Experimental and Molecular Pathology 96 (2014) 207–211

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Experimental and Molecular Pathology

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Recombinant adenovirus encoding FAT10 small interfering RNA inhibitsHCC growth in vitro and in vivo

Jingxiang Chen a,1, Li Yang b,1, Hongxu Chen a, Tao Yuan a, Menggang Liu a, Ping Chen a,⁎a Department of Hepatobiliary Surgery, Daping Hospital, Third Military Medical University, Chongqing 400042, Chinab Department of Cardiology, Daping Hospital, Third Military Medical University, Chongqing 400042, China

⁎ Corresponding author. Tel./fax:+86 23 68757966.E-mail address: pingchen1956@163.com (P. Chen).

1 These authors contributed equally to this study.

0014-4800/$ – see front matter © 2014 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.yexmp.2014.01.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 December 2013Available online 14 January 2014

Keywords:HCCFAT10RNA interferenceCarcinogenesisStrategy

Hepatocellular carcinoma is an aggressive and rapidly fatal malignancy representing the common cancerworldwide. The specific cellular gene involved in carcinogenesis has not been fully characterized. Theubiquitin-like modifier FAT10, recently reported to be overexpressed in 90% of hepatocellular carcinomacarcinomas, was attributed to transcriptional upregulation upon the loss of p53 and induced chromosomeinstability in long-term in vitro culture. However, the exact function of FAT10 in hepatocellular carcinoma isnot clear. In the present study, we utilized adenovirus-mediated RNA interference to knock down FAT10expression in hepatocellular carcinoma cells and observed its effects on hepatocellular carcinoma cell growthin vitro and in vivo. The results demonstrated that interference of FAT10 could inhibit cell proliferation byinhibiting the cell cycle S-phase entry and inducing cell apoptosis. In addition, in vivo experiments showedthat adenovirus Ad-siRNA/FAT10 significantly suppressed tumor growth and prolonged the lifespan of tumor-bearingmice. These results suggest that knockdownof FAT10 by adenovirus-delivered siRNAmay be a promisingtherapeutical strategy for treatment of hepatocellular carcinoma.

© 2014 Elsevier Inc. All rights reserved.

Introduction

Hepatocellular carcinoma accounts for N90% of all primary livercancers and has a dismal prognosis with a median life expectancy of 6to 9 months. It ranks fifth in frequency among all malignancies world-wide and causes nearly 1 million deaths annually (El-Serag, 2002;Mulcahy, 2005; Yu and Keeffe, 2003). Surgery is the mainly choice forHCC treatment. However, the long-term prognosis after resection ofHCC remains unsatisfactory as a result of a high incidence of recurrence(Mann et al., 2009; Nakamura et al., 2005; Ziparo et al., 2002). To solvethis problem, many biologic therapies have been investigated.

The ubiquitin (Ub)–proteasome pathway is the main system for thetargeted degradation of intracellular proteins (Dai and Li, 2001; Loveet al., 2007; Murray and Norbury, 2000). Members of ubiquitin-relatedfamily have also been implicated to be involved in the regulation ofcell cycle as well as apoptosis (Bayer et al., 1998; Reverter and Lima,2005; Su and Li, 2002). One member of this family is FAT10, which isalso known as diubiquitin. Role of FAT10 in cell-cycle regulation hasbeen suggested by its ability to bind to MAD2, a spindle checkpointprotein (Ren et al., 2006; Zhang et al., 2006). In addition, it is recentlyreported that the FAT10 gene is upregulated in various cancers, impli-cating its role in tumorigenesis (Ji et al., 2009; Lim et al., 2006; Oliva

ghts reserved.

et al., 2008; Zhang et al., 2006). Whether function of FAT10 contributesto cancer development remains unclear and warrant further research.

Therefore, in this study, we employed the adenovirus deliveredsmall interfering RNA (siRNA) technique to study the effects of knock-down of FAT10 on HCC cell growth in vitro and in vivo.

