G Model
ARTICLE IN PRESS
PRP 51188 1–5
Pathology – Research and Practice xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original Article
1
Low expression of activating transcription factor 3 in human hepatocellular carcinoma and its clinicopathological significance
2
3
4 5 6 7 8
Q1
Li Xiaoyan a,c,∗ , Zang Shengbing a , Zhang Yu a , Zheng Lin a , Lin Chengjie b , Liu Jingfeng b,∗∗ , Huang Aimin a,∗ ∗ ∗ a
Department of Pathology and Institute of Oncology, Fujian Medical University, Fuzhou 350004, PR China Liver Center, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, PR China c Department of Pathology, Fujian Medical University Union Hospital, Fuzhou 350001, PR China b
9
10 22
a r t i c l e
i n f o
a b s t r a c t
11 12 13 14 15
Article history: Received 10 September 2013 Received in revised form 10 February 2014 Accepted 19 March 2014
16
21
Keywords: Carcinoma Hepatocellular Activating transcription factor 3 (ATF3) Gene expression
23
Introduction
17 18 19 20
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
Background and Aim: To explore the expression and role of Activating Transcription Factor 3 in Human hepatocellular carcinoma. Methods: Immunohistochemistry, Western blot assay and Real-time PCR were used to evaluate Activating Transcription Factor 3 protein and gene level in HCC clinical samples. Results: Activating Transcription Factor 3 expression is lowest in HCC, and the protein level is lower in patients with capsule invasion, while no association with other main clinical pathological features. Conclusions: Low expression of ATF3 may function as a tumor suppressor during human hepatocellular oncogenesis and targeting ATF3 pathway might be beneficial for anti-HCC therapy. © 2014 Published by Elsevier GmbH.
Human hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is responsible for approximately one million deaths each year [1]. Because of a high prevalence of chronic HBV infection, HCC has been ranked as the second most frequent fatal cancer since the 1990s in China [2]. Within this context is the growing effort being undertaken to diagnose and treat HCC as effectually as possible. The occurrence process of HCC is as complicated as other malignant tumors. It is influenced by various factors in the internal and external environment, such as virus infection, genetic variation, and alterations in molecular cellular pathways. According to previous researches, dysregulation of transcription factors like NF-B, AP – 1, Ets et al., is believed to play an important role in the tumorigenesis, where each transcription factor can mediate multiple signaling pathways and have the coupling effect in the interaction of different signaling ways. Therefore interposing regulatory mechanism of
Q2 ∗ Corresponding author at: Department of Pathology and Institute of Oncology, Q3 Fujian Medical University, Fuzhou 350004, PR China. Tel.: +86 13509332104.
∗∗ Corresponding author. Tel.: +86 13905029580; fax: +86 59183722198. ∗ ∗∗Corresponding author. Tel.: +86 13960818461; fax: +86 59122862869. E-mail addresses: [email protected], [email protected] (L. Xiaoyan), [email protected] (L. Jingfeng), [email protected] (H. Aimin).
transcription factors in signaling ways might present a novel target for the prevention and treatment of HCC. The activating transcription factor 3 (ATF3), a member of the ATF/CREB subfamily, is a stress-inducible gene, and its protein is a basic leucine zipper transcription factor [3]. The induction of ATF3 is a common cellular response to many stress signals; it is neither tissue- nor stress-specific [4]. ATF3 acts as a transcriptional repressor as a homodimer, although the same protein functions as a transcriptional repressor or activator in heterodimeric form [5]. In some instances ATF3 has been shown to activate transcription [6], while in other cases ATF3 has been found to suppress transcription of target genes [7]. The effects of ATF3 on cellular physiology appear to be very context- and cell type-dependent. So far, studies have been done in different laboratories in America, Europe and Asia, most of the studies have focused on ATF3’s precise role in oncogenesis and tumor progression. Some of the results could be coincident, but the diversity is still distinct due to the differences in tumor types and cell lines. And in most of these studies cell lines and animal models were used as the subjects frequently, while human tissues were rarely studied. These results may serve as reference for our research. For further guidance in clinical practice, more human tissue samples should need to be studied. Until now, the relationship between ATF3 expression and HCC has remained uncertain and unclear, and rare experiments have been put on the liver tissues. In order to better understand the role by which ATF3 acts in HCC, the expression of ATF3 was
http://dx.doi.org/10.1016/j.prp.2014.03.013 0344-0338/© 2014 Published by Elsevier GmbH.
