin intake, as well as alcoholic liver disease, the detailed pathological processes underlying HCC development remains largely elusive [3]. In addition, aberrant cell cycle regulation has been long viewed as a fundamental feature underlying HCC progression. Therefore, it is important to clarify the molecular mechanism underlying cell cycle dysregulation during HCC development. Cell cycle progression is initiated by serial activation of different members of cyclin-dependent kinases (CDKs). G1/S transition, a critical event during cell cycle progression, is mainly controlled by several CDK-Cyclin
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complexes, including Cyclin D1-CDK4/CDK6 and Cyclin E-CDK2 complexes [4, 5]. Cyclin D1 is a crucial G1 Cyclin that plays an integral role in cell cycle progression and tumor growth. Overexpression of Cyclin D1 has been intensively reported in a wide range of tumor types, including breast cancer, esophageal carcinoma, lung cancer, nasopharyngeal carcinoma, oral verrucous carcinoma as well as HCC [6]. SYF2, also referred to as CBPIN or NTC31, is a putative homolog of human p29, that encodes a small protein of 243 amino acids [7, 8]. Through yeast two-hybrid screening and the in vitro GST pull-down assay, human p29 has been identified to be a novel binding partner of GCIP (Cyclin D-interacting protein) [9, 10]. Recent investigations revealed that SYF2 depletion led to upregulated expression of p21 and p107 and increased cell proportion in G1 phase [11, 12]. It was also reported that SYF2 was involved in cell cycle progression by regulating several cell cycle regulators, such as RB protein and atubulin [7, 13]. However, whether SYF2 contributes to cell cycle dysregulation and HCC development has not yet been clarified. In this study, we examined the expression of SYF2 in fresh HCC tissues by Western blot and in HCC specimens using immunohistochemistry. We also investigated the involvement of SYF2 in regulating Cyclin D1 and cell proliferation of HCC cells. Our findings showed that depletion of SYF2 critically attenuated the expression of Cyclin D1 and the proliferation of HCC cells. These results showed SYF2 may be a novel prognostic marker and may play a potential role in anti-proliferative therapy of HCC.
Table 1 SYF2 or Ki-67 expression and clinicopathological parameters in 92 patients with HCC Characteristics
Total
SYF2 expression Low
High
Sex
0.921
Female
19
7
12
Male Age (years)
73
26
47
B45
32
14
18
[45
60
19
41
\50
34
14
20
C50
58
19
39
I–II
54
25
29
III–IV
38
8
30
Absent
19
9
10
Present
73
24
49
0.250
Serum AFP (ng/ml)
0.416
Histological grade
0.013*
Cirrhosis
0.241
Tumor size (cm) \ 4.5
0.035* 45
21
24
47
12
35
Absent
79
31
48
Present
13
2
11
Single
63
23
40
Multiple
29
10
19
Absent
18
9
9
Present
74
24
50
Absent
66
24
42
Present
26
9
17
Low
33
18
15
C 4.5 Tumor metastasis
0.097
Tumor number
0.851
HBsAg
0.163
Microvascular invasion
Materials and methods Patients and tissue samples All paired samples of HCC and their corresponding peritumoral tissues were obtained from patients who newly received partial hepatic resection at the Affiliated Hospital of Nantong University between the year of 2005 and 2008. All HCC tissues were approved by the research ethics committee of the institute, and written informed consent was obtained from every patients. The 92 patients included 73 men and 19 women. The age of the patients ranged from 21 to 75 years, with an average age of 48.47 years. Histological grades were classified into well-differentiated (Edmondson’s grade I–II; n = 54) and poorly differentiated HCC (Edmondson’s grade III–IV; n = 38). The total follow-up time of the patients was 5 years, with a range of 1–60 months. The main clinical and pathologic variables of patients are listed in Table 1.
