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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2010 May 26;137(3):533–541. doi: 10.1007/s00432-010-0920-x

Overexpression of SUMO-1 in hepatocellular carcinoma: a latent target for diagnosis and therapy of hepatoma

Wu-hua Guo 1,3, Li-hua Yuan 2,3, Zhi-hua Xiao 2,3, Dan Liu 3, Ji-xiang Zhang 2,3,✉
PMCID: PMC11957374  PMID: 20502916

Abstract

Purpose

To investigate the expression of SUMO-1 in human hepatocellular carcinoma (HCC) cell lines and clinical HCC samples.

Methods

RT–PCR and Western blot were used to detect the expressions of SUMO-1 in HCC cell lines, clinical HCC samples,and the non-neoplastic liver tissues adjacent to HCC. After transfection of SUMO-1 siRNA into HCC cell line SMMC-7721, the expression levels of Bcl-2, c-Myc and α-tubulin were examined, and MTT assay and cell cycle analysis were carried out as well.

Results

Overexpressions of SUMO-1 were detected in HCC cell lines and clinical HCC samples, while the expression level of SUMO-1 in the non-neoplastic liver tissues was significantly lower (P < 0.001). Transfection of SUMO-1 siRNA resulted in 73.43% of maximal silencing efficiency of SUMO-1 in 48 h. The expressions of Bcl-2 and c-Myc were down-regulated coincidentally. SUMO-1 siRNA notably inhibited SMMC-7721 cells proliferation in vitro and increased the ratios of G2 phase and S phase in the cells.

Conclusions

Owing to overexpression of SUMO-1 in HCC and its important role in the development of HCC, SUMO-1 could be a latent target in diagnosis and therapy of HCC.

Keywords: SUMO-1, Hepatocellular carcinoma (HCC), siRNA, Bcl-2 α-tubulin

Introduction

SUMO (small ubiquitin-like modifier) is a member of the ubiquitin-like protein superfamily that consists of four proteins, SUMO-1, SUMO-2, SUMO-3, and SUMO-4. SUMO plays a vital role in numerous biological processes (Jones et al. 2006; Martin et al. 2007). Although SUMO-2 and SUMO-3 are closely related to each other and share 96% sequence identity in vertebrates, either of them shares only about 46% sequence identity to SUMO-1 (Di Bacco et al. 2006). Like ubiquitination, the SUMO modification (SUMOylation) processes include several sequential actions and are mediated by three factors such as E1-activating enzyme, E2-conjugating enzyme, and E3-ligating enzyme (Kim et al. 2000). SUMOylation is a dynamic and reversible process (Itahana et al. 2006). Many proteins, including proliferating cell nuclear antigen (PCNA) (Pfander et al. 2005), murine double minute (Mdm2) (Buschmann et al. 2000), promyelocytic leukemia (PML) (Weidtkamp-Peters et al. 2008), SP100(12), and X-ray cross-complementation group 4(XRCC4)(Yurchenko et al. 2006), are confirmed to be the substrates of SUMO-1.

A growing body of evidence shows that SUMOylation is involved in development and metastasis of cancers (Kang et al. 2008; Kim and Baek 2009; Park et al. 2007). The regulation of p53 by SUMO system has been studied considerably, and Mdm2 has been found to be one of the most important regulators of p53. Mdm2 is a ring domain E3 ligase that ubiquitinates p53, leading to p53 ubiquitination and degradation (Carter et al. 2007). Mdm2 is also a substrate of ubiquitin, and its self-ubiquitination results in itself degradation (Song et al. 2008). Once Mdm2 comes into SUMOylation, the SUMO molecule will protect Mdm2 from ubiquitination, and the expression level of Mdm2 in cells will increase consequentially. The increased Mdm2 would bind more p53 to ubiquitin, which leads to a decline of p53 level in cells (Lee et al. 2006). Therefore, SUMO-1 is a negative regulatory factor of p53, and its modification is inversely correlated with the level of p53.