Please cite this article in press as: L. Xiaoyan, et al., Low expression of activating transcription factor 3 in human hepatocellular carcinoma and its clinicopathological significance, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.03.013
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
G Model PRP 51188 1–5
L. Xiaoyan et al. / Pathology – Research and Practice xxx (2014) xxx–xxx
2 66 67 68
ARTICLE IN PRESS
evaluated in HCC clinical samples using Real-time PCR and Western blot assay, and its expression was correlated with HCC clinicopathological parameters in this study.
were similarly generated to ensure that equal amounts of protein were loaded in the wells. Reverse transcription and real-time RCR using SYBR green
69
70
71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93
94
Methods Patient tissue samples Samples were taken from 84 HCC and 10 hepatic hemangioma patients at the Liver Center, First Affiliated Hospital of Fujian Medical University, between 2009 and 2010. All HCC tissue samples and corresponding peritumoral liver tissue samples were obtained from patients who had undergone surgical hepatectomy (72 men and 12 women, age range 30–81 years, median age 51 years). Ten normal liver tissue samples were obtained from patients who underwent liver resection due to hepatic hemangiomas. None of the patients received preoperative chemotherapy or radiation therapy. Peritumoral liver tissues were obtained from regions more than 5 cm in distance from the tumors. The tissue samples were immediately fixed in neutral buffered formalin and embedded in paraffin for HE staining. The diagnoses of HCC were confirmed by pathological studies, as was the normalcy of the peritumoral liver tissue samples. In addition, the peritumoral tissue samples were evaluated to ensure the absence of tumor and inflammation. Clinical information was collected from patient records and incorporated in Tables 1–3, tumor differentiation was graded by the Edmondson grading system [8], and tumor stage was determined by the Barcelona Clinic Liver Cancer (BCLC) staging system [9]. This study was approved by the Institute Research Ethics Committee of Fujian Medical University, and informed consent was obtained from each patient according to the committee’s regulations. Immunohistochemistry
104
Immunohistochemistry was performed using the EliVision Plus Two-step System (Maixin Incorporation, Fuzhou, China) according to the manufacturer’s instructions. The paraffin sections were repaired with heat induced epitope retrieval and incubated with a 1:100 dilution of mouse monoclonal antihuman ATF3 antibody (Sigma, USA) for 1 h at 37 ◦ C, followed by 3 washes with phosphatebuffered saline. Then the sections were incubated with poly-HRP anti-mouse/rabbit IgG (ZSbio, Beijing, China), followed by DAB and counterstained with hematoxylin. Negative control sections were incubated with preimmune serum.
