Volume 139, Issue 6, Pages 2183-2194.e5 (December 2010) Increased Expression of iASPP, Regulated by Hepatitis B Virus X Protein-Mediated NF- κB Activation, in Hepatocellular Carcinoma  Bin Lu, Huaizu Guo, Jian Zhao, Chong Wang, Guobin Wu, Mingshu Pang, Xin Tong, Fangfang Bu, Anmin Liang, Sheng Hou, Xiaoyu Fan, Jianxin Dai, Hao Wang, Yajun Guo  Gastroenterology  Volume 139, Issue 6, Pages 2183-2194.e5 (December 2010) DOI: 10.1053/j.gastro.2010.06.049 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 The aberrant expression levels of iASPP in HCC tissues. (A) iASPP mRNA was increased in paired tumorous and nontumorous tissues, HBV-infected liver, and liver cirrhosis (LC) tissues, compared with normal liver tissues (NL), using real-time RT-PCR analysis. (B) Representative immunostaining analyses of iASPP expression in normal liver, HBV-infected and liver cirrhosis tissues, and HCC tissue. Original magnification, 200×. Gastroenterology 2010 139, 2183-2194.e5DOI: (10.1053/j.gastro.2010.06.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 The identification of putative sequence and transcriptional activity of iASPP promoter. (A) Agarose gel electrophoresis of nested PCR products from the 5'-RACE procedure. Molecular weight (MW) markers (base pairs) were indicated on the left. The main PCR product was marked by an arrow on the right. (B) Sequencing of second-round PCR products showed the boundary between universal anchor primer and iASPP mRNA sequences. The thymidine marked by a red arrow indicated a putative transcriptional start site. (C) Various truncated iASPP promoters were inserted into reporter vector pGL3. These constructs were cotransfected with an internal control vector (Renilla) into HepG2 cells in triplicates. The luciferase activities were analyzed 48 hours after transfection and expressed as folds over that of pGL3. Data are means ± SEs of triplicate determinations. (D) Schematic representation of the 5′-flanking iASPP genomic region: the cloned promoter (shaded box), the 2 first exons (solid boxes), and the first intron (open box). NF-κB (p65 subunit) binding site, corresponding to nucleotides −428 to −420 (relative to putative transcription start site +1), was found with the use of the TFSEARCH program. Gastroenterology 2010 139, 2183-2194.e5DOI: (10.1053/j.gastro.2010.06.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Transcriptional factor NF-κB binds to and activates iASPP promoter. (A) Decreased luciferase activity of the iASPP promoter by mutation of the putative NF-κB binding site in HepG2 cells (*P < .05). Data are means ± standard errors of triplicate determinations. (B) Gel shift analysis of transcriptional factors binding to the nt −436 to −415 cis-regulatory domain. A synthetic 22-bp oligonucleotide with (ProbeMut) or without (Probe) NF-κB binding site mutation was incubated with nuclear extract (NE) purified from HepG2 cells. SC, specific competitor. (C) Identification of the trans-regulatory protein and confirmation of binding. Gel shift assays with the use of Probe were repeated in the presence of antibody to p65 or p50. Supershift bands were detected with increasing concentration of anti-p65 (lane 5 and 6) and anti-p50 (lane 8 and 9). Gel shown is representative of 3 independent experiments. (D) ChIP analysis of p65/p50 binding to the iASPP promoter in HepG2 cells. The blot is representative of 3 experiments. Gastroenterology 2010 139, 2183-2194.e5DOI: (10.1053/j.gastro.2010.06.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 NF-κB activates the iASPP promoter in HCC cell lines. (A) HL-7702 and HepG2 cells 24 hours after being transfected with iASPP-Luc plasmid were serum starved for at least 6 hours, treated with 20 ng/mL TNF-α in serum-free Dulbecco's modified Eagle's medium, and harvested at the indicated time for luciferase assay. (B) iASPP transcripts were detected by quantitative RT-PCR at the indicated times after being treated with 20 ng/mL TNF-α. *P < .05; **P < .01 vs 0 hour group. (C) Protein were extracted from HL-7702 and HepG2 cells 24 or 36 hours after treatment with TNF-α and were subjected to immunoblot analysis for the detection of iASPP expression. *P < .05; **P < .01 vs untreated group (UT). (D) CH-Hep1 and PLC/PRF/5 cells were transfected with iASPP-Luc plasmid for 24 hours. Then cells were treated with Bay 11-7802 (500 ng/mL) for 48 hours, and luciferase activities were analyzed. CH-Hep1 and PLC/PRF/5 cells were treated with Bay 11-7802 for 48 hours, the mRNA (E) and protein (F) levels were detected. *P < .05; **P < .01. Experiments were performed in triplicate for each treatment and repeated 3 times. Gastroenterology 2010 139, 2183-2194.e5DOI: (10.1053/j.gastro.2010.06.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 HBx up-regulates the expression of iASPP by activating NF-κB. (A) NF-κB activity was determined by a gel shift assay with nuclear extracts prepared from HL-7702 and HepG2 cells transfected with HBx expression plasmid or empty vector. Gel shown is representative of 3 independent experiments. (B) ChIP analysis of p65 binding activity to iASPP promoter region affected by HBx gene transfection. The input fraction corresponded to 0.1% of the chromatin solution before immunoprecipitation. The blot is representative of 3 experiments. (C) HL-7702 and HepG2 cells were cotransfected with iASPP-Luc and HBx expression plasmid for 24 hours, with or without treatment of Bay 11-7802 for another 48 hours before being subjected to luciferase assay. Ctrl, pGL3-enhancer vector. *P < .05, **P < .01. HL-7702 and HepG2 cells were transfected with HBx expression plasmid or empty vector for 72 hours; the mRNA (D) and protein (E) levels were detected. *P < .05, **P < .01 vs pcDNA transfection group. The experiment was repeated 3 times. Gastroenterology 2010 139, 2183-2194.e5DOI: (10.1053/j.gastro.2010.06.