J Mol Hist (2014) 45:59–68 DOI 10.1007/s10735-013-9529-0

ORIGINAL PAPER

Decreased expression of SERPINB1 correlates with tumor invasion and poor prognosis in hepatocellular carcinoma Xiaopeng Cui • Yanhua Liu • Chunhua Wan • Cuihua Lu • Jing Cai • Song He • Tingting Ni • Junya Zhu • Lixian Wei • Yixin Zhang • Haixin Qian

Received: 29 May 2013 / Accepted: 25 July 2013 / Published online: 9 October 2013 Ó Springer Science+Business Media Dordrecht 2013

interfering RNA targeting SERPINB1 was used to knock down the expression of SERPINB1 in Huh7 and BEL-7404 cells. We showed that interference of SERPINB1 promoted migration and invasion of HCC cells, while cell proliferation was not affected. Finally, we observed an apparent increase in the level of active matrix metalloproteinase-2 (MMP2) after SERPINB1 knockdown, implying that SERPINB1 might participate in the regulation of HCC metastasis through modulating the activation of matrix metalloproteinases. Overall, our results suggested an inhibitory role of SERPINB1 in the migration and invasion of HCC, implying that SERPINB1 might be a potential prognostic indicator of HCC metastasis.

Abstract SERPINB1 (serine protease inhibitor, clade B, member1) is a member of the SERPINB family. Recent studies suggested that SERPINB1 may suppress the migration and invasion of lung and breast cancers. In this study, we investigated a possible involvement of SERPINB1 in the regulation of hepatocellular carcinoma metastasis (HCC). The expression of SERPINB1 was evaluated using western blot analysis in 8 paired fresh HCC specimens and immunohistochemistrical assay on 67 paraffin-embedded HCC slices. SERPINB1 was downregulated in HCC specimens and correlatively related with two clinicopathologic features of HCC, metastasis (P = 0.000) and vein invasion (P = 0.006). Univariate and multivariate survival analyses showed a lower level of SERPINB1 expression is associated with poor prognosis and clinical outcome (P = 0.001). In addition, small

Keywords SERPINB1  Hepatocellular carcinoma  Invasion  Prognosis

Xiaopeng Cui and Yanhua Liu contributed equally to this work.

Introduction

X. Cui  H. Qian (&) Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, People’s Republic of China e-mail: [email protected] X. Cui  Y. Liu  C. Lu  T. Ni  J. Zhu  L. Wei Department of General Surgery, Affiliated Hospital of Nantong University, Medical College of Nantong University, Nantong, Jiangsu 226001, People’s Republic of China C. Wan School of Public Health, Nantong University, Nantong, Jiangsu 226001, People’s Republic of China J. Cai  S. He  Y. Zhang (&) Department of Pathology, Nantong University Cancer Hospital, Nantong, Jiangsu 226001, People’s Republic of China e-mail: [email protected]

Hepatocellular carcinoma (HCC) represents one of the most common malignant tumors and the third leading cause of cancer mortality in the world, accounting for approximately 750,000 new cases and more than 670,000 cancer-related deaths annually worldwide (Jemal et al. 2011). Despite improvements in therapy for HCC, the prognosis of HCC remains unsatisfactory, mainly due to the high metastasis and recurrence rate of HCC cells (Tung-Ping Poon et al. 2000). Metastasis causes most cancer-related deaths in HCC patients. The pathogenesis of cancer metastasis is a highly complicated multi-step process and involves various factors, whose detailed mechanism remains one of the most enigmatic aspects of the disease (Chaffer and Weinberg 2011). Therefore, a better knowledge with the molecular mechanism of cancer

