| Background and Objective：Hepatocellular carcinoma (HCC) accounts for>90%of all primary liver cancers andhas a dismal prognosis with a median life expectancy of6to9months. It ranks fifth infrequency among all malignancies worldwide and causes nearly1million deaths annually.Despite great progress in diagnosis and management of HCC, the exact biology of thetumor remains poorly understood overall limiting the patients’ outcome. Surgery is themainly choice for HCC treatment. However, because of the high recurrence rate, long-termprognosis after resection of liver cancer is still not satisfactory. To solve this problem, manybiologic therapies have been investigated.The ubiquitin (Ub)-proteasome pathway is the main system for the targeteddegradation of intracellular proteins. Members of ubiquitin-related family have also beenimplicated to be involved in the regulation of cell cycle as well as apoptosis. One memberof this family is FAT10, which is also known as diubiquitin. Role of FAT10in cell-cycleregulation has been suggested by its ability to bind to MAD2, a spindle checkpoint protein.In addition, it is recently reported that the FAT10gene is upregulated in various cancers,implicating its role in tumorigenesis. FAT10was reported to be overexpressed in90%ofhepatocellular carcinoma carcinomas, was attributed to transcriptional upregulation uponthe loss of p53and induced chromosome instability in long-term in vitro culture. However,the exact function of FAT10in Hepatocellular carcinoma is not clear and warrant furtherresearch.Therefore, in this study, we employed the adenovirus delivered small interfering RNA(siRNA) technique to study the effects of knock down of FAT10on HCC cell growth invitro and in vivo.The study was divided into four parts:1. Generation and appraisal of recombinant adenovirus encoding FAT10small interfering RNA.2. Gene and protein expression in HCC cells after FAT10gene silence mediated byadenovirus.3. Inhibitory effect of Ad-siRNA/FAT10on HCC cell growth in vitro.4.Tumor challenge assay in vivo.Main methods1.Mice and cellsNude mice were used at6weeks of age. Animals were bred in the Laboratory AnimalCenter and all studies were performed in agreement with the local ethics committee.Hepatocellular carcinoma cells (Hep3B) were maintained as monolayers in DMEMcontaining10%heat-inactivated FBS,200units/mL penicillin, and100μg/mL streptomycinand then were kept at37C in a humidified atmosphere of5%CO2in air.2.Generation and appraisal of recombinant adenovirus encoding FAT10smallinterfering RNAThe target sequence was subcloned into shuttle vector pDC315and sequenced. Thedesired replication-deficient adenovirus containing the full-length cDNA of RNAi wasgenerated by homologous recombination through co-transfection of plasmidspDC315-RNAi and pBHG1oXE1,3Cre in HEK293cells using the DOTAP liposomereagent. After several rounds of plaque purification, the adenovirus was amplified andpurified from cell lysates by banding twice in CsCl density gradients. Viral products weredesalted and stored at-80C in PBS containing10%glycerol (v v). The infectious titer wasdetermined by a standard plaque assay. and then, the mRNA and protein levels weredetected by RT-PCR and western blot assays.3. Inhibitory effect of Ad-siRNA/FAT10to HCC cell growth in vitro.Hepatocellular carcinoma cells Hep3B were purchased from American Type CultureCollection. The cell line was maintained as monolayers in DMEM containing10%heat-inactivated FBS,200units/mL penicillin, and100μg/mL streptomycin and was kept at37C in a humidified atmosphere of5%CO2in air.(1)Cell proliferation was measured by a colorimetric assay using MTT. Hep3B cellswere seeded in96-well plates cells well and incubated in culture medium overnight. Thecells were treated with Ad-siRNA/FAT10or controls for24h. Then MTT solution (5 mg mL) was added and cultured for48h. The colored formazan crystal produced fromMTT was dissolved with0.15mL DMSO then the optical density (OD) value A490wasmeasured.