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p value
0.875
Ki-67 expression
0.005*
High
59
15
44
Survival Died
57
13
44
Alive
35
20
15
0.002*
Statistical analyses were performed by the Pearson v2 test * p \ 0.05 was considered significant
Immunohistochemistry staining In order to analysis SYF2 and Ki-67, serial sections of five micrometer thick were mounted on glass slides coated with 10 % polylysine. These sections were dewaxed in xylene and rehydrated through graded alcohol. Immunoreactivity
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was enhanced by high temperature and pressure, and these sections boiled in 0.01 M citrate buffer (pH 6.0) for 20 min in an autoclave to retrieve the antigen. Thereafter, endogenous peroxidase activity was blocked using hydrogen peroxide (0.3 %). Anti-SYF2 antibody (Sigma– Aldrich, MO, USA, diluted 1:200) overnight at 4 °C and anti-Ki-67 antibody (Millipore, Bedford, MA, USA diluted 1:500) for 2 h at room temperature were incubated in the sections after rinsing in PBS (pH 7.2). Negative control slides were incubated in parallel using a nonspecific immunoglobulin IgG (Sigma–Aldrich, MO, USA) at the same concentration as the primary antibody. All slides were processed using the peroxidase-anti-peroxidase method (Dako, Hamburg, Germany). Finally, slides were counterstained with hematoxylin, dehydrated, and mounted in resin mount.
0.05 % Tween-20) was used to block the on-specific binding site; after 2 h, the membranes were incubated overnight at 4 °C with the corresponding primary antibody. The antibodies used in this study include anti-SYF2 (1:500; Santa Cruz, CA, USA), anti-Cyclin D1 (1:400; Santa Cruz, CA, USA), anti-PCNA (1:1000; Abcam, Cambridge, UK), antiactive Caspase-3 (1:500; Santa Cruz, CA, USA), anticleaved caspase 9 (1:1000; Santa Cruz, CA, USA), and antiGAPDH (1:2000; Santa Cruz, CA, USA). Then the membranes were washed three times 5 min, after that the membranes were incubated with horseradish peroxidase-linked IgG as the secondary antibody at room temperature. Two hours later, the membranes were washed three times 15 min and visualized using enhanced chemiluminescence system (ECL; Pierce Co., Wisconsin, USA). Cell culture and cell cycle analysis
Immunohistochemistry evaluation Two independent pathologists (TP and XM) evaluated the immunostaining. Five high-power fields were randomly chosen for assessment of SYF2 and Ki-67, and at least 300 cells were counted per field. Each tumor section was assigned a score according to the intensity of the nucleic staining and the proportion of stained tumor cells [14]. The intensity of staining was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The extent of staining was scored based on the percentage of positive tumor cells: 0 (B10 %), 1 (10–30 %), 2 (30–50 %), 3 (50–75 %), and 4 (75–100 %). The two scores were added, and resulting in scores of 0 and 2–7. As in statistical analysis, 0–3 were counted as low expression, while 4–7 were counted as overexpression [15]. Western blot analysis Western blot analysis was conducted as previously described [16]. Briefly, fresh frozen tissue and cell protein were promptly homogenized in a homogenization buffer containing 1 M Tris HCl pH 7.5, 1 %Triton X-100, 1 % Nonidet p-40 (NP-40), 10 % sodium dodecyl sulfate (SDS), 0.5 % sodium deoxycholate, 0.5 M EDTA, leupeptin 10 lg/ ml, aprotinin 10 lg/ml, and 1 mM PMSF, then centrifuged at 10,0009g for 30 min to collect the supernatant liquid. Total protein concentrations were detected by Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Before gel electrophoresis, the supernatant diluted in 29 SDS loading buffer and boiled for 15 min. The Equal amounts of lysate were selected for SDS-PAGE. Then the proteins were resolved by 12 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride filter (PVDF) membranes (Millipore, Bedford, MA, USA). 5 % dried skim milk in PBST (20 mM Tris, 150 mM NaCl,