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metastasis would provide novel therapeutic opportunities for HCC and other types of cancers. Serine protease inhibitors (serpins) are a superfamily of proteins that employ a unique suicide substrate-like inhibitory mechanism to inhibit serine-type proteases. Over 1,500 serpin genes have been identified in the genomes of organisms across all kingdoms of life, in which 36 genes are human serpins. Although a few serpins have been found to exert diverse functions such as hormone transport (Pemberton et al. 1988) and blood pressure regulation (Potempa et al. 1994), most members of this family functions as serine protease inhibitors (Irving et al. 2000; Rawlings et al. 2004). Serpins share a highly conserved core structure that is critical for their function as serine protease inhibitors. Human serpins are involved in many biological processes, including blood clotting, fibrinolysis, inflammation, complement activation and cell migration (Law et al. 2006; Gettins 2002). According to phylogenic analysis, the superfamily is divided into 16 different clades (A–P), among which human serpins are the first 9 clades (A–I) (Irving et al. 2000). The clade B serpins (SERPINB family) contain 13 members, in which three genes (SERPINB1, B6, and B9) localize on 6p25 and the others (SERPINB2, B4, B5, B7, B8, B10, B11, B12, and B13) localize on 18q21.3 (Askew et al. 2001; Bartuski et al. 1997; Coughlin et al. 1995; Evans et al. 1995; Eyre et al. 1996;). The N and C terminus of the SERPINB family are shorter than other serpins, and they also lack a classical secretory signal peptide (Remold-O’Donnell 1993). SERPINB1 is a member of the SERPINB family, and functions as a fast-acting stoichiometric proteinase inhibitor that regulates the activity of neutrophil elastase (NE), cathepsin-G and proteinase-3 (Zeng et al. 1998). In a rat lung injury model, recombinant SERPINB1 was capable of blocking both the hemorrhagic and epithelial permeability changes associated with instillation of neutrophil elastase (Rees et al. 1999). SERPINB1 may enhance or delay apoptosis depending on the mode of induction in BHK cells (Silverman et al. 2004). Besides, recent studies have found that overexpression of SERPINB1 significantly suppressed invasion and migration of CL1-5 and MDAMB231 cells (Chou et al. 2012). However, the role of SERPINB1 in the pathogenesis and development of HCC has never been explored. The invasion and metastasis of cancer cells is a complicated event that involves many factors (Zheng et al. 2011). To initiate the process of metastasis, cancer cells must degrade the ECM barrier. The MMPs family is critically involved in the tumor cell migration and invasion by degrading all essential components of the extracellular matrix (Deryugina and Quigley 2006; Egeblad and Werb 2002). Amongst the MMPs, MMP-2/9 play determinant roles in ECM degradation and cancer metastasis. Some

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studies have shown that the invasive and metastatic capabilities of HCC were restrained by suppression of MMP2 and MMP9 (Kessenbrock et al. 2010; Yang et al. 2009). These studies indicated MMP-2/9 are promising therapeutic targets in the prevention of cancer metastasis. Normally, MMPs are secreted as inactive zymogens into the extracellular space. The activation of pro matrix metalloproteinases (pro-MMPs) by sequential proteolysis of the propeptide blocking the active site cleft is regarded as one of the key events for the regulation of these proteinases. The activation of soluble pro-MMPs is mediated by serine proteases, including plasmin, by MT-MMPs (e.g., activation of the MMP-2 proenzyme by MT1-MMP) or by other active MMPs (e.g., activation of the proenzymes of MMP1 and -9 by MMP-3) (Murphy et al. 1999; Chernov and Strongin 2011). However, the regulatory mechanism of how pro-MMP2/9 were cleaved in cancer cells remains to be clarified. Identification of factors involved in MMP2/9 activation is of important therapeutic value. In this study, we determined the SERPINB1 expression in HCC and found that SERPINB1 was remarkably downregulated in human HCC specimens, compared with adjacent normal liver tissues. Besides, we also evaluated its associations with clinicopathological variables and the prognostic significance. Moreover, we showed that interference of SERPINB1 promoted the migration and invasion of HCC cells, as well as the expression of acitve MMP2. Our findings implicated that SERPINB1 might be a repressor of HCC metastasis.