Growing evidence shows that SUMOylation has many crucial positively regulatory effects on the telomere length maintenance (Xhemalce et al. 2007). SUMOylation elongates the telomere length by increasing telomerase activity, promoting alternative lengthening of telomeres (ALT), and regulating the functions of telomere proteins. Telomerase reactivation and telomere maintenance are crucial in carcinogenesis and tumor progression.

The proto-oncogene c-Myc is a transcription factor that forms a heterodimer with Max and activates the genes involved in proliferation, which promotes progression through G1 into S phase of the cell cycle(Amati et al. 1993). The tumor cell lines proliferation is markedly inhibited by means of short hairpin RNA (shRNA) for reducing c-Myc expression (Wang et al. 2008). Deregulated expression of c-Myc can sensitize cells to a variety of death stimuli, including inactivity of growth factors and loss of oxygen (Brunelle et al. 2004). The Bcl-2 protein family is the key regulators of the apoptotic process, which include the anti-apoptotic protein Bcl-2 and apoptotic proteins Bax and Bak (Cao et al. 2008). Bcl-2 that acts upstream of caspase activation prevents apoptosis by inhibiting the release of Ca2+ from the thapsigargin-sensitive Ca2+ store(Okuno et al. 1998; Reynolds and Eastman 1996). Therefore, the inhibition of both expression and function of Bcl-2 would lead to apoptosis of malignant cells (Duan et al. 2005; Sutter et al. 2004).

In the present study, we investigated the expression levels of SUMO-1 gene in human hepatocellular carcinoma (HCC) cell lines and clinical HCC samples. Using RNAi method, we further studied the role of SUMO-1 in the proliferation of HCC cell line SMMC-7721 by means of MTT assay. The expressions of Bcl-2 and c-Myc were examined to interpret the mechanisms by which SUMO-1 controlled the proliferation of SMMC-7721. We detected overexpressions of SUMO-1 in HCC cell lines as well as in clinical HCC samples. Following the transfection of SUMO-1 siRNA into SMMC-7721, the expressions of Bcl-2 and c-Myc in the cell line were down-regulated coincidentally. Our data confirmed that SUMO-1 could serve as a latent diagnostic marker for hepatocellular carcinoma.

Materials and methods

Cell culture

Three human hepatocellular carcinoma (HCC) cell lines, Hep3B, HepG2, and SMMC-7721 were used in this study, which were all obtained from China Center for Type Culture Collection (CCTCC, China) and cultured in a humidified incubator at 37°C with 5% CO2. Hep3B and HepG2 were cultured in minimum essential medium (MEM, Gibco), and SMMC-7721 was in RPMI-1640 (HyClone). The culture media above were supplemented with 10% fetal bovine serum (HyClone), 50 U/ml penicillin, and 50 μg/ml streptomycin.

Patients and tissue samples

A total of 26 consecutive patients with HCC who underwent surgical resection in the Department of Hepatobiliary Surgery, the Second Affiliated Hospital of Nanchang University, were enrolled in this study. The ages of the 22 males and 4 females ranged from 31 to 67 (means, 46.4 years). The maximal diameters of these masses were from 3.2 to 10.8 cm according to computed tomography scan. The serum levels of alpha fetoprotein (AFP) in peripheral venous blood from the patients varied from 3.32 to 122, 732.14 ng/ml (Table 1). The normal range of serum level of AFP was 0–8 ng/ml. All the patients showed the evidence of suffering from hepatitis B virus (HBV) infection which is the predominant carcinogenic agent in Chinese patients with HCC. The study was approved by Ethics Committee of the second affiliated hospital of Nanchang University, and all the patients signed informed consent for tissue studies. The partial cancer tissues and the non-neoplastic liver tissues adjacent to HCC from surgical resection were reserved in liquid nitrogen. The HCC tissue samples of these patients were confirmed by pathological diagnosis.

Table 1.