The HCC cell lines (Huh7, HepG2, Hep3B) and normal hepatocyte cell lines (LO2 and HL-7702) were purchased from the Shanghai Institute of Cell Biology. The cells cultured in DMEM that contains 10 % fetal bovine serum, 100 units/ml penicillin, and 100 lg/ml streptomycin and incubated at 37 °C in a humidified chamber containing 5 % CO2. For cell cycle analysis, cells were harvested at the proper time, then fixed with 70 % methanol, and their nuclei were labeled with propidium iodide as described [17]. At last, we analyzed the cells using a Becton–Dickinson BD FACScan flow cytometer and Cell Quest acquisition and analysis software. siRNA and transfection The sequences of siRNA targeting SYF2 are as follows: 5-GUG GUU UCU UAA ACG UAU ATT-3, 5-GAG ACG UCC UUA UAA UGA UTT-3, 5-GAA UGA AGC UCG UAA AUU TT-3. The transfections of SKIP siRNA and control siRNA were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were collected 48 h later for Western blot, CCK8, and flow cytometry assays. Immunofluorescence assay Forty-eight hours after transfection, cells were fixed with 4 % paraformaldehyde in PBS for 1 h at room temperature and later permeabilized with 1 % Triton X-100 in PBS for 20 min. The cells were then blocked with 1 % BSA in PBS for 1 h and incubated with primary antibodies overnight at 4 °C. Secondary antibody incubation was performed using Alexa Fluor 568 or Alexa Fluor 488-conjugated goat antimouse, or Alexa Fluor 568-conjugated goat anti-rabbit antibodies (Invitrogen) for 1 h at room temperature. In all,
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5 mg/ml DAPI was added to stain the nucleus. The slides were mounted and visualized using a Nikon confocal microscope (Nikon, Melville, NY, USA). Cell proliferation assay To evaluate cell viability, we use Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) assay following the manufacturer’s instructions. Huh7 cells were inoculated into 96-well plates at a density of 2 9 104/well to allow 24 h incubation. Cell Counting Kit-8 reagents were added to each well at due time, and the cells were incubated for an additional 2 h at 37 °C, and the absorbance at the wavelength of 490 nm was read in an automated plate reader. Flow cytometry analysis of cell apoptosis Huh7 cells were cultured for 48 h and harvested. Then add 60 ll of MuseTM Annexin V & Dead Cell Reagent (Part No.4700-1485, 100 tests/bottle) to each tube and add 60 ll of cells in suspension to each tube. After incubated for 20 min at room temperature in the dark, then the apoptosis assay was performed by MuseTM Cell Analyser (Millipore, Bedford, MA, USA) according to the manufacturer’s instructions. Colony formation assays Cells were inoculated into 6-well plates at a density of 200 cells/well and following si-RNA transfection according to the manufacturer’s instructions. We analyzed the surviving colonies (C50 cells/colony) using 0.5 % crystal violet stain after 10 days of the cells cultured. Statistical analysis The SPSS 17.0 statistical program was used for statistical analysis. All values were performed as mean ± SEM [18]. The v2 test was used to analyze the association between SYF2 and Ki-67 expression and the clinicopathological features. For multivariate analysis, Cox’s proportional hazards model was used and p \ 0.05 was considered to be statistically significant.
Results The expression of SYF2 in HCC tissues, cell lines, and specimens To determine the expression pattern of SYF2 in HCC tissues, 8 paired fresh HCC and non-tumorous adjacent tissues were analyzed using Western blot analysis.
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Interestingly, it was revealed that the expression of SYF2 was significantly higher in tumor tissues than in adjacent non-tumor ones (Fig. 1a, b). Moreover, we examined the expression profile of SYF2 in HCC cell lines and normal hepatocyte cell lines. Compared with two normal hepatocyte cell lines, SYF2 was highly expressed in HCC cell lines, especially in Huh7 cells (Fig. 1c). Therefore, these observations revealed that SYF2 may be interrelated with the development of HCC. To estimate the relationship of SYF2 with clinicopathologic variables, 92 samples from patients with HCC was analyzed by immunohistochemistry. We found that both of SYF2 and Ki-67 were highly expressed in HCC tissues. However, the expression of SYF2 and Ki-67 was rarely detectable in the adjacent non-tumorous liver tissues (Fig. 1d). For statistical analysis, HCC specimens were divided into high or low groups by cutoff value that has been mentioned in ‘‘Materials and methods’’ section. The clinicopathological data are summarized in Table 1. As shown in Table 1, high expression of SYF2 was apparently associated with Ki-67 (p = 0.005), histological grade (p = 0.013), and tumor size (p = 0.035). There was no statistical correlation between SYF2 expression and other prognostic factors. Survival information was