Materials and methods

Mice, cells, and other reagents

Nude mice were purchased from the Laboratory Animal Institute ofBeijing Medical University and were used at 6 weeks of age. Animalswere bred in the Laboratory Animal Center and all studies wereperformed in agreement with the local ethics committee. Hepatocellularcarcinoma cells Hep3B were purchased from the American Type CultureCollection. The cell line was maintained as monolayers in Dulbecco'smodified Eagle's medium containing 10% heat-inactivated fetal bovineserum, penicillin (200 units/mL), and streptomycin (100 μg/mL) andwas kept at 37 °C in a humidified atmosphere of 5% CO2 in air.

Recombinant adenovirus generation

The complementary DNA sequence of FAT10 was obtained fromGenBank. The potential target sequence for RNA interference (RNAi)were scanned with the siRNA Target Finder and Design Tool availableat the Ambion Web site. The target sequence selected 5′-GCUCAGUG

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GC ACAAGUGAATT-3′ (sense) and 5′-UUCACUUGUGCCACUGAGCTG-3′(antisense). The target sequence was subcloned into shuttle vectorpDC315 and sequenced. The desired replication-deficient adenoviruscontaining the full-length cDNA of RNAi was generated by homologousrecombination through co-transfection of plasmids pDC315-RNAi andpBHG1oXE1, 3Cre in HEK 293 cells using the DOTAP liposome reagent(Roche, Mannheim, Germany). After several rounds of plaque purifica-tion, the adenovirus was amplified and purified from cell lysates bybanding twice in CsCl density gradients. Viral products were desaltedand stored at −80 °C in PBS containing 10% glycerol (v⁄v). The infec-tious titer was determined by a standard plaque assay.

Adenovirus infection

Transduction of cells with adenoviruswas done in 6-well plateswith1 × 106 cells⁄well in 3 mL RPMI-1640 medium containing 10% FBS.Adenovirus was added to the wells at an MOI of 100 and the cellswere harvested after 48 h of incubation.

Reverse transcription PCR analysis

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad,CA) according to the protocol of the manufacturer. Two micrograms ofRNAwas subjected to reverse transcription. The polymerase chain reac-tion (PCR) primers used were as follows: for FAT10, 5′-CAATGC TTCCTGCCTCTGTG-3′ (forward), 5′-TGCCTCTTTGCCTCATCACC-3′ (reverse) andfor β-actin, 5′-ATG ATATCG CCG CGC TCG TC-3′ (forward), 5′-CGC TCGGTG AGG ATC TTC A-3′ (reverse). PCR products were separated on a1% agarose gel, visualized and photographed under ultraviolet light.

Western blot analysis

For Western blot assay, proteins of the cell extracts were separatedby SDS-PAGE and transferred onto a nitrocellulose membrane. Themembrane was incubated with 5% non-fat milk in PBS and then withanti-FAT10 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA)for 2 h at room temperature. After washing, themembranes were incu-bated with an alkaline phosphatase-conjugated goat antimouse IgGantibody (Amersham Biosciences, Little Chalfont, UK) for 1 h at roomtemperature. Immunoreactive bands were detected using the ECLWestern blot analysis system (Amersham Biosciences). Densitometryanalysis of protein levels was performed by using Scion-Image 4.0.2software (Scion Corporation, Frederick, MD).

Inhibitory effect of cell proliferation

Cell proliferation was measured by a colorimetric assay using MTT.In brief, the Hep3B cells were seeded in 96-well plates in triplicate at5 × 103 cells⁄well and incubated in culture medium overnight. Thenthe cells were treated with Ad-siRNA/FAT10 (1 × 109 pfu) or controlsin a total volume of 0.2 mL each well for 24 h. Thereafter, 20 μL of theindicator dye MTT solution (5 mg⁄mL) was added to each well andcultures were continued for 48 h at 37 °C, 5% CO2. After centrifugation,the supernatant was removed from each well. The colored formazancrystal produced from MTT was dissolved with 0.15 mL DMSO,then the optical density (OD) value A490 was measured by themultiscanner autoreader (Dynatech MR 5000; Dynatech Laborato-ries, Chantilly, VA, USA). The following formula was used:cell proliferation inhibited (%) = [1 − (OD of the experimentalsamples ⁄ OD of the control) × 100%].