The levels of serum AFP and the ratios of SUMO-1/β-actin in HCC and in non-neoplastic liver tissues in 26 patients

Case Gender Age (years) AFP (ng/ml) SUMO-1/β-actin (HCC) SUMO-1/β-actin (non-neoplastic liver)
1 Male 51 13548.22 2.01 0.35
2 Male 53 5.41 1.65 0.23
3 Male 67 23147.36 1.78 0.35
4 Male 51 86445.95 2.15 0.11
5 Male 44 6.23 1.89 0.26
6 Male 41 531.24 1.82 0.31
7 Male 37 1561.92 1.85 0.27
8 Male 32 2698.23 1.95 0.18
9 Male 36 136.43 1.53 0.35
10 Male 45 122732.14 1.67 0.16
11 Male 42 890.17 1.77 0.19
12 Male 55 10.15 1.87 0.21
13 Male 51 45873.15 1.73 0.33
14 Male 55 63.22 1.55 0.29
15 Male 43 85431.21 1.78 0.24
16 Male 38 56756.13 1.56 0.33
17 Female 54 3.32 1.97 0.18
18 Female 47 1278.20 1.74 0.27
19 Male 39 1554.29 1.76 0.16
20 Male 40 897.51 1.86 0.36
21 Male 52 1212.62 1.83 0.21
22 Male 31 445.34 1.61 0.31
23 Female 42 5445.22 1.66 0.18
24 Female 40 1590.70 1.85 0.35
25 Male 55 964.38 1.74 0.16
26 Male 66 6.92 1.77 0.32
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RNAi experiments

The cDNA sequence of SUMO-1 was obtained from GenBank. Three short interfering RNA (siRNA) duplexes targeting human SUMO-1, NO.001(5′-CAA GAA ACT CAA AGA ATC A-3′), NO.002(5′-GGA AGA AGA TGT GAT TGA A-3′), and NO.003(5′-CAA TGA ATT CAC TCA GGT T-3′), were provided by RiboBio Co., Ltd (Guangzhou, China). Negative control siRNA, scrambled sequences from RiboBio which were confirmed not to interact with any mRNA sequence else, was used to balance siRNA where necessary. SMMC-7721 cell line was used to examine the interfering efficiency of the three siRNA duplexes. The transfections of SUMO-1 siRNA were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. SMMC-7721 cell that was not treated served as control (non-treated cells).

RNA extraction and cDNA synthesis

Total RNAs were extracted with Trizol (Invitrogen) from the HCC cell lines, the HCC tissues and non-neoplastic liver tissues adjacent to HCC. The concentration of RNA was determined by absorption measurements at 260 nm of a UV–visible spectrophotometer (Bio-Rad). Two micrograms of total RNA was used as the template for synthesis of first-strand cDNA with an M-MLV RT kit (Promega, USA). The reaction mixture was incubated for 10 min at 70°C and cooled down rapidly on ice water for 5 min. Then, 1 μl M-MLV reverse transcriptase (Promega), 1 μl Rnase inhibitor(Promega), 1 μl dNTP (Generay Biotech, China), 4 μl M-MLV RT 5 × buffer (Promega), and Rnase free water were added into each reaction mixtures, giving a final volume of 20 μl. The reaction mixture was incubated at room temperature for 10 min before RT, which was performed at 42°C for 60 min. Finally, the mixture was incubated at 95°C for 5 min to terminate the reaction.

Semi-quantitative RT–PCR

For amplification of specific cDNAs, Oligomer primers were designed for the following genes: SUMO-1 (sense: 5′-AGG AGG CAA AAC CTT CAA CT-3′; antisense: 5′-TTC TTC CTC CAT TCC CAG TT-3′), Bcl-2 (Sense 5′-GGT GAA CTG GGG GAG GAT TG-3′; antisense 5′-ACT TGT GGC TCA GAT AGG CA-3′), c-Myc (5′-ACA GCG TCT GCT CCA CCT-3′; antisense:5′-CCT CAT CTT CTT GTT CCT CCT-3′), and β-actin (sense: 5′-ACA CTG TGC CCA TCT ACG AGG-3′; antisense: 5′-AGG GGC CGG ACT CGT CAT ACT-3′). The above primers were synthesized by Sangon biological engineering technology & services CO., Ltd (Shanghai, China). PCR was performed in a reaction mixture containing 1 μl sense primer, 1 μl antisense primer, 500 ng cDNA, 12.5 μl 2 × Reaction Mix (TIANGEN, China), and dH2O, giving a final volume of 25 μl. Thirty cycles of denaturation (35 s at 94°C), annealing (35 s, at 51°C for SUMO-1, 53°C for Bcl-2, 54°C for c-Myc or β-actin), and extension (45 s at 72°C) were carried out, followed by a final extension at 72°C for 7 min in a PCR thermal cycler (GeneAmp® PCR System 9600). PCR products were electrophoresed on a 1.5% agarose gel containing ethidium bromide and visualized with UV light. The quantity of band intensity was carried out using GeneGenius Match systems (Syngene).