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 The expression of iASPP promotes cell proliferation and its resistance to chemotherapeutic drug in human HCC cells. PLC/PRF/5 and HepG2 cells were stably transfected with lentivirus encoding shRNA or cDNA as indicated for 72 hours, and the mRNA (A) and protein (B) levels of iASPP were detected. (C) PLC/PRF/5 or HepG2 cells treated as described above were seeded in 96-well plates, and cell proliferation was assessed every day for 5 days with the use of the MTS assay. (D) The cells handled as above were plated in semisolid soft agar medium to monitor anchorage-independent growth. The numbers represent the mean ± SD of 3 independent experiments. Photographs are representative images from each group as indicated under the microscope. (E) The cells above were treated with 0.5 μg/mL cisplatin for 24 hours. The percentage of apoptotic cells were detected by fluorescence flow cytometry with annexin V and propidium iodide (PI) staining. The bar graphs show the average number of apoptotic cells (right). The experiments were repeated 3 times. *P < .05 vs control lentivirus group. Gastroenterology 2010 139, 2183-2194.e5DOI: (10.1053/j.gastro.2010.06.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 The expression of iASPP promoted tumorigenicity of HCC xenografts in nude mice. (A) Male Balb/c nude mice were injected subcutaneously with 1 × 107 PLC/PRF/5 or HepG2 cells into the right flank of each animal. One week after injection, the mice were dosed intraperitoneally with (solid line) or without (dotted line) cisplatin at a concentration of 5 mg/kg of body weight weekly for 3 weeks. Tumor size was monitored and recorded every 5 days. Data are the means ± standard deviations of the tumor volumes derived from each group. (B) Total proteins were extracted from tumor tissues by 1× SDS buffer and subjected to Western blotting with anti-iASPP antibody. *P < .05 vs control lentivirus group. (C) In situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling apoptosis analysis was performed in tumor sections derived from the same mice as in panel A. The apoptotic nuclei were seen as a green color excited under fluorescence microscopy. Cell nuclei were counterstained with DAPI and seen as blue (magnification ×200). The percentage of apoptotic cells was calculated by counting green-stained nuclei compared with blue-stained nuclei from 6 randomly chosen fields in each section. Data are means ± standard deviations. *P < .05, **P < .01. Gastroenterology 2010 139, 2183-2194.e5DOI: (10.1053/j.gastro.2010.06.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 The aberrant expression levels of iASPP in HCC cell lines. Expressions of iASPP mRNA (A) and protein (B) in HCC cell lines were detected by quantitative RT-PCR and Western blotting. The signal in each lane was quantified by Quantity One software (Bio-Rad, Hercules, CA), and the ratio of iASPP to actin protein was determined. Gastroenterology 2010 139, 2183-2194.e5DOI: (10.1053/j.gastro.2010.06.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 Nucleotide sequence of the 5′ portion of human iASPP gene. The DNA sequence of the 2000-bp fragment is shown as iASPP putative promoter, where +1 corresponds to the most downstream RNA start sit, and residues preceding this position are represented by negative numbers. The TATA and CAAT sequences are boxed, and the putative NF-κB binding sequences are underlined. Gastroenterology 2010 139, 2183-2194.e5DOI: (10.1053/j.gastro.2010.06.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 3 (A) Map of predicted CpG islands of the 5′ upstream region of the iASPP gene. Content of G+C and Obs/Exp CpG were calculated with the use of the CPGPLOT (EMBOSS) program. One main CpG island was identified (nt −569 to −347). (B) Representative examples of bisulfite genomic sequencing in cancer cell lines and tissues. One microgram of genomic DNA was incubated with 3 mol/L sodium bisulfite, and 50 ng of bisulfite-modified DNA was subjected to PCR amplification of the iASPP promoter region with the use of primer sets iABSPF (sense, 5′-TTTGTAGTAGAAGGGGAAATGG-3′) and iABSPR (5′-ATACCCTACCCCTTTAAATCC-3′) for nucleotides −744 to −262 (30 CpG sites). The PCR products were cloned into pGEM-T easy vectors (Promega), and 5 clones of each specimen were sequenced by automated fluorescence-based DNA sequencing to determine the methylation status. The white circles indicate an unmethylated CpG (0 of 5 clones), and the gray squares indicate a partially methylated CpG (≤3 of 5 clones). Gastroenterology 2010 139, 2183-2194.e5DOI: (10.1053/j.gastro.2010.06.049) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 4 iASPP is overexpressed in response to NF-κB activation. (A) HepG2 cells were treated with increasing amounts of TNF-α as indicated for 24 hours in the absence of serum, and iASPP relative expression was calculated. (B) HL-7702 cells were transfected with the p65, IKβ, or IKKβ expression plasmid, alone or combined together. Transfected cells were incubated in a serum-free media and were harvested 48 hours after transfection. *P < .05. Gastroenterology 2010 139, 2183-2194.e5DOI: (10.1053/j.gastro.2010.06.049) Copyright © 2010 AGA Institute Terms and Conditions