available in these cases. In addition, Kaplan–Meier survival curves showed that high SYF2 level was significantly associated with poor overall survival (p \ 0.001, Fig. 2). Only 15 of 56 (27 %) patients in the SYF2 high expresser group were alive versus 20 of 33 (61 %) in the SYF2 low expresser group (Table 1). Furthermore, using the Cox proportional hazards model, we demonstrated that SYF2 (p = 0.038), as well as Ki-67 (p = 0.008), histological grade (p = 0.006), and Microvascular invasion (p = 0.024), were independent prognostic indicators for patients’ overall survival (Table 2). SYF2 expression correlated with the cell cycle progression of HCC cells SYF2 was reported to play an important role in cell cycle progression in several cancer types. As shown in our aforementioned data, SYF2 and Ki-67 were significantly highly expressed in HCC tissues and positively associated with poor overall survival. On the basis of these results, we speculated that SYF2 expression might facilitate cell cycle progress in HCC cells. To verify the hypothesis, we analyzed SYF2 expression during serum starvation and refeeding experiment in Huh7 cells. After serum deprivation for 72 h, Huh7 cells were arrested in G1 phase (Fig. 3a). Following serum refeeding, Huh7 cells were released from G1 phase and reentered into S phase, and the G1 phase obviously downregulated from 70.02 to 52.56 % (Fig. 3a). Furthermore, we detected the expression of
Mol Cell Biochem Fig. 1 The expression of SYF2 in HCC tissues, cell lines, and specimens. a Western blot analysis revealed that the expression of SYF2 is upregulated in HCC tissues (T) compared with adjacent peritumor tissues (N). b The bar chart shows the ratio of SYF2 protein to GAPDH by densitometry. The data are mean ± SEM of three independent experiments. (*p \ 0.05, tumor tissues compared with adjacent nontumorous). c The SYF2 expression is increased in HCC cells compared with the normal liver cell lines (LO2 and HL7702). d Immunohistochemical evaluation the SYF2 and Ki-67 levels, the results showed that expression of SYF2 and Ki-67 significantly strong in HCC tissues
SYF2, PCNA, and Cyclin D1 using Western blot analysis at the indicated time periods and found that their expression was coordinately upregulated by serum stimulation (Fig. 3b, c). The results suggested that the expression of SYF2 was strongly stimulated following serum-stimulated cell cycle progression. Depletion of SYF2 downregulates Cyclin D1 and inhibits cell proliferation in HCC
Fig. 2 Accumulated survival curves for high SYF2 expression versus low SYF2 expression in 92 patients of HCC. Patients with high expression of SYF2 group show poor prognosis (p \ 0.01)
Based on the above results, we conjectured that SYF2 could promote cell proliferation through the modulation of Cyclin D1. To further determine the role of SYF2 in HCC development, we analyzed the association between SYF2 and Cyclin D1 in HCC cells. We found that SYF2 could interact with Cyclin D1 in HCC tissue and Huh7 cells via immunoprecipitation (Fig. 4a). To determinate the involvement of SYF2 in HCC proliferation, Huh7 cells was transfected with SYF2 siRNAs to knockdown endogenous
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Mol Cell Biochem Table 2 Contribution of various potential prognostic factors to survival by Cox regression analysis in HCC specimens
Hazard ratio
95 % confidence interval
p value
Sex
2.245
0.988–5.100
0.053
Age (years)
1.196
0.654–2.185
0.561
Serum AFP (ng/ml)
1.274
0.717–2.265
0.409
Histological grade
2.265
1.267–4.047
0.006*
Cirrhosis
1.798
0.822–3.931
0.142 0.139
Tumor size(cm)
1.567
0.865–2.839
Tumor metastasis
0.759
0.256–2.248
0.618
Tumor number
1.502
0.815–2.771
0.192
HBsAg
1.153
0.521–2.554
0.725
Microvascular invasion
2.913
1.149–7.381
0.024*
SYF2 expression
2.100
1.041–4.234
0.038*
Ki-67 expression
2.686
1.295–5.572
0.008*
Statistical analyses were performed using log-rank test * p \ 0.05 was considered significant
Fig. 3 SYF2 expression correlated with the cell cycle progression of HCC Cells. a. Flow cytometry quantitation of cell cycle progress in Huh7 cells. After serum starvation for 72 h, the Cells were arrested in G1. Then serum re-addition, Huh7 cells were released from G1 phase and reentered S phase for the indicated times (R4 h, R8 h, R12 h, R24 h). Mean ± SEM of three independent experiments (*, #p \ 0.05, compared with control cells serum starved for 72 h).