Western blot analysis

The grinded HCC tissues and non-neoplastic liver tissues adjacent to HCC were resuspended in lysis buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, and pH 8.0). The cultured cells were washed twice with PBS, scraped, and resuspended in the lysis buffer. All the lysates were clarified by centrifugation at 4°C, 12,000 rpm for 15 min followed by desolation and denature in 4% sodium dodecyl sulphate. Forty milligram of protein of each sample was separated by SDS–polyacrylamide gel electrophoresis, followed by being transferred to nitrocellulose membrane (0.45 micrometer, Bio-Rad) which was then blocked for 3 h at 4°C with 0.5% non-fat milk in PBS buffer. The membrane was incubated overnight at 4°C with an appropriate dilution of each primary antibody for SUMO-1 (ABZOOM), Bcl-2(Santa Cruz), c-Myc (Santa Cruz), α-tubulin (Santa Cruz), or β-actin (Santa Cruz). After being washed three times with PBS buffer, the membrane was incubated for 2 h with a 1:1,000 dilution of appropriate horseradish peroxidase-conjugated secondary antibody. After the membrane was washed three times, the immunoblot signals were visualized through enhanced chemiluminescence. The membrane was then exposed to X-ray film. The scan densitometry analysis was carried out by scanning the film on GDS-8000 UVP photo scanner and LAB WOEK45 Image software (Bio-rad). The monoclonal anti-β-actin antibody was used as an internal control for samples loading.

MTT assay

In order to assess cell viability following a treatment with efficient SUMO-1 siRNA duplex, 5 × 103 SMMC-7721 cells were plated in each well of a 96-well plate and treated with SUMO-1 siRNA. The cells treated with negative control siRNA or Lipofectamine alone and non-treated cell served as control groups. The cells were treated in triplicate for each group. After incubation for 12 h in incubator, the cells were transfected according to the manufacturer’s instructions. Following a 48-h treatment with various reagents, 20 ml of MTT (Sigma) solution (5.0 mg/ml) was added to each well and the cells were incubated again for 4 h in a humidified incubator at 37°C with 5% CO2. Then, 200 μl of dimethylformamide solution was added to each well, and cells were incubated for 20 min at 37°C with 5% CO2. The absorbance at 450 nm was measured by ELISA plate reader (DENLEY DRAGON MK2).

Cell cycle analysis

The cell cycle analysis was performed by propidium iodide staining. SMMC-7721 cells were incubated in 10-cm plates for 12 h at 37°C with 5% CO2 before transfection of SUMO-1 siRNA duplex according to the manufacturer’s instructions. The cells trasfected with negative control siRNA or treated by Lipofectamine alone served as controls. After incubation for 48 h, the cells were trypsinized and collected by centrifugation. The cells were then stained by propidium iodide (Sigma, 40 μg/ml in PBS) after washing twice with PBS. Data were collected and analyzed with BD FACSCalibur System. Each experiment was performed in triplicate.

Statistical analysis

All experiments were done at least in triplicate. Data were presented as mean ± SD, and statistical significances were analyzed by paired-samples t-test or one-way analysis of variance (ANOVA), and then the least significant difference method (LSD) was used to compare each of the treatment groups and the control group. The P-values less than 0.05 were considered to be statistically significant.

Results

Expressions of SUMO-1 gene in HCC cell lines, clinical HCC samples, and the non-neoplastic liver tissues adjacent to HCC

In RT–PCR analysis, the SUMO-1 gene was overexpressed in Hep3B, HepG2, SMMC-7721, and 26 clinical HCC samples. The expression levels of SUMO-1 in SMMC-7721, Hep G2, Hep3B, and the clinical HCC samples were elevated by 4.96-, 4.82-, 4.28-, and 6.96-fold than that in non-neoplastic liver tissues adjacent to HCC, respectively (Table 1, P < 0.001). The Western blot analyses all conformed the RT–PCR results coincidentally (Fig. 1). In this study, the positive rate of AFP (≥8.00 ng/ml) was 22 of 26 patients (84.6% positive), while all the 26 patients (100%) showed SUMO-1 levels in clinical HCC samples remarked higher than those in matched non-neoplastic livers. So the expression level of SUMO-1 may be an interesting clue to diagnose clinical hepatocellular carcinoma.

Fig. 1.

Fig. 1

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Expressions of SUMO-1 in HCC cell lines, HCC tissues, and the non-neoplastic liver tissues adjacent to HCC. Total RNA and protein were extracted from above tissues, and RT–PCR and Western Blot were performed as described under “Materials and methods”. Three gels on the left side represent RT–PCR results, and the other three gels on the right side represent Western blots results. a HCC cell lines. b HCC tissues. c The non-neoplastic liver tissues adjacent to HCC. d The bar graph represents the mean ± standard deviation of the expression of SUMO-1 mRNA in the HCC cell lines, HCC tissues, and the non-neoplastic liver tissues adjacent to HCC

Silencing efficiency of SUMO-1 induced by siRNA duplexes in SMMC-7721

At a final concentration of 100 nM, the maximal silencing efficiency of siRNA duplexes NO.001, which was determined by RT–PCR, was 9.80% at 24 h, 73.43% at 48 h, and 46.56% at 72 h after transfection. The expression levels of SUMO-1 proteins were coordinated with the RT–PCR examination (Fig. 2). NO.001 was an ideal siRNA duplex targeting to SUMO-1 gene in SMMC-7721. The concentration of 100 nM and 48 h after transfection were good choices for the next experiments.

Fig. 2.

Fig. 2

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The silencing efficiency in SMMC-7721 at 24, 48, and 72 h after transfection with SUMO-1 siRNA. Total RNA and protein were extracted from SMMC-7721. RT–PCR and Western Blot were performed as described under “Materials and methods”. a RT–PCR analysis. b Western blotting analysis

Expressions of Bcl-2 and c-Myc in SMMC-7721 transfected with SUMO-1 siRNA

Oncogenes, Bcl-2 and c-Myc, both were overexpressed in SMMC-7721. At 48 h after transfection with SUMO-1 siRNA, the mRNA and protein expressions of both Bcl-2 and c-Myc in SMMC-7721 were down-regulated (Fig. 3). The P-values were all less than 0.001 for Bcl-2 and c-Myc. There was a significant positive correlation between the expression of SUMO-1 and that of either Bcl-2 or c-Myc.

Fig. 3.

Fig. 3

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Expressions of SUMO-1, Bcl2 and c-Myc in SMMC-7721 treated with efficient SUMO-1 siRNA, negative control siRNA, or Lipofectamine. RNA and protein were extracted, and RT–PCR and Western Blot were performed as described under “Materials and methods”. a RT–PCR results b Western blots results. c The bar graph represents the mean ± standard deviation of the expression of SUMO-1, Bcl-2 and c-Myc in different groups

Growth inhibition of SMMC-7721 induced by SUMO-1 siRNA

To investigate the role of SUMO-1 siRNA in the growth of SMMC-7721 cells, we transfected SUMO-1 siRNA into SMMC-7721 cells and detected the cells growth using MTT assay. As shown in Fig. 4, at 48 h after transfection, the growth of SMMC-7721 cells was inhibited significantly compared with those of the cells treated with negative control siRNA or Lipofectamine and non-treated cells (each P ≤ 0.001). These results suggest that down-regulation of SUMO-1 could strongly suppress the proliferation of SMMC-7721 cells.

Fig. 4.