Materials and methods Patients and tissue samples Specimens of HCC tissues were obtained from 67 patients who underwent hepatic surgical resection without preoperative systemic chemotherapy at the Surgery Department, the Affiliated Hospital of Nantong University. The main clinical and pathologic variables of the patients are shown in Table 1. Fifty-six patients were male and eleven were female; their ages ranged from 31 to 72 years, with an average age of 53.33 years. A total of 58 patients were positive for the HBV surface antigen, 36 were positive for cirrhosis. Tumors were classified to well (grade I; n = 19), mood differentiated cancers (grade II; n = 35), and poor differentiated tumors (grade III; n = 13). The follow-up time for 67 patients ranged from 1 to 102 months from the date of surgery. None of the patients received postoperative adjuvant therapy. Informed consent was obtained from all patients. Tissue specimens were immediately processed after surgical removal. For histological examination, all

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Table 1 SERPINB1 expression and clinicopathologic parameters in 67 HCC specimens Parameters

Total

SERPINB1 expression Low

High

P value

Age (year) B50

31

11

20

[50 Gender

36

10

26

Male

51

14

37

Female

16

7

9

0.600

0.234

20 min to retrieve the antigen. Tissue sections were incubated with overnight at 4 °C with rabbit anti-SERPINB1 polyclonal antibody. Similar tissue sections immunostained with nonspecific immunoglobulin G were used as negative controls. After washed in phosphate-buffered saline (PBS), tissues were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse Ig polymer as a second antibody (Envision kit, Dako) for 30 min at room temperature, according to the manufacturer’s instructions. After rinsing in water, slides were counterstained with hematoxylin, dehydrated, and mounted in resin mount.

Histological grade Well

19

5

14

Mod

35

12

23

Poor

13

4

9

Presence

11

10

1

Absence

56

11

45

0.833

Metastasis 0.000*

Vein invasion Presence

12

8

4

Absence

55

13

42

0.006*

53 14

15 6

38 8

0.340

Presence

58

16

42

0.126

Absence

9

5

4

Presence

36

12

24

Absence

31

9

22

B50

33

10

23

[50

34

11

23

Tumor size B5 [5 HBsAg

Cirrosis 0.795

AFP 1.000

Statistical analyses were performed by the Pearson v2 test HBsAg hepatitis B surface antigen, AFP alphafetoprotein * P \ 0.05 was considered significant

tumorous and surrounding non-tumorous tissue portions were processed into 10 % buffered formalin-fixed, paraffin-embedded blocks. Protein was analyzed in 8 snap-frozen tumorous and adjacent nontumorous tissue samples that were stored at -80 °C. All human tissue was collected using protocols approved by the Ethics Committee of Affiliated Hospital of Nantong University. Immunohistochemistry staining In brief, sections were dewaxed in xylene and rehydrated in graded ethanol. Endogenous peroxidase activity was blocked by immersion in 3 % methanolic peroxide for 10 min. Then the slides were processed in 10 mmol/L citrate buffer (pH6.0) and heated to 121 °C in an autoclave for

Immunohistochemical evaluation All of the immunostained sections were evaluated in a blinded manner without knowledge of the clinical and pathological parameters of the patients. For assessment of SERPINB1, five highpower fields in each specimen were selected randomly, and nuclear staining was examined under high power magnification. More than 500 cells were counted to determine the mean percent, which represented the percentage of immunostained cells relative to the total number of cells. Expression score was determined by staining intensity and immunoreactive cell percentage. Tissues with no staining were rated as 0, with a faint staining or moderate to strong staining in B25 % of cells as 1, with moderate staining or strong staining in 25–50 % of cells as 2, strong staining in C50 % of cells as 3. For statistical analysis, \2 were counted as low expression, while C2 were counted as high expression. Cell culture All of the cell lines were obtained from the Shanghai Institute of Cell Biology, Academic Sinica. The LO2, Huh7, HepG2, and BEL7404 cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 100 U/mL penicillin, and 100 l g/m L streptomycin in 5 %CO2 at 37 °C. Western blot analysis and antibodies Western blot experiments were used to measure certain proteins. Briefly, tissue and cell protein were promptly homogenized in a homogenization buffer containing 1 M Tris–HCl pH 7.5, 1 % Triton X-100, 1 % NP-40 (non-idet p-40), 10 % sodium dodecyl sulfate (SDS), 0.5 % sodium deoxycholate, 0.5 M EDTA, 10 mg/mL leu-peptin, 10 mg/mL aprotinin, and 1 mM PMSF, then centrifuged at 10,000g for 30 min to collect the supernatant. Protein concentrations were determined with a Bio-Rad protein assay (Bio-Rad, Hercules, CA). The supernatant was diluted in 2 9 SDS loading buffer and boiled. The proteins extracted were