(2)For colony forming assay,3×102cells were seeded into10cm culture dishes, thenthe cells were treated with Ad-siRNA/FAT10(1×109pfu) or controls for18days of culture,cell colonies were fixed with glutaraldehyde, and stained with0.1%crystal violet andvisible colonies were manually counted.(3)Standard fluorescence-activated cell sorter analysis was used to determine thedistribution of cells in cell cycle and apoptosis rate of the cells.3×102Hep3B cells wereseeded into6-well plates, then the cells were treated with Ad-siRNA/FAT10(1×109pfu) orcontrols for2days. For cell cycle analysis, adherent cells then were collected bytrypsinization and fixed with70%ethanol overnight at4°C. After washing with PBS, thecells were treated with100μg/mL RNase,50μg/mL of propidium iodide and0.05%TritonX-100and incubated at roomtemperature for45minutes. The samples were analyzed usingFCM. For cell apoptosis analysis, the cells were seeded in100ml bottles and incubateduntil there was80–85%confluence.Then the cells were resuspended in binding buffer afterharvested, washed twice with cold PBS. AnnexinV (0.5mg/ml) and PI (0.5mg/ml) werethen added to a250ml aliquot of this cell suspension. After a15min incubation in the darkat room temperature, stained cells were immediately analyzed by FCM.4.Tumor challenge assay in vivo.Nude mice were used at6weeks of age. Animals were bred in the Laboratory AnimalCenter and all studies were performed in agreement with the local ethics committee. Nudemice were challenged with subcutaneous injection of1×105Hep3B cells into the left flankto induce primary tumors. Two weeks after tumor cell inoculation, mice were dividedrandomly into three groups (ten mice per group) and were received an intratumor injectionof of Ad-siRNA/FAT10(1×109pfu) or Ad-siRNA/LacZ (1×109pfu). The control micereceived100μL PBS. Tumor volume and mean lifespan of the mice were observed. Tumorvolume was measured in two dimensions and calculated as follows: length/2×width2.Main results1.Construction of Ad-siRNA/FAT10and its effects on FAT10expressionTo examine whether adenovirus mediated siRNA can be used to specifically inhibit target gene expression, the adenovirus encoding siRNA targeted against FAT10was usedto infect HCC cell lines. The expression of FAT10was inhibited48h after infection.However, the expression of FAT10was not decreased in the control groups. In addition,Ad-siRNA/FAT10did not induce a non-specific downregulation of β-actin gene expression.To examine the reduction level of target mRNA induced by Ad-siRNA/FAT10, the real-timereverse transcription-PCR was also performed. Ad-siRNA/FAT10caused obvious reductionof FAT10mRNA level, whereas control groups had not the effect. Altogether, these dataindicated that the Ad-siRNA/FAT10could specific suppress the FAT10expression in HCCcells.2.Inhibitory effect of Ad-siRNA/FAT10to HCC cell growth in vitro.(1)Ad-siRNA/FAT10inhibites HCC cell growth and colony formation in vitro.We first tested the effect of Ad-siRNA/FAT10on the proliferation of hepatoma cells incell culture. MTT colorimetric method was adopted to measure proliferation ratio, and theinhibition rate was calculated with the method mentioned above. In addition, we stillobserved the effect of Ad-siRNA/FAT10on cell colony formation. The results showed thatAd-siRNA/FAT10could not only significantly inhibit the proliferation of Hep3B cells butalso reduce the cell colony formation compared with control groups.(2)Ad-siRNA/FAT10inhibits S-phase entry of HCC cells in vitroIn addition, to determine whether Ad-siRNA/FAT10could change the cell cycledistribution in Hep3B cells, we performed flow cytometry at96h after virus infection. Ourresults showed a decreased cell population in the S phase after Ad-siRNA/FAT10infectioncompared with control groups. The data indicated that the Ad-siRNA/FAT10exhibited aspecific inhibitory effect on the HCC cell growth through inhibition of S-phase entry inHCC cells.(3)Ad-siRNA/FAT10induces apoptosis of HCC cells.To detect whether Ad-siRNA/FAT10could induce apoptosis of HCC cells, two daysafter infection of Ad-siRNