Int J Clin Exp Pathol 2016;9(10):10373-10381 www.ijcep.com /ISSN:1936-2625/IJCEP0031063 Original Article High expression of Sp1 is significantly correlated with tumor progression and poor prognosis in patients with hepatocellular carcinoma Yan Lin1, Jia-Zhou Ye1, Xue-Xin Yan1, Zhi-Hui Liu1, Yong-Qiang Li1, Xiao-Ling Luo1, Rong Liang2 1Department of First Chemotherapy, Affiliated Cancer Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, P. R. China; 2Department of Chemotherapy, Affiliated Minzu Hospital of Guangxi Medical University, Naning 530001, Guangxi Zhuang Autonomous Region, P. R. China Received April 22, 2016; Accepted June 17, 2016; Epub October 1, 2016; Published October 15, 2016 Abstract: This study aimed to investigate the relationship between specificity protein 1 (Sp1) expression and clini- copathological feature and prognosis of hepatocellular carcinoma (HCC). We detected Sp1 mRNA and protein ex- pressions in 12 fresh HCC tissues by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis, respectively. Immunohistochemical assay was performed in 127 paraffin-embedded HCC tissues. The mRNA and protein expressions of Sp1 were significantly higher in HCC tissues than in matched adjacent normal tissues. Sp1 high expression was detected in the cytoplasm and nuclei of carcinoma cells from 68 (53.5%) patients. In addition, high expression of Sp1 was significantly correlated with HBsAg infection (P = 0.036), liver cirrhosis (P = 0.024), tumor differentiation (P = 0.010), vascular invasion (P = 0.035) and metastasis (P = 0.029). Furthermore, Kaplan-Meier analysis showed that patients with high Sp1 expression had significantly lower overall survival (OS) and disease free survival (DFS) rates, compared with patients with low Sp1 expression. Sp1 is an independent prognostic factor for HCC patients. In conclusion, Sp1 expression is significantly up-regulated in HCC and could identify a subset of HCC with aggressive clinical behavior and poor clinical outcome. Sp1 is a potential independent prognostic biomarker for HCC patients after surgery. Keywords: Hepatocellular carcinoma (HCC), specificity protein 1 (Sp1), prognostic marker, invasion Introduction population screening, clinical diagnosis, prog- nosis prediction, and treatment efficiency eval- Hepatocellular carcinoma (HCC) is one com- uation. Therefore, it is important to identify mon cancer and the third leading cause of can- novel biomarkers for HCC and help clinicians cer-related deaths worldwide and China [1]. assess the clinicopathological feathers of the HCC is highly prevalent in China and its mortal- malignancy and decrease the rate of unfavor- ity rate has been increasing since the 1990s able outcomes. [2]. Despite the significant improvements in diagnostic and therapeutic methods in recent Specificity protein 1 (Sp1) protein is a well-char- years, the prognosis of HCC still remains unsat- acterized transcription factor of Sp/Kruppel- isfactory, which is largely caused by its high like factor (KLF) family that can bind to recurrence and invasion rates [3]. The molecu- sequence-specific promoter elements of its tar- lar mechanisms underlying HCC have not been get genes through C-terminal zinc fingers fully known. However, the initiation and devel- domains [5]. Sp1 plays important regulatory opment of HCC are associated with multiple roles in multiple biological functions, including risk factors, including chronic hepatitis virus cell cycle regulation [6], cell growth [7], apopto- infection, aflatoxin, alcohol abuse, and diabe- sis [8], metabolism [9], angiogenesis [10] and tes mellitus [4]. These risk factors can not only metastasis [11]. Growing evidence showed that facilitate deeper understanding mechanisms of Sp1 is overexpressed in a variety of tumors, HCC, but also play important roles in high-risk including lung cancer [12], glioma [13], pancre- High expression of Sp1 in HCC Table 1. Association of Sp1 expression with clinico- In this study, we investigated the expres- pathological features in HCC sion levels of Sp1 in HCC and their paired Sp1 expression adjacent nontumor tissues, and further P Clinical features Cases High level investigated whether the Sp1 expression Low level value (n = 59) (n = 68) levels correlate with HCC clinical parame- ters and prognosis. Age (Years old) 0.183 ≤50 44 24 20 Patients and methods >50 83 35 48 Gender 0.816 Patients and tissue samples Male 81 37 44 Female 46 22 24 This study consists of 127 primary HCC HBsAg 0.036 tumor tissues collected from patients who Negative 50 29 21 underwent hepatectomy at the Affiliated Cancer Hospital of Guangxi Medical Positive 77 30 47 University between July 2005 and AFP (μg/L) 0.955 December 2010. All HCC was diagnosed ≤400 52 24 28 by pathology based on WHO criteria, and >400 75 35 40 none of these patients received chemo- Liver cirrhosis 0.024 therapy or radiotherapy before surgery. No 41 25 16 Their complete clinicopathological data Yes 86 34 52 were recorded and summarized in Table Tumor size (cm) 0.230 1. Tumor differentiation was based on the ≤5 51 27 24 