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separated with SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidine difluoride filter (PVDF) membranes (Millipore, Bedford, MA). The membranes were blocked with 5 % nonfat dry milk in TBST (20 mM Tris, 150 mM NaCl, 0.05 % Tween-20). After 2 h at room temperature, the membranes were incubated overnight with antibodies against anti-SERPINB1 (anti-rabbit, 1:500; Santa Cruz Biotechnology); and anti-GAPDH (antirabbit, 1:1,000, Sigma). The secondary antibodies were visualized using LumiGLO Regent and Peroxide (Cell Signaling). The optical density on the film was measured with a computer imaging system (Imaging Technology, Ontario, Canada). SiRNA and transfection Small interference RNAs (siRNA) were chemically synthesized from Biomics (Nantong, China). The human SERPINB1-siRNA targeting the nucleotide residues 50 -CG GGCAUGGUUGAUAACAU-30 , The negative control siRNA targeting the nucleotide residues 50 -UUCUCCGA ACGUGUCACGU-30 , was used as a control. HuH7 and BEL-7404 cells were seeded the day before transfection using the Dulbecco modified Eagle medium with 10 % FBS but without antibiotics. Transient transfection of siRNA vectors and the nonsilence vectors was performed using Lipofectamine 2000 (Invitrogen, USA) Plus reagent in OptiMEM, as suggested by the manufacturer. Cells were incubated for 4 h at 37 °C, and FBS was then added to achieve a final concentration of 10 %. Transfected cells were used for subsequent experiments 48 h after transfection.

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each bottom chamber, 1 9 105 HCC cells in 0.2 mL serumfree media were loaded onto the upper chamber. After incubating for 24 h at 37 °C, top cells were removed, and bottom cells were fixed with 4 % paraformaldehyde and stained with crystal violet before being counted under an inverted microscope. All experiments were performed in triplicate. Data represent the average number of cells from three filters (Tao et al. 2012). Cell proliferation assay Cell proliferation was measured using the CCK-8 (cell counting Kit-8) assay following the manu- facturer’s instructions. In brief, cells were seeded on a 96-well cluster plate (Corning Inc, Corning, NY) at a concentration of 2 9 104/well in a volume of 100lL and grown overnight. Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) reagents were added to a subset of wells under different treatments and incubated for 2 h at 37 °C, after which absorbance was quantified on an automated plate reader. Statistical analysis Data were presented as (mean ± SD). Statistical analysis was performed using SPSS 17.0 statistical software (SPSS, Inc. Chicago, IL, USA). For analysis of survival data, Kaplan–Meier curves were constructed, and the log-rank test was performed. Multivariate analysis was performed using Cox’s proportional hazards model, with P \ 0.05 considered statistically significant.

Wound-healing assay

Results

Equal numbers of stably transfected cells were seeded in density of 5 9 105 per well into six-well plates. When the cells reached 90 % confluence, a scratch wound was created in the center of the cell monolayer by gently removing the attached cells with a 200 lL pipette tip. The debris was removed by washing the cells with serum-free culture medium. The cells were then cultured for 0, 24 h and inspected under an inverted microscope. The distances cells migrated into the wounded area were calculated by subtracting the distance 24 h after wound healing from the initial distance.

Decreased expression of SERPINB1 in HCC tissues and cells

In vitro invasion assay A 24-well transwell plate (8-lm pore size, Corning, USA) was used to measure the invasive ability of each cell line. Transwell filters were coated on the upper side with 30 ug of Matrigel for 2 h at 37 °C. After 500 lL DMEM medium supplemented with 10 % fetal bovine serum was placed into

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To explore a possible role of SERPINB1 in HCC, we examined the expression of SERPINB1 in 8 paired specimens using western blot analysis and found that SERPINB1 expression was remarkably downregulated in tumor tissues compared with the adjacent noncancerous liver tissues (Fig. 1a). We next examined the basal abundances of SERPINB1 in normal hepatocyte cell line LO2 and the human HCC cells HuH7, HepG2, BEL-7404. As shown in Fig. 1b, the level of endogenous SERPINB1 in the three HCC cell lines was lower than that in the normal hepatocyte cell line LO2. Correlation between SERPINB1 expression and clinicopathological parameters in HCC We further investigated the expression of SERPINB1 in HCC by immunohistochemical staining. As shown in