105
Western blot
95 96 97 98 99 100 101 102 103
106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123
Frozen tissue was homogenized and thawed in ice-cold lysis buffer (150 mM NaCl, 100 mM Tris (pH 8.0), 1%Tween 20, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml trypsin, and 10 g/ml leupeptin). The lysate was left on ice for 20 min and then centrifuged at 12,000 rpm for 10 min. The clarified supernatant was collected, and the protein concentration was measured by using a Biotek protein assay kit (Biotek, Elx800, USA). Protein lysate (60 g per well), was separated by 12% SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with 5% non-fat milk for 1 h at room temperature and then with mouse monoclonal anti-human ATF3 antibody (at 1:100 dilution, Abcam, USA) at 4 ◦ C overnight. The membranes were washed and stained with a horseradish-peroxidase-conjugated secondary antibody (at 1:1000 dilution, Beyotime, Shanghai, China). Proteins were visualized using Enhanced Chemiluminescence Plus system (Beyotime, Shanghai, China) and exposed to autoradiography film (Kodak, Japan). Blots with mouse monoclonal -actin antibody (at 1:1000 dilution, Beyotime, Shanghai, China)
Total RNA was isolated from the liver tissue following the TriPure Isolation Reagent (Roche, CH) protocol and reverse transcribed using Prime ScriptTM RT reagent kit (TaKaRa, Dalian). The cDNA was then amplified with primers for the ATF3 gene (Forward 5 -GTG TCC ATC ACA AAA GCC GA-3 , Reverse 5 -TGA GCC TTC AGT TCA GCA TTC-3 , 173bp) and the human -actin gene (Forward 5 -GCG TGA CAT TAA GGA GAA GC-3 , Reverse 5 -CCA CGT CAC ACT TCA TGA TGG-3 , 230bp). Real-time PCR was done using ABI Step-One system (Applied Biosystems, US) and SYBR Green qPCR premix (TaKaRa, Dalian), and -actin for standardization. The thermal cycling conditions composed of an initial denaturation step at 95 ◦ C for 3 min followed by 40cycles of PCR using the following profile: 95 ◦ C for 5 s, 60 ◦ C for 34 s. There is inverse relationship between threshold cycle (Ct value) and gene starting copy numbers, the Ct value was estimated after adjustment of baseline cycles and calculation of the Ct value. Statistical analysis The statistical package SPSS Version 19.0 was used to calculate the P value for the association between variables. The Western Blot (WB) results were expressed as median, and the PCR results were expressed as x¯ ± s. Significance was declared when P was 3 With Without With Without I–II III–IV Solid Pseudoglandular Trabecular Sclerosis Positive Negative ≥400
ARTICLE IN PRESS
PRP 51188 1–5
Pathology – Research and Practice xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original Article
1
Low expression of activating transcription factor 3 in human hepatocellular carcinoma and its clinicopathological significance
2
3
4 5 6 7 8
Q1
Li Xiaoyan a,c,∗ , Zang Shengbing a , Zhang Yu a , Zheng Lin a , Lin Chengjie b , Liu Jingfeng b,∗∗ , Huang Aimin a,∗ ∗ ∗ a
Department of Pathology and Institute of Oncology, Fujian Medical University, Fuzhou 350004, PR China Liver Center, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, PR China c Department of Pathology, Fujian Medical University Union Hospital, Fuzhou 350001, PR China b
9
10 22
a r t i c l e
i n f o
a b s t r a c t
11 12 13 14 15
Article history: Received 10 September 2013 Received in revised form 10 February 2014 Accepted 19 March 2014
16
21
Keywords: Carcinoma Hepatocellular Activating transcription factor 3 (ATF3) Gene expression
23
Introduction
17 18 19 20
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
Background and Aim: To explore the expression and role of Activating Transcription Factor 3 in Human hepatocellular carcinoma. Methods: Immunohistochemistry, Western blot assay and Real-time PCR were used to evaluate Activating Transcription Factor 3 protein and gene level in HCC clinical samples. Results: Activating Transcription Factor 3 expression is lowest in HCC, and the protein level is lower in patients with capsule invasion, while no association with other main clinical pathological features. Conclusions: Low expression of ATF3 may function as a tumor suppressor during human hepatocellular oncogenesis and targeting ATF3 pathway might be beneficial for anti-HCC therapy. © 2014 Published by Elsevier GmbH.