Fig. 4

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MTT toxicity assays. 5 × 103 SMMC-7721 cells were plated in each well of the 96-well plates and treated in triplicate with efficient SUMO-1 siRNA, negative control siRNA, or Lipofectamine. After treated with MTT reagents, the optical density was measured at 450 nm

Increases of ratios of G2 and S phases induced by SUMO-1 siRNA

There were interesting variances found in the cell cycle analysis (Fig. 5). As showed in Table 2, the ratios of G1, S, and G2 in SUMO-1 siRNA group showed significant difference from that in control groups. The number of cells in G1 phase decreased at 48 h after transfection with SUMO-1 siRNA compared with those in negative control siRNA group, Lipofectamine and non-treated cell groups, respectively (each P ≤ 0.001). However, the numbers of cell in S and G2 phases increased reversely in SUMO-1 siRNA group compared with those in three control groups (P = 0.007, 0.009, and 0.003 for S phase and each P < 0.001 for G2 phase). These observations indicate that SUMO-1 siRNA could induce a growth arrest of the cells at the G2 and S phases.

Fig. 5.

Fig. 5

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Cell cycle analysis performed by propidium iodide staining. a Efficient SUMO-1 siRNA. b Negative control siRNA. c Lipofectamine alone. d Non-treated cell

Table 2.

The ratios of G1, S, and G2 in SMMC-7721 cells treated with SUMO-1 siRNA, negative control siRNA, or Lipofectamine and in non-treated cells

Groups G1 S G2
siRNA① 71.29 ± 2.46 21.90 ± 3.05 6.81 ± 0.73
NControl② 84.67 ± 1.40 12.45 ± 1.21 2.88 ± 0.30
Lipofectamine③ 84.13 ± 0.82 13.11 ± 1.01 2.76 ± 0.19
Non-treated cells④ 87.46 ± 0.88 10.03 ± 0.87 2.52 ± 0.35
F 65.764** 64.460** 25.772**
t
 ①:② 10.638** 6.533** 10.881**
 ①:③ 10.209** 6.079** 11.213**
 ①:④ 12.851** 8.211** 11.878**
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** P < 0.001

The level of α-tubulin in SMMC-7721 after transfection with SUMO-1 siRNA

In order to elucidate the mechanism by which the transfection of efficient SUMO-1 siRNA increased the ratio of G2 phase in SMMC-7721, Western blot was employed to detect the protein expression levels of α-tubulin in SMMC-7721 cells treated with SUMO-1 siRNA, negative control siRNA, or Lipofectamine as well as in non-treated SMMC-7721 cells. As showed in Fig. 6, the level of α-tubulin in SMMC-7721 transfected with efficient SUMO-1 siRNA was significantly declined, in contrast to those of control groups (each P < 0.001). These results reveal that SUMO-1 could suppress the expression of α-tubulin in SMMC-7721 cells.

Fig. 6.

Fig. 6

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Expression of α-tubulin in SMMC-7721 cells treated with efficient SUMO-1 siRNA, negative control siRNA, or Lipofectamine. Proteins were extracted and Western Blot was performed as described under “Materials and methods”. Top figure: representative Western blot results; Bar graph: the mean ± standard deviation of the level of α-tubulin in different groups

Discussion

A growing body of evidence shows that SUMO-1 plays an important role in the regulation of many cancer-related proteins (Buschmann et al. 2000; Carter et al. 2007; Karamouzis et al. 2008; Pfander et al. 2005). SUMO-1 is a characteristic marker of cancer which is involved in development and metastasis of cancers. In order to elucidate the role of SUMO-1 in the development of HCC, we investigated firstly the expression of SUMO-1 in HCC cell lines and clinical HCC tissues. We found that SUMO-1 was overexpression in all three HCC cell lines experimented in this study and all clinical HCC samples. There was a significant difference of expression levels of SUMO-1 gene between clinical HCC samples and the non-neoplastic liver tissues adjacent to HCC. In contrast, the expression levels did little difference among the non-neoplastic liver tissues adjacent to HCC. Interestingly, the positive rate of AFP in the 26 patients of our study was 84.6%, while all the 26 patients showed much higher levels of SUMO-1 in clinical HCC compared with those in the matched non-neoplastic liver tissues adjacent to HCC. This could illustrate that SUMO-1 makes a vital contribution to development of HCC, which could be a potential diagnostic target for clinical HCC.