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Fig. 1 The expression of SERPINB1 in human hepatocellular carcinoma tissues and cell lines. a Expression of SERPINB1 in eight representative paired hepatocellular carcinoma tissue (T) and adjacent noncancerous liver tissues (N). b The SERPINB1 protein levels were significantly lower in the HCC cells lines: HuH7, HepG2, BEL-7404 than in the normal liver cell line LO2. GAPDH was used as a loading control. The same experiment was repeated at least three times

Fig. 2, increased expression of SERPINB1 was found in the sample tissues with no lymph node metastasis. Then, we analyzed the association between SERPINB1 expression and clinicopathologic variable of the patients, which are summarized in Table 1. For statistical analysis of the expression of SERPINB1, the patients were divided into

Fig. 2 Immunohistochemical analysis of the SERPINB1 protein expression in paraffin-embedded HCC tissues. a1, a2, a3 Immunostaining of an HCC tumor (T) and the adjacent non-tumorous area (N). 9100 (a1) and 9400 (a2 and a3). b1, b3 Cancer tissues with no

63

Fig. 3 Accumulated survival curves according to SERPINB1 expression. On the basis of the results of immunohistochemistry, HCC patients were divided into high SERPINB1 expressers (score C 2) and low EHD2 expressers (score \ 2). Kaplan–Meier survival curves for high SERPINB1 expression versus low SERPINB1 expression in 67 patients of HCC showed a highly significant separation (P = 0.001, log-rank test)

two groups: high SERPINB1 expressers (score C 2) and low SERPINB1 expressers (score \ 2). SERPINB1 expression was significantly correlated with metastasis (P = 0.000) and invasion (P = 0.006) (Table 1), while there was no significant association between expression of

lymph nodes metastases showed high SERPINB1 expression (9200 and 9400). b2, b4 Cancer tissues with lymph nodes metastases showed low SERPINB1 expression (9200 and 9400)

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Table 2 Survival status and clinicopathologic paremeters in 67 HCC specimens Parameters

Total

Survival status Dead

P value

Alive

Age

Age (year) B50

31

21

10

[50 Gender

36

22

14

Male

51

31

20

Female

16

12

4

19

10

9

0.617

0.379

Histological grade Well Mod

35

22

13

Poor

13

11

2

Presence

11

11

0

Absence

56

32

24

0.175

95.0 % CI

P

1.183

0.594–2.355

0.632

Gender

1.084

0.455–2.584

0.856

Histological grade

1.490

0.884–2.512

0.135

Metastasis

0.917

0.297–2.833

0.881

Vein invasion

2.612

0.914–7.467

0.073

Tumor size

0.791

0.355–1.762

0.566

AFP HBsAg

0.749 1.956

0.341–1.643 0.609–6.290

0.470 0.260

Cirrhosis

0.850

0.433–1.667

0.636

SERPINB1 expression

0.339

0.133–0.862

0.023*

HBsAg hepatitis B surface antigen, AFP alphafetoprotein 0.005*

Vein invasion Presence

12

11

1

Absence

55

32

23

0.044*

53 14

32 11

21 3

0.347

Presence

58

37

21

1.000

Absence

9

6

3

Tumor size

HBsAg

Cirrosis Presence

36

21

15

Absence

31

22

9

B50

33

23

10

[50

34

20

14

0.317

AFP 0.447

SERPINB1 Low expression

21

19

2

High expression

46

24

22

0.002*

Statistical analyses were performed by the Pearson v2 test HBsAg hepatitis B surface antigen, AFP alphafetoprotein * P \ 0.05 was considered significant

SERPINB1 and other clinicopathological parameters, such as gender, age, histological grade, tumor size, HBsAg, cirrhosis and the level of AFP. Prognostic significance of SERPINB1 expression We further carried out Kaplan–Meier analysis to study the correlation between SERPINB1 expression and patients’ survival. At the end of clinical follow-up, survival analysis was restricted to 67 patients with follow-up data and immunohistochemistrical results of SERPINB1. Kaplan–Meier survival curves showed that the HCC patients in the low