Edmondson and Steiner classification. >5 76 32 44 These patients were followed up after sur- Tumor number 0.853 gery every three months, and the follow- Solitary 57 27 30 up deadline was October 2015. Overall Multiple 70 32 38 survival (OS) was defined as the interval between surgery and death or the last Tumor differentiation 0.010 follow-up (Censored data for living Well + Moderate 64 37 27 patients). Disease free survival (DFS) was Poor 63 22 41 defined as the interval between surgery Vascular invasion 0.035 and the date of relapse. This study was No 52 30 22 approved by the Medical Ethics Committee Yes 75 29 46 of the Affiliated Hospital of Guangxi Metastasis 0.029 Medical University. Written informed con- Negative 60 34 26 sent was signed and obtained from all Positive 67 25 42 patients. HCC, hepatocellular carcinoma; AFP, AFP, α-fetal protein; HR haz- ard ratio; CI confidence interval; Sp1, Specificity protein 1. Quantitative real-time reverse transcrip- tion polymerase chain reaction (qRT-PCR) atic adenocarcinoma [14], thyroid tumor [15], A total of 12 fresh tumorous and adjacent non- nasopharyngeal cancer [16] and gastric cancer tumorous tissue samples were acquired and [17]. However, Sp1 overexpression can lead to immediately frozen at -80°C after resection. apoptosis in multiple cancer cells [18, 19]. The Total RNA was extracted from clinical tissues apparently opposing effects of Sp1 might be using TRIzol reagent (Invitrogen, Grand Island, associated with its regulated multiple genes NY, USA) according to the manufacturer’s involved in essential cellular functions, includ- instructions. Complementary DNA (cDNA) was ing proliferation, apoptosis, differentiation, the synthesized from 1 μg of the total RNA using a DNA damage response, inflammation, senes- reverse transcriptase (RT)-for-PCR Kit (Clon- cence and angiogenesis [20]. Therefore, it is ur- tech Laboratories). The mRNA levels of Sp 1 gent to indentify the clinicopathological feath- and glyceraldehyde-3-phosphate dehydroge- ers and prognostic values of Sp1 in HCC. nase (GAPDH) was determined using aSYBR 10374 Int J Clin Exp Pathol 2016;9(10):10373-10381 High expression of Sp1 in HCC adjacent nontumorous tissues. Protein was extracted using cell lysis buffer and their concentra- tions were determined by BCA method (Pierce, Rockford, IL, USA). The protein (50μg) was electropho- retically separated by 12% SDS- polyacrylamide gel electrophoresis (SDS-PAGE) gels, and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). Then PVDF membrane was blocked with 3% BSA and incubat- ed with primary mouse anti-human monoclonal antibody Sp1 (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 37°C overnight. After washing, the PVDF membrane was incubated with horseradish peroxidase (HRP)-conjugated sec- ond rabbit anti-mouse monoclonal antibody (1:1000 dilutions) to for 2 h at room temperature. The signal was detected by enhanced chemi- luminescence (Pierce® ECL Plus Western Blotting Substrate, Pierce Figure 1. Sp1 was up-regulated in HCC tissues. A. Sp1 mRNA was Biotechnology, IL, USA). β-actin was markedly increased in tumor tissues than that in the paired adjacent used as an internal control. To con- nontumor tissues by qRT-PCR. B. Sp1 protein was markedly increased firm equal loading of the samples, in tumor tissues than that in the paired adjacent nontumor tissues by western blot. β-actin protein served as an internal control. Repre- the same membrane was stripped sentative pictures from three independent experiments are shown. and incubated with mouse mono- C. Increased relative expression of Sp1 protein in HCC normalized to clonal antibodies against β-actin. β-actin. Data were expressed as mean ± SD and a two-tailed, paired t-test was performed. Significant difference from the control group is denoted by ‘‘*’’ (P<0.05). N, normal adjacent nontumor tissues; T, Immunohistochemical (IHC) assay HCC tumor tissue. Immunohistochemical staining was performed as previously described Green PCR Kit (Applied Biosystems) and Light- with slight modifications [16]. Briefly, paraffin- Cycler480 384-well PCR system (Roche Dia- embedded samples were cut into 5 μm sec- gnostics). The GAPDH was used as an internal tions and placed on polylysine-coated slides. control for ITPKA. Primers for Sp1 were 5’- Paraffin sections were baked for 2 h at 58°C, TCCAGACCATTAACCTCAGTGC-3’ (forward) and dewaxed in xylene and rehydated with ascend- 5’-TGTATTCCATCACCACCAGCC-3’ (reverse). Pri- ing graded ethanol. Endogenous peroxidase mers for GAPDH were 5’-CTCCTCCTGTTCGAC- activity was quenched by incubating sections in AGTCAGC-3’ (forward) and 5’-CCCAATACGACCA- 0.3% hydrogen peroxide for 10 min. Antigen AATCCGTT-3’ (reverse). The value of relative retrieval was performed by high pressure cook- expression for each sample was determined ing (700 W microwave oven) sections in 10 mM using the Ct method, and was normalized to the citrate antigen retrieval solution (0.1 mM citric average expression value of 12 adjacent nontu- acid, 0.1 M sodium citrate; pH = 6.0) for about morous tissues.