Small interfering RNA (siRNA) has become one of the primary means by which most researchers attempt to target specific genes for silencing (Bantounas et al. 2004). In present study, synthetical siRNA duplexes targeting human SUMO-1 was used to clarify the special functions of SUMO-1 in development of HCC. MTT assay was employed to examine the proliferation of SMMC-7721 after transfection by SUMO-1 siRNA duplexes. Interestingly, MTT assay revealed that the proliferation of SMMC-7721 was apparently slower after transfection by siRNA than that of control groups. The cell proliferation is a sophisticated process. In general, once a gene exerts an effect on the potential for a cell to become transformed, the gene can be classified as either a tumor suppressor or an oncogene. There are a variety of tumor suppressors or oncogenes according to their different potencies to suppress or promote cancer cell proliferation. The tumor suppressor, wild-type p53, could inhibit the growth of cancer cell after being transfected into tumor cell (Pataer et al. 2006; Xu et al. 1996). While Bcl-2 and c-Myc, which are two dominant oncogenes, could promote the cancer development and growing, the inhibition of expression and function of Bcl-2 and c-Myc would inhibit tumor growth and induce apoptosis of malignant cells (Brunelle et al. 2004; Duan et al. 2005; Sutter et al. 2004). In this study, following the down-regulation of SUMO-1, the oncogenes Bcl-2 and c-Myc were down-regulated coincidentally in SMMC-7721. Although there may be some other mechanisms to explain growth inhibition of SMMC-7721 induced by SUMO-1 siRNA, the down-regulation of Bcl-2 and c-Myc following SUMO-1 silencing should be at least an important reason.

In this study, cell cycle analysis detected a significant difference of ratios of the cells number in G1, G2, and S phases in SMMC-7721 treated with SUMO-1 siRNA from that in control cells. The cell number increased significantly in the G2 and S phases and decreased in the G1 phase in SUMO-1 siRNA group compared with those in control groups. Many factors, such as Bcl-2 and c-Myc, have been found to play a critical role in the regulation of cell cycle progression, and p53 may lead to a G1/S cell cycle arrest in response to DNA damage (Iyer et al. 2004). Ubc9 is an E2-conjugating enzyme for SUMOylation, which plays a key role in sumoylation-mediated cellular pathways (Wu et al. 2009). Ubc9 is essential for the viability of yeast cells with a specific role in the G2-M transition of the cell cycle (Saitoh et al. 1998). Depletion of Ubc9 arrests cells in G2 or early M phase (Seufert et al. 1995). Our study reveals that down-regulation of SUMO-1 arrests cells in G2 phase, which suggests that Ubc9 and SUMO-1 may share the same pathways to control the cell cycle. SUMO modification regulates the activity of numerous transcription factors that play a direct role in cell cycle progression, apoptosis, cellular proliferation, and development; however, its role in differentiation processes is less clear (Deyrieux et al. 2007). There are many proteins that have been identified as the substrates of SUMO-1 modification. Tubulin, putative SUMO-1 substrates identified by LC-MALDI/MS/MS analysis of TAP-purified proteins (Rosas-Acosta et al. 2005), plays a key role in the G2 phase of cell cycle. Dysfunction of tubulin protein would lead to a G2 cell cycle arrest. In present study, the level of α-tubulin was significantly down-regulated in SUMO-1 siRNA group in contrast with other control groups, which maybe an important mechanism by which SUMO-1 controls cell cycles. These results indicate that SUMO-1 maybe an essential regulator in SMMC-7721 cell cycle progression. Taking together, our experimental results suggest that SUMO-1 could be a latent target in diagnosis and therapy of clinical HCC.

Acknowledgments

We thank Jiang-jing Xu and Dong Yin for their technical assistance. This work has been funded by grants from National nature science grant of China (No. 303200067, for J. Zhang) and from Department of Education of Jiangxi province, China (No. GJJ09107, for W. Guo).

Conflict of interest statement

None.

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