S serum starvation, R serum release. b Western blot analysis the expression of SYF2, PCNA, Cyclin D1 in Huh7 cells that serum starvation for 72 h and serum re-addition in indicated times (R4 h, R8 h, R12 h, R24 h). c The bar chart shows the ratio of SYF2, PCNA, Cyclin D1 protein to GAPDH by densitometry. The data are mean ± SEM of three independent experiments. (*, ^, #p \ 0.05 compared with control cells treated with serum starvation for 72 h)
SYF2. At the same time, the control groups were established with negative control siRNA. 48 h after transfection, we analyzed the levels of cellular proteins. SYF2-siRNA#3 was found to obviously downregulate the level of SYF2, as well as PCNA and Cyclin D1 levels. Therefore, SYF2siRNA#3 was used for further experiments (Fig. 4b, c). In addition, immunofluorescence assay showed that SYF2 and Cyclin D1 could co-localization in nucleus. Depletion of SYF2 resulted in decreased Cyclin D1 level in Huh7 cells (Fig. 4d). Moreover, using CCK-8 assay, we found that the proliferation of huh7 cells was obviously reduced after the transfection with SYF2-siRNA (Fig. 4e). Furthermore, flow cytometry analysis of cell cycle revealed depletion of SYF2 caused cell cycle arrest in huh7 cells (Fig. 4f). To sum up, SYF2 depletion inhibits the proliferation of HCC cells.
High expression of SYF2 in Huh7 cells resulted in doxorubicin resistance
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Doxorubicin (DOX) is one of the most potent antitumor agents known, displaying clinical activity against a wide variety of tumors [17]. To investigate the sensitivity of HCC cells to doxorubicin, we perform cell proliferation assays in Huh7 cells following the addition of various concentrations of doxorubicin. Cell proliferation was deceased in a dose-dependent manner following doxorubicin exposure (Fig. 5a). To further study the contribution of SYF2 to doxorubicin resistance in HCC cells, we determined the proliferation and apoptosis rate of Huh7 cells following transfection of SYF2-siRNA#3 and doxorubicin exposure. As shown in Fig. 5b, incubation with doxorubicin (1 lmol/l) resulted in obviously attenuated
Mol Cell Biochem
Fig. 4 Depletion of SYF2 downregulates Cyclin D1 and inhibits cell proliferation in HCC. a Western blot analyzed SYF2 interacted with Cyclin D1 in HCC tissues and Huh7 cells by Immunoprecipitation. b Determination of the efficiency of SYF2-siRNAs in Huh7 cells. c The bar chart shows the ratio of SYF2 and Cyclin D1 to GAPDH by densitometry. The data are mean ± SEM of three independent experiments. (*, #, p \ 0.05, SYF-siRNAs group compared with Con-siRNA group). d Immunofluorescence assay showed that SYF2
and Cyclin D1 could co-localization in nuclear and knockdown SYF2 result in Cyclin D1 levels decreased in Huh7 cells. e CCK-8 assay measured the viability of Cells transfected with SYF2-siRNA#3 and control siRNA. The data are mean ± SEM (*, #, p \ 0.05, compared with control cells). f Flow cytometric analysis the Huh7 cells of transfected with con-siRNA and SYF2-siRNA#3 revealed Depletion of SYF2 inhibits cell cycle
cell growth. Notably, the proliferation of HCC cells was further decreased by SYF2 depletion. Furthermore, cell apoptosis assay revealed that downregulated SYF2 could be contributed to augmented cell apoptosis (Fig. 5c). To verify the above results, Western blot assay was performed to detect the levels of apoptotic proteins, active caspase 3 (Fig. 5d), and cleaved caspase 9 (Supplementary Fig. S1). In this way, we found that depletion of SYF2 increased the levels of cellular active caspase 3 and cleaved caspase 9. In addition, colony formation assays revealed that the colony formation capacity of DOX-treated group is much lower than control group, and knockdown SYF2 further decreased the number of colonies in Huh7 cells (Fig. 5e). On the basis of the results, high SYF2 expression might contribute to doxorubicin resistance.
