Volume 128, Issue 7, Pages 2029-2041 (June 2005) Use of Adenovirus-Delivered siRNA to Target Oncoprotein p28GANK in Hepatocellular Carcinoma  Honghai Li, Xiaoyong Fu, Yao Chen, Yi Hong, Yexiong Tan, Huifang Cao, Mengchao Wu, Hongyang Wang  Gastroenterology  Volume 128, Issue 7, Pages 2029-2041 (June 2005) DOI: 10.1053/j.gastro.2005.03.001 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 In vitro transcription of the T7-siRNA system was established and a specific p28GANK RNAi target site was identified. (A) Different amounts of T7-siRNA against GFP along with pEGFP-C1 and HA-tagged p28GANK plasmids were cotransfected into 293T cells. Cell lysates were harvested after 48 hours and subjected to anti-GFP and anti-HA Western blot. (B) Fluorescence photomicrographs of 293T cells 48 hours after transfection of T7-siRNA against GFP (1.0 μL) and pEGFP-C1. (C) Three T7-siRNAs targeting different coding regions of p28GANK were transfected individually into 293T cells along with HA-tagged p28GANK and pEGFP-C1 plasmids. Cell lysates were blotted at 48 hours after transfection with anti-HA and anti-GFP antibodies. (D) Real-time RT-PCR for p28GANK transcripts was performed using extracted mRNA from 293T cells transfected with p28GANK siRNA against different target sites. β-actin was used as the inner control and the mRNA amplitude in mock group was set as 1. (E) The sequence of nucleotide-2 was used as the template for siRNA transcription under the T7 promoter. Annealed 22-bp siRNA duplex was used to target p28GANK most effectively. Gastroenterology 2005 128, 2029-2041DOI: (10.1053/j.gastro.2005.03.001) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Adenovirus packaging of vectors capable of producing short-hairpin RNA targeting p28GANK was constructed and proved to be specific and potent in silencing p28GANK expression in 4 HCC cell lines. (A) The stem-loop-producing oligonucleotides with palindrome structure were cloned into the unique Xho I and Xba I sites in linearized pSuppressorAdeno shuttle vector. The target sequence was selected as the nucleotide-2 transcripted siRNA. (B) Indicated cells were infected with AdSiGFP (control) or AdSip28GANK. Cell lysates were harvested at 48 or 96 hours after infection and detected by anti-p28GANK and anti-actin antibodies. (C) Quantification of protein expression shown in B. Densitometric analysis of the p28GANK protein bands was normalized to the β-actin bands. □, Mock; ■, AdSiGFP; , AdSip28GANK 2d; , AdSip28GANK 4d. (D) Real-time RT-PCR of p28GANK mRNA from HuH-7 cells harvested 2 days after infection at the indicated MOIs showed a dose-dependent decrease in mRNA levels. At an MOI of 10, the AdSip28GANK suppresses the expression of p28GANK mRNA by up to 80% compared with the mock group (data set as 1). Gastroenterology 2005 128, 2029-2041DOI: (10.1053/j.gastro.2005.03.001) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Adenovirus-delivered p28GANK siRNA inhibits HCC cell growth, tumorigenesis, and S-phase entry. (A) The growth of HCC cells and normal PHHs infected with adenovirus was measured by MTT assay. Values were given at the indicated time points as the mean absorbance with an SD of 4 wells. ◊, Mock; ■, AdSiGFP; ▴, AdSip28GANK. (B) Colony formation assay was performed by using SMMC-7721 cells infected with adenovirus for 96 hours. The cells without any infection (mock) were used as control. Upper panels, cell growth morphologies under light-phase microscopy; lower panels, crystal violet-stained colonies 14 days after plating. (C) Quantitation of colony formation shown in B. The data were the averages from 2 independent triplicate experiments. *P < .001 relative to the mock group. (D) Ex vivo assay for the tumor suppression effect of AdSip28GANK. SMMC-7721 cells were infected with adenoviruses or untreated cells (mock) in culture plates for 96 hours. Then viable cells (3 × 106) were injected subcutaneously into the dorsal flanks of 5 mice in each group. Photographs of a representative mouse in each group are shown. (E) Quantitation of tumor volume shown in D. Tumor formation was scored weekly. Data were expressed as means ± SD (n = 5). ◊, Mock; ■, AdSiGFP; ▴, AdSip28GANK. (F) AdSip28GANK decreased S-phase population in HCC cells after