Colony formation assay

For colony forming assay, 3 × 102 cells were seeded into 10 cmculture dishes, then the cells were treated with Ad-siRNA/FAT10(1 × 109 pfu) or controls for 18 days of culture, cell colonies were

fixedwithmethanol, and stainedwith 0.1% crystal violet and visible col-onies were manually counted (the colonies containing at least 50 cellswere considered viable).

Cell cycle analysis

Standard fluorescence-activated cell sorter analysis was used todetermine the distribution of cells in cell cycle of the cells. Briefly, thecells were infected with Ad-siRNA/FAT10 (1 × 109 pfu) or controls for2 days. Adherent cells then were collected by trypsinization and fixedwith 70% ethanol overnight at 4 °C. After washing with phosphate-buffered saline (PBS), the cells were treated with 100 μg/mL RNase A(Roche Diagnostics), 50 μg/mL of propidium iodide (Sigma) and 0.05%(vol/vol) Triton X-100 and incubated for 45 min at room temperature.The sampleswere analyzed using a FACSCaliburflow cytometer (BectonDickinson, San Jose, CA). The cell cycle distribution was established byplotting the intensity of the propidium iodide signal, which reflectsthe cellular DNA content. Findings from at least 1 × 104 cells werecollected and analyzed with CellQuest software (Becton Dickinson).

Flow cytometric analysis of apoptosis

An Annexin V-fluorescein isothiocyanate kit (Oncogene) was usedto detect apoptosis. Briefly, the cells were infected with Ad-siRNA/FAT10 (1 × 109 pfu) or controls for 2 days. The cells were seeded in100 mL bottles and incubated until there was 80–85% confluence inRPMI-1640 containing 10% bovine serum. Then the cells were harvest-ed, washed with ice-cold PBS twice, and resuspended in binding buffer(10 mM of HEPES, pH 7.4, 150 mM of NaCl, 2.5 mM of CaCl2, 1 mM ofMgCl2, 4% bovine serum albumin). Annexin V-fluorescein isothiocya-nate (0.5 mg/mL) and propidium iodide (0.5 mg/mL) were thenadded to a 250 mL aliquot (5 × 106 cells) of this cell suspension. Aftera 15 min incubation in the dark at room temperature, stained cellswere immediately analyzed by Flow Cytometry (Coulter Biosciences).Apoptotic cell were determined by Annexin V positive and propidiumiodide (PI) negative cells. All of the samples were assayed in triplicate,and the cell apoptosis rate calculated as: apoptosis rate = (apoptoticcell number / total cell number) × 100%.

Tumor challenge assay

6-Week-old nude mice were challenged with subcutaneous (s.c.)injection of 1 × 105 Hep3B cells into the left flank to induce primarytumors. Two weeks after tumor cell inoculation, mice were dividedrandomly into three groups (ten mice per group) and received anintratumor injection of Ad-siRNA/FAT10 (1 × 109 pfu) or Ad-siRNA/LacZ (1 × 109 pfu). The control mice received 100 μL PBS. Tumor volumeand mean lifespan of the mice were observed. Tumor volume was mea-sured in two dimensions and calculated as follows: length / 2 × width2.

Statistical analysis

All the experiments were done in triplicate, and the results are givenasMeans ± S.E.M. of triplicate determinations. Statistical analyseswereperformed using the Student's t test. The difference was consideredstatistically significant when the P value was b0.05.

Results

Construction of Ad-siRNA/FAT10 and its effects on FAT10 expression

To examine whether adenovirus mediated siRNA can be used tospecifically inhibit target gene expression, the adenovirus encodingsiRNA targeted against FAT10 was used to infect HCC cell lines. Asshown in Fig. 1A, the expression of FAT10was inhibited 48 h after infec-tion. However, the expression of FAT10was not decreased in the control

Fig. 2. Inhibitory effect of Ad-siRNA/FAT10 on cell proliferation and colony formation.(A). Hep3B cells were seeded in 96-well plates at 5 × 103 cells⁄well and incubated inculture medium overnight. The cells were treated with Ad-siRNA/FAT10 (1 × 109 pfu)or controls for 24 h. Then MTT solution (5 mg⁄mL) was added and cultured for 48 h.The colored formazan crystal produced from MTT was dissolved with 0.15 mL DMSOthen the optical density (OD) value A490 was measured. (B). 3 × 102 Hep3B cells wereseeded into 10 cm culture dishes, then the cells were treated with Ad-siRNA/FAT10(1 × 109 pfu) or controls for 18 days of culture, cell colonies were fixed with methanol,and stained with 0.1% crystal violet and visible colonies were manually counted. Thedata were the averages from three independent triplicate experiments.

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groups. In addition, Ad-siRNA/FAT10 did not induce a non-specificdownregulation of β-actin gene expression. To examine the reductionlevel of target mRNA induced by Ad-siRNA/FAT10, the real-time reversetranscription-PCR was also performed. As shown in Fig. 1B, Ad-siRNA/FAT10 caused obvious reduction of FAT10 mRNA level, whereas controlgroups had not the effect. Altogether, these data indicated that the Ad-siRNA/FAT10 could specifically suppress the FAT10 expression in HCCcells.

Ad-siRNA/FAT10 inhibits HCC cell growth and colony formation in vitro

We first tested the effect of Ad-siRNA/FAT10 on the cell proliferationof Hep3B in cell culture. Cell proliferationwasmeasured by a colorimet-ric assay using MTT, and the inhibition rate was calculated with themethod mentioned above. In addition, we still observed the effect ofAd-siRNA/FAT10 on cell colony formation. The results showed thatAd-siRNA/FAT10 could not only significantly inhibit the proliferationof Hep3B cells but also reduce the cell colony formation comparedwith control groups (Figs. 2A and B).

Ad-siRNA/FAT10 inhibits S-phase entry of HCC cells in vitro

In addition, to determine whether Ad-siRNA/FAT10 could changethe cell cycle distribution in Hep3B cells, we performed flow cytometryat 96 h after virus infection. Our results showed a decreased cellpopulation in the S phase after Ad-siRNA/FAT10 infection comparedwith control groups (Fig. 3). The data indicated that the Ad-siRNA/FAT10 exhibited a specific inhibitory effect on the HCC cell growththrough inhibition of S-phase entry in HCC cells.

Ad-siRNA/FAT10 induces of apoptosis of HCC cells

To detect whether Ad-siRNA/FAT10 could induce apoptosis of HCCcells, 2 days after infection of Ad-siRNA/FAT10, Ad-siRNA/LacZ or PBS,cell apoptosis ratio was detected by flow cytometry. Apoptosis cellswere determined by Annexin V positive and propidium iodide (PI)negative cells. The results demonstrated that the apoptosis rate ofHep3B cells obviously increased compared with control groups(Fig. 4). The results suggested that Ad-siRNA/FAT10 had potential toinduce the apoptosis of HCC cells.

Fig. 1. The effects of Ad-siRNA/FAT10 on FAT10 expression. Ad-siRNA/FAT10 wasconstructed and transduced with Hep3B cells at an MOI of 100, and then cells wereharvested after 48 h of incubation. (A). Cell lysates were harvested at 48 h after infectionand detected by anti-FAT10 and anti-β-actin antibodies with Western blot assay.(B). FAT10 mRNA from Hep3B cells was harvested at 48 h after infection, and was alsoanalyzed with RT-PCR assay. The results showed that Ad-siRNA/FAT10 could specificallysuppress the expression of FAT10.