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Hazard ratio

Statistical analyses were performed by the Cox regression analysis

Metastasis

B5 [5

Table 3 Contribution of various potential prognostic factors to survival by Cox regression analysis in 67 HCC specimens

* P \ 0.05 was considered significant

expression group had poorer overall survival than those with high SERPINB1 expression (P = 0.001; Fig. 3). Moreover, in univariate analysis, when all variables were compared separately to survival status, it was found that SERPINB1 (P = 0.002), metastasis (P = 0.005) and vein invasion (P = 0.044) significantly influenced patients’ survival (Table 2). Multivariate analysis using the Cox’s proportional hazards model showed that SERPINB1 was an independent prognostic factor in patients with HCC (Table 3). SERPINB1 affects migration and invasion of HCC cells To determine the detailed function of SERPINB1 in HCC development, we prepared SERPINB1-targeting siRNA and transfected it into HuH7 and BEL-7404 cells which have higher expression of SERPINB1. The knocking-down efficiency of siRNA oligo was confirmed in the two HCC cell lines using western blot analysis (Fig. 4a). Since results on clinical specimens pointed an involvement of SERPINB1 in HCC metastasis and vein invasion, we then examined the potential role of SERPINB1 on the invasiveness of HCC cells by cell migration and invasion assays. As shown in Fig. 4b, the wound healing assay was conducted at different confluence levels of mock, control, and si-SERPINB1 cells. and representative photo-micrographs were taken at 0, 24 h after damaging cells using a 2-mm-wide tips. We observed that the relative migrating distance in si-SERPINB1-transfected cells was significantly longer than other groups. The matrigel invasion assay revealed that the invasiveness of siRNA-treated Huh7 and BEL-7404 cells was increased compared with the non-interfered and control-siRNA transfected cells (Fig. 4c). These results suggested that SERPINB1 could inhibit the migration and invasion in HCC cells.

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Fig. 4 Knockdown of SERPINB1 affects migration and invasion of HCC cells. a SERPINB1 protein expression detected by Western blot 48 h after siRNA transfection of HuH7 and BEL-7404 cells. b A wound-healing assay was used to determine the migration of cells treated with si-SERPINB1 (or negative control). Migration of the cells to the wound was visualized at 0 and 24 h with an inverted Leica phase-contrast microscope. The data are mean ± SEM *P \ 0.05. c Invasion activity of HuH7 and BEL-7404 cells transfected with

indicated expression vectors. Cells with si-SERPINB1 showed more prominent invasion activity. The data are mean ± SEM (*P \ 0.01, compared with Mock). All of above assays were repeated 3 times. d Cell proliferation was measured by CCK-8 assay. Cell Counting Kit-8 reagents were added to the medium and incubated for additional 2 h. Absorbance was measured at each indicated time (0, 1, 2, and 3 d). The data are mean ± SEM. (P [ 0.05, compared with Mock). The results are responsible for three independent experiments

Furthermore, we also examined the effects of SERPINB1 knockdown on cell proliferation. HuH7 and BEL7404 cells transfected with si-SERPINB1 and control siRNA were subjected to CCK-8 assay. As shown in Fig. 4d, the proliferation of HuH7 and BEL-7404 cells was unchanged after SERPINB1 knockdown, suggesting that SERPINB1 could specifically influence the migration and invasion in HCC cells, but not cell proliferation.

extracellular matrix and are tightly related to tumor cell migration and invasion (Bjorklund and Koivunen 2005; Canel et al. 2008; Zheng et al. 2006). Since it was reported that SERPINB1 could inhibit the activity of neutrophil elastase (NE), cathepsin-G and proteinase-3, all of which played critical roles in the cleavage and activation of MMP2 and other MMP members, we investigated whether SERPINB1 knockdown in Huh7 and BEL-7404 cells had an effect on the level of the active form of MMP2. Huh7 and Bel-7404 cells were mock-transfected, or transfected with control siRNA or siRNA targeting SERPINB1, and then subjected to western blot analysis. As shown in Fig. 5a, we found that knockdown of SERPINB1 significantly increased the level of the active MMP2. The finding

Effects of altered SERPINB1 expression on the level of active MMP2 MMP2 is a member of the matrix metalloproteinase (MMP) family, which play crucial roles in degradation of

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Fig. 5 Expressions of MMP2 in SERPINB1-silenced HCC cells. MMP2 protein expression was detected by Western blot 48 h after siRNA transfection of HuH7 and BEL-7404 cells. The expressions of MMP2 in HCC si-SERPINB1 cells were increased dramatically. The data are mean ± SEM. (*P \ 0.01, compared with Mock)

suggested that elevated activity of MMP2 might contribute to the enhanced migration and invasion of SERPINB1knockdown cells.