infection for 96 hours. The S-phase population in the indicated cells was determined by flow cytometry and experiments were repeated 3 times. □, Mock; ■, AdSiGFP; , AdSip28GANK. Error bars: SD. Gastroenterology 2005 128, 2029-2041DOI: (10.1053/j.gastro.2005.03.001) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Suppression of p28GANK expression enhances dephosphorylation of RB1 and decreases E2F-1 transcription activity in HuH-7 cells. (A) HuH-7 cell lysates were harvested at 48 or 96 hours after infection and probed with anti-RB1, anti-phosph-Ser780-RB1/phosph-Ser807-RB1, and anti-actin antibodies. ppRB1 and pRB1 indicate the hyper- and hypophosphorylation form of RB1, respectively. (B) HuH-7 cells were infected with adenovirus or were untreated for 48 hours and then transfected with the E2F-1 luciferase reporter gene along with Renilla luciferase plasmid (inner control). Cells were harvested 36 hours later and the values of luciferase activity driven by the E2F-1 promoter were normalized for Renilla luciferase readings in the same extract. The data were the averages from 2 independent triplicate experiments; error bars: SD. *P < .05 relative to the mock group. Gastroenterology 2005 128, 2029-2041DOI: (10.1053/j.gastro.2005.03.001) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 AdSiRNA targeting p28GANK induces cell apoptosis. (A) Down-regulation of p28GANK promoted apoptosis of the 4 indicated HCC cell lines. Ninety-six hours after the virus infection, the adherent cells were collected by trypsinization and determined with flow cytometry. Three individual experiments were performed, and the cell distribution in the cell cycle was determined by standard fluorescence-activated cell sorter analysis. The cell population in sub-G1 was shown. The X-axis and Y-axis represent DNA content and the cell number, respectively. (B) 4′,6-diamidino-2-phenylindole staining showed the apoptotic cells in HCC cells infected with AdSip28GANK for 96 hours, whereas this apoptosis cannot be seen in normal PHHs being infected. The arrows indicated the blue color in apoptotic nuclei with condensed chromatin or nuclear membrane contiguousness morphology. (C) TUNEL assay was used to detect the cell apoptosis. Four HCC cell lines were infected with AdSiGFP or AdSip28GANK or untreated (mock) for 96 hours and subjected to TUNEL assay. Brown nuclei staining indicated apoptosis in HCC cells induced by AdSip28GANK. (D) AdSip28GANK induced the cleavage of PARP in HCC cells. Western blot showed the cleaved 85-kilodalton fragment of PARP emerged in AdSip28GANK-infected HCC cells at 48 hours and was increased at 96 hours after infection. (E) AdSip28GANK infection enhanced the cleavage of caspase-8 and caspase-9 in HuH-7 cells. HuH-7 cell lysates harvested at 24, 30, 36, 42, and 48 hours after infection were immunoblotted with antibodies against caspase-8, caspase-9, and β-actin (loading control). Uncleaved procaspases and cleavage products were indicated at the left margin, the molecule weight at the right margin. Gastroenterology 2005 128, 2029-2041DOI: (10.1053/j.gastro.2005.03.001) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Local injection of adenovirus-delivered p28GANK siRNA suppresses the established tumor growth in nude mice. (A) Two weeks after subcutaneous inoculation of SMMC-7721 cells, 3 groups of nude mice were treated every other day for 10 days by intratumor injection of AdSip28GANK or AdSiGFP at 2 × 108 plaque-forming units per animal or PBS as a control (arrow). Representative photographs of a mouse in each group 6 weeks later are shown. (B) Quantitation of tumor volume shown in A. The tumor size was measured and the tumor volume was calculated weekly. ◊, PBS; ■, AdSiGFP; ▴, AdSip28GANK. Data are presented as means ± SD (n = 6). Error bars: SD. (C) Tissue slices of established tumors from the killed mice were subjected to fluorescein-labeled TUNEL assay. Apoptotic nuclei are seen as green color excited in fluorescence microscopy. 4′,6-diamidino-2-phenylindole staining showed the morphologic changes of cell nuclei. Original magnification, 400×. Gastroenterology 2005 128, 2029-2041DOI: (10.1053/j.gastro.2005.03.001) Copyright © 2005 American Gastroenterological Association Terms and Conditions