Ad-siRNA/FAT10 inhibits tumor growth and improves the lifespan ofnude mice

To determine whether the Ad-siRNA/FAT10 could serve as a thera-peutic agent against HCC growth, we established a tumor model innude mice bearing Hep3B xenografts. Two weeks after inoculation ofHep3B, the nude mice received an intratumor injection of Ad-siRNA/

Fig. 3. Ad-siRNA/FAT10 inhibits S-phase entry of HCC cells and induces cell apoptosisin vitro. Hep3B cells were infected with Ad-siRNA/FAT10 (1 × 109 pfu) or controls for2 days. Adherent cells then were collected by trypsinization and fixed with 70% ethanolovernight at 4 °C. After washing with phosphate-buffered saline (PBS), the cells weretreated with 100 μg/mL RNase A (Roche Diagnostics), 50 μg/mL of propidium iodide(Sigma) and 0.05% (vol/vol) Triton X-100 and incubated for 45 min at room temperature.The samples were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, SanJose, CA). The cell cycle distribution was established by plotting the intensity of thepropidium iodide signal, which reflects the cellular DNA content. The data were the aver-ages from three independent triplicate experiments.

Fig. 4.Ad-siRNA/FAT10 induces HCC cell apoptosis in vitro. Two days after infection of Ad-siRNA/FAT10, Ad-siRNA/LacZ or PBS, Annexin V-fluorescein isothiocyanate (0.5 mg/mL)and propidium iodide (0.5 mg/mL) were then added to a 250 mL aliquot (5 × 106 cells)of this cell suspension. After a 15 min incubation in the dark at room temperature, stainedcells were immediately analyzed by Flow Cytometry (Coulter Biosciences). Apoptosis cellswere determined by Annexin V positive and propidium iodide (PI) negative cells. The datawere the averages from three independent triplicate experiments.

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FAT10, Ad-siRNA/LacZ or PBS. In addition, tumor volume and lifespan ofnude mice were observed. The result demonstrated that the tumorgrowth was obviously inhibited after treatment with Ad-siRNA/FAT10comparedwith the Ad-siRNA/LacZ or PBS group (Fig. 5A). Furthermore,thedeath only occurred later in 40 days after tumor challenge in theAd-siRNA/FAT10 group. However, all control mice died within 40 days. Inaddition, the mean lifespan of nude mice in the Ad-siRNA/FAT10group was prolonged remarkably, with approximately half of micesurviving beyond60 days (Fig. 5B). Taken together, these data indicatedthat targeting FAT10 by Ad-siRNA could exert a robust antitumor effect

Fig. 5. Ad-siRNA/FAT10 inhibits tumor growth and improves the lifespan of nudemice. 6-Week-oldnudemicewere challengedwith subcutaneous (s.c.) injection of 1 × 105Hep3Bcells into the left flank to induce primary tumors. Two weeks after tumor cell inoculation,mice were divided randomly into three groups (ten mice per group) and received anintratumor injection of Ad-siRNA/FAT10 (1 × 109 pfu) or Ad-siRNA/LacZ (1 × 109 pfu).The control mice received 100 μL PBS. Tumor volume and mean lifespan of the micewere observed. Tumor volume was measured in two dimensions and calculated as fol-lows: length / 2 × width2.

in vivo which could inhibit tumor growth and prolong the lifetime oftumor bearing nude mice.

Discussion

In this study,we employed the adenovirus delivered small interferingRNA (siRNA) technique to study the effects of knockdown of FAT10 onHCC cell growth in vitro and in vivo. Our results demonstrated that Ad-siRNA/FAT1 could suppress the FAT10 expression of HCC cell, which(1) inhibitedHCC cell growth and colony formation in vitro; (2) inhibitedS-phase entry and induced the apoptosis of HCC cells in vitro; and (3)inhibited tumor growth and improved the lifespan of nude mice.