Discussion Currently, various SERPINB genes have been reported to play important roles in the tumorigenesis and progression of many types of cancers. Although previous studies have suggested that SERPINB1 might function as a tumor suppressor in breast and lung cancers (Chou et al. 2012), its role in HCC still remains virtually unknown. In the present study, the expression of SERPINB1 was found significantly decreased in HCC tissues and cell lines using western blot analysis. Moreover, Immunohistochemistrical analysis confirmed the reduced expression of SERPINB1 in the HCC samples, compared with the corresponding nontumorous liver tissues. Further, we showed that interference of SERPINB1 promoted the expression of active MMP2 and the invasion of HCC cells. However, the proliferation of HCC cells didn’t alter signficantly when SERPINB1 was interfered. Based on these findings, we infered that SERPINB1 might play a crucial role in repressing the recurrence and metastasis of hepatocellular carcinoma. Epidemiologic and molecular studies have suggested that 80 % of the HCC cases worldwide correlate with chronic hepatitis B virus (HBV) and hepatitis C virus

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(HCV) infection, while 20 % are ascribed to alcoholic cirrhosis, metabolic disorders, and environmental factors such as aflatoxin B1 exposure (Ferlay et al. 2010). The factors may cause genetic alterations in liver cells, such as activation of proto-oncogenes and inactivation of tumorsuppressor genes, providing chances for neoplastic transformation and progression (Feitelson et al. 2002). Since HCC exhibits a high rate of metastasis, it would be of great importance to understand the molecular alterations that affect the process of HCC metastasis. Although some proteins have been reported to be involved in the metastasis of HCC, such as AhR, NET-1 and VEGF (Liu et al. 2013; Wu et al. 2013), much remains unclear with this regard. In the study, we showed that SERPINB1 was downregulated in HCC specimens and negatively correlated with the metastasis and vein invasion of the tumor. We linked depressed expression of SERPINB1 with enhanced MMP2 activity and cell invasion in HCC cells. These results indicated that downregulated SERPINB1 might be a novel molecular alteration involved in HCC metastasis. Matrix metalloproteinases have been heavily implicated in tumor metastasis. Some serine-type proteases, such as neutrophil elastase (NE), cathepsin-G and proteinase-3, has been implicated in the cleavage and activation of MMP-2, MMP-9 and other MMP members (Shamamian et al. 2001). The acitivity of the serine proteases are tightly regulated by serine protease inhibitors that are also distributed in the extracellular space, such as SERPINB1, SERPINB2 and SERPINB3, etc. (Cooley et al. 2001; Baker

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et al. 1990; Schick et al. 1998). Therefore, the decreased levels of serine protease inhibitors in tumor tissues might associate with higher activation of MMPs and tumor metastasis. In keeping with the notion, we showed that SERPINB1 expression was negatively correlated with HCC metastasis, as well as the migration and invasion of HCC cells, which could at least partially attribute to the hyperactivation of MMP-2. Since these serine proteases are also responsible for the activation of other MMPs, such as MMP-9 and MMP-3, further investigation regarding the issue would provide a more detailed and convincing knowledge to our study. In conclusion, this study showed for the first time that SERPINB1 was downregulated in hepatocellular carcinoma. Further, we demonstrated that interference of SERPINB1 profoundly enhanced the migration and invasion of HCC cell lines, as well as the activity of MMP2. Further studies are needed to clarify the underlying molecular mechanism of SERPINB1 in the regulation of HCC metastasis. Acknowledgments This work was supported by a Grant of Administration of Science and Technology of Nantong (BK2012090). Conflict of interest

All the authors declare no conflict of interest.

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