Human hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is responsible for approximately one million deaths each year [1]. Because of a high prevalence of chronic HBV infection, HCC has been ranked as the second most frequent fatal cancer since the 1990s in China [2]. Within this context is the growing effort being undertaken to diagnose and treat HCC as effectually as possible. The occurrence process of HCC is as complicated as other malignant tumors. It is influenced by various factors in the internal and external environment, such as virus infection, genetic variation, and alterations in molecular cellular pathways. According to previous researches, dysregulation of transcription factors like NF-B, AP – 1, Ets et al., is believed to play an important role in the tumorigenesis, where each transcription factor can mediate multiple signaling pathways and have the coupling effect in the interaction of different signaling ways. Therefore interposing regulatory mechanism of
Q2 ∗ Corresponding author at: Department of Pathology and Institute of Oncology, Q3 Fujian Medical University, Fuzhou 350004, PR China. Tel.: +86 13509332104.
∗∗ Corresponding author. Tel.: +86 13905029580; fax: +86 59183722198. ∗ ∗∗Corresponding author. Tel.: +86 13960818461; fax: +86 59122862869. E-mail addresses: [email protected], [email protected] (L. Xiaoyan), [email protected] (L. Jingfeng), [email protected] (H. Aimin).
transcription factors in signaling ways might present a novel target for the prevention and treatment of HCC. The activating transcription factor 3 (ATF3), a member of the ATF/CREB subfamily, is a stress-inducible gene, and its protein is a basic leucine zipper transcription factor [3]. The induction of ATF3 is a common cellular response to many stress signals; it is neither tissue- nor stress-specific [4]. ATF3 acts as a transcriptional repressor as a homodimer, although the same protein functions as a transcriptional repressor or activator in heterodimeric form [5]. In some instances ATF3 has been shown to activate transcription [6], while in other cases ATF3 has been found to suppress transcription of target genes [7]. The effects of ATF3 on cellular physiology appear to be very context- and cell type-dependent. So far, studies have been done in different laboratories in America, Europe and Asia, most of the studies have focused on ATF3’s precise role in oncogenesis and tumor progression. Some of the results could be coincident, but the diversity is still distinct due to the differences in tumor types and cell lines. And in most of these studies cell lines and animal models were used as the subjects frequently, while human tissues were rarely studied. These results may serve as reference for our research. For further guidance in clinical practice, more human tissue samples should need to be studied. Until now, the relationship between ATF3 expression and HCC has remained uncertain and unclear, and rare experiments have been put on the liver tissues. In order to better understand the role by which ATF3 acts in HCC, the expression of ATF3 was
http://dx.doi.org/10.1016/j.prp.2014.03.013 0344-0338/© 2014 Published by Elsevier GmbH.
Please cite this article in press as: L. Xiaoyan, et al., Low expression of activating transcription factor 3 in human hepatocellular carcinoma and its clinicopathological significance, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.03.013
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
G Model PRP 51188 1–5
L. Xiaoyan et al. / Pathology – Research and Practice xxx (2014) xxx–xxx
2 66 67 68
ARTICLE IN PRESS
evaluated in HCC clinical samples using Real-time PCR and Western blot assay, and its expression was correlated with HCC clinicopathological parameters in this study.