multiple molecular mechanisms, while the underlying mechanism has not yet been fully elucidated [20]. Thus, the identification of novel molecular markers related with the prognosis of HCC is a primary task in the field of HCC research. SYF2 is widely regarded as an interacting protein of CCNDBP1, which modulates the function of Cyclin D1 [21–23]. Mounting evidence shows that SYF2 participates in diverse tumor progress, such as breast cancer, prostate cancer, colon cancer, and esophageal cancer [24]. However, the role of SYF2 in HCC development remains obscure. In the study, we delineated the potential role of SYF2 in the development of HCC. First of all, the expression of SYF2 was examined in eight paired fresh HCC tumor and adjacent non-tumor tissues, HCC cell lines and Normal hepatocyte cell lines using Western blot analysis. We found that SYF2 was upregulated in the most of clinical HCC tissues as well as HCC cell lines. Next, 92 samples of HCC specimens were analyzed using immunohistochemistry, which revealed that SYF2 expression was obviously correlated with several clinicopathological parameters, such as tumor grade, tumor size, and Ki-67 expression. Univariate and multivariate analyses demonstrated that SYF2 was an
Discussion HCC is one of the most common cancer types worldwide. Despite the improvement in HCC diagnosis and therapy, the rate of HCC mortality remains considerably unfavorable [19]. The development of HCC is associated with
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Mol Cell Biochem Fig. 5 High expression of SYF2 in Huh7 cells resulted in doxorubicin resistance. a CCK8 assays showing cell growth rate of Huh7 cells following addition of DOX for the indicated concentration. b 48 h after SYF2-siRNA#3 and ConsiRNA transfection, cells were treated with DOX stimulation (1 lmol/L) or not. Then CCK-8 assays were performed to compare cell viability of SYF2siRNA#3 group with ConsiRNA group. c Tunnel assay showing cell apoptosis of Huh7 cells. d Western blot analysis active caspase 3 expression in Huh7 cells that transfected with SYF2-siRNA#3 and control siRNA, meanwhile with or without treatment of DOX. e Colony formation analysis of Huh7 cells transfected with Con-siRNA and SYF2siRNA#3 and treated with DOX stimulation(1 lmol/L) or not. The results showed that knockdown SYF2 increased the efficiency of DOX
independent prognostic factor for the survival of HCC patients. In addition, serum starvation and refeeding assay showed that the level of SYF2 along with PCNA and Cyclin D1 was upregulated after cell entrance into S phase. Moreover, we found SYF2 might interact with Cyclin D1. To further clarify the involvement of SYF2 in cell cycle progression, we knockdown SYF2 expression using siRNA oligos, SYF2 depletion lead to downregulate the expression of PCNA and Cyclin D1. CCK-8 assay, Colony formation analysis, and flow cytometry analysis also suggested that silencing SYF2 inhibited cell cycle progression and cell proliferation in HCC cells. Furthermore, drug resistance experiment showed high expression of SYF2 might confer doxorubicin resistance in HCC cells. SYF2 encodes a nuclear protein that interacts with Cyclin D-type binding-protein 1 (GICP), which is considered to be involved in pre-mRNA splicing and cell cycle arrest [12, 25].
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Previous research reported that upregulated expression of GICP impaired the expression of Cyclin D1 though inhibiting the transcriptional activity of Cyclin D1 promoter [21, 26]. As we all know, Cyclin D1 and its catalytic partners (Cdk4/6) induce cell cycle transition of G1-to-S phase to promote cell proliferation [4, 5]. One study suggested that SYF2 is associated with neuronal apoptosis by GCIP modulating the expression of Cyclin D1 [27]. On this account, we supposed that SYF2 plays a part in regulating HCC cell proliferation in a cell cycle-dependent pathway. Our research revealed the function of SYF2 and the relationship between SYF2 and Cyclin D1. Despite these findings, further investigations must be conducted to clarify the explicit role of SYF2 in HCC development. To summarize, our study first revealed that SYF2 was overexpressed in HCC, and upregulated SYF2 was correlated with the histological grade and predicted poor
Mol Cell Biochem
prognosis of HCC. Moreover, the expression SYF2 could promote cell cycle progression in HCC cells. In addition, knockdown SYF2 resulted in cell cycle arrest at G1 phase and inhibited cell proliferation. Furthermore, we found SYF2 might interact with Cyclin D1 and could induce doxorubicin resistance in HCC cells. Collectively, these findings implicated that SYF2 might play a significant role in the development of HCC. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81272708). Conflict of interest of interests.
14.
15.
16.
The authors declare that they have no conflict 17.
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