Surgery is themainly choice of treatment for HCC. Yet, nomore than20% of patients with hepatocellular carcinoma have opportunity to un-dergo surgery procedures. General treatment or combined treatmentshave drawn great attention due to the poor prognosis of hepatocellularcarcinoma (Fatourou and Koskinas, 2009; Niguma et al., 2005; Zhu,2006). Palliative treatments including chemoembolization, hepatic ar-tery infusion, percutaneous interstitial ablation, cryosurgery, radiationtherapy, and systemic chemotherapy have been beneficial approachesfor liver cancer treatment (Cabibbo et al., 2009; Carr, 2004; Hoffmannand Schmidt, 2009). Recently, with the expectation of increasing thera-peutic efficacy, gene therapy is being investigated as an promisingtherapeutical strategy (Huang et al., 2008; Mucha et al., 2009; Palmeret al., 2005; Shen et al., 2008; Zhao et al., 2008).

FAT10, also known as diubiquitin, is a ubiquitin-like modifier (UBL)of the ubiquitin protein family, first discovered by in mapping HLA-Fgene in 1996 (Liu et al., 1999). The evidence suggested that FAT10 isexpressed in mature B cells and dendritic cells (Bates et al., 1997). Inaddition, it has been reported that FAT10 regulates cell-cycle and non-covalently binds to the human spindle assembly checkpoint protein(MAD2) that is responsible for the maintenance of spindle integrityduringmitosis. Inhibition ofMAD2may lead to chromosomal instability,a common feature of tumorigenesis (Liu et al., 1999; Raasi et al., 2001).Moreover, it was reported that that FAT10 is up-regulated in liver,uterine cervix, ovarian, rectal, and pancreatic cancers and small intesti-nal adenocarcinoma, suggesting that FAT10 plays an important role intumorigenesis (Lee et al., 2003). However, up to date, its biologicalfunction is far from being fully elucidated. Therefore, in this study, weemployed the adenovirus delivered small interfering RNA (siRNA)technique to study the effects of knockdown of FAT10 on HCC cellgrowth in vitro and in vivo.

Firstly, the Hep3B cells were transduced with Ad-siRNA/FAT10, andthen, themRNAandprotein levelswere detected by RT-PCR andWesternblot assays. The results demonstrated that Ad-siRNA/FAT10 caused obvi-ous reductionof FAT10mRNA level and gene expression,whereas controlgroups had not the effect. These data indicated that the Ad-siRNA/FAT10could specifically suppress the FAT10 expression in HCC cells.

Secondly, the effect of Ad-siRNA/FAT10 on the HCC cell proliferationand cell colony formation was detected. The results showed that Ad-siRNA/FAT10 could not only significantly inhibit the proliferation ofHep3B cells but also reduce the cell colony formation compared withcontrol groups. To further identify the biological function of FAT10 onHep3B cells, after Ad-siRNA/FAT10 transduction, the cell cycle andapoptosis were analyzed. The results indicated that the Ad-siRNA/FAT10 exhibited a specific inhibitory effect on the HCC cell growththrough inhibition of S-phase entry and induction of apoptosis in HCCcells.

Lastly, to explore the therapeutical potential of Ad-siRNA/FAT10in vivo, nude mice were challenged with subcutaneous (s.c.) injectionof 1 × 105 Hep3B cells into the left flank to induce primary tumors andreceived an intratumor injection of Ad-siRNA/FAT10 (1 × 109 pfu) orcontrols. Tumor volume andmean lifespan of nudemice were observed.The result demonstrated that the tumor growthwas obviously inhibitedand the mean lifespan of nude mice was prolonged remarkably after

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treatment with Ad-siRNA/FAT10 compared with the Ad-siRNA/LacZ orPBS group.

In summary, our results demonstrated that the knockdown of FAT10expression in HCC cells could inhibit cell growth in vitro and in vivothrough inhibiting the cancer cell S-phase entry and inducing cell apo-ptosis. Moreover, Ad-siRNA/FAT10 could obviously inhibit the tumorgrowth and prolong the mean lifespan of nude mice. Therefore, ourfindings suggested that knockdown of FAT10 by adenovirus-deliveredsiRNA might be a promising therapeutic strategy in the treatment ofHCC in clinic.

Conflict of interest

The authors have no conflicts of interest to declare.

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