were similarly generated to ensure that equal amounts of protein were loaded in the wells. Reverse transcription and real-time RCR using SYBR green
69
70
71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93
94
Methods Patient tissue samples Samples were taken from 84 HCC and 10 hepatic hemangioma patients at the Liver Center, First Affiliated Hospital of Fujian Medical University, between 2009 and 2010. All HCC tissue samples and corresponding peritumoral liver tissue samples were obtained from patients who had undergone surgical hepatectomy (72 men and 12 women, age range 30–81 years, median age 51 years). Ten normal liver tissue samples were obtained from patients who underwent liver resection due to hepatic hemangiomas. None of the patients received preoperative chemotherapy or radiation therapy. Peritumoral liver tissues were obtained from regions more than 5 cm in distance from the tumors. The tissue samples were immediately fixed in neutral buffered formalin and embedded in paraffin for HE staining. The diagnoses of HCC were confirmed by pathological studies, as was the normalcy of the peritumoral liver tissue samples. In addition, the peritumoral tissue samples were evaluated to ensure the absence of tumor and inflammation. Clinical information was collected from patient records and incorporated in Tables 1–3, tumor differentiation was graded by the Edmondson grading system [8], and tumor stage was determined by the Barcelona Clinic Liver Cancer (BCLC) staging system [9]. This study was approved by the Institute Research Ethics Committee of Fujian Medical University, and informed consent was obtained from each patient according to the committee’s regulations. Immunohistochemistry
104
Immunohistochemistry was performed using the EliVision Plus Two-step System (Maixin Incorporation, Fuzhou, China) according to the manufacturer’s instructions. The paraffin sections were repaired with heat induced epitope retrieval and incubated with a 1:100 dilution of mouse monoclonal antihuman ATF3 antibody (Sigma, USA) for 1 h at 37 ◦ C, followed by 3 washes with phosphatebuffered saline. Then the sections were incubated with poly-HRP anti-mouse/rabbit IgG (ZSbio, Beijing, China), followed by DAB and counterstained with hematoxylin. Negative control sections were incubated with preimmune serum.
105
Western blot
95 96 97 98 99 100 101 102 103
106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123
Frozen tissue was homogenized and thawed in ice-cold lysis buffer (150 mM NaCl, 100 mM Tris (pH 8.0), 1%Tween 20, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml trypsin, and 10 g/ml leupeptin). The lysate was left on ice for 20 min and then centrifuged at 12,000 rpm for 10 min. The clarified supernatant was collected, and the protein concentration was measured by using a Biotek protein assay kit (Biotek, Elx800, USA). Protein lysate (60 g per well), was separated by 12% SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with 5% non-fat milk for 1 h at room temperature and then with mouse monoclonal anti-human ATF3 antibody (at 1:100 dilution, Abcam, USA) at 4 ◦ C overnight. The membranes were washed and stained with a horseradish-peroxidase-conjugated secondary antibody (at 1:1000 dilution, Beyotime, Shanghai, China). Proteins were visualized using Enhanced Chemiluminescence Plus system (Beyotime, Shanghai, China) and exposed to autoradiography film (Kodak, Japan). Blots with mouse monoclonal -actin antibody (at 1:1000 dilution, Beyotime, Shanghai, China)
Total RNA was isolated from the liver tissue following the TriPure Isolation Reagent (Roche, CH) protocol and reverse transcribed using Prime ScriptTM RT reagent kit (TaKaRa, Dalian). The cDNA was then amplified with primers for the ATF3 gene (Forward 5 -GTG TCC ATC ACA AAA GCC GA-3 , Reverse 5 -TGA GCC TTC AGT TCA GCA TTC-3 , 173bp) and the human -actin gene (Forward 5 -GCG TGA CAT TAA GGA GAA GC-3 , Reverse 5 -CCA CGT CAC ACT TCA TGA TGG-3 , 230bp). Real-time PCR was done using ABI Step-One system (Applied Biosystems, US) and SYBR Green qPCR premix (TaKaRa, Dalian), and -actin for standardization. The thermal cycling conditions composed of an initial denaturation step at 95 ◦ C for 3 min followed by 40cycles of PCR using the following profile: 95 ◦ C for 5 s, 60 ◦ C for 34 s. There is inverse relationship between threshold cycle (Ct value) and gene starting copy numbers, the Ct value was estimated after adjustment of baseline cycles and calculation of the Ct value. Statistical analysis The statistical package SPSS Version 19.0 was used to calculate the P value for the association between variables. The Western Blot (WB) results were expressed as median, and the PCR results were expressed as x¯ ± s. Significance was declared when P was 3 With Without With Without I–II III–IV Solid Pseudoglandular Trabecular Sclerosis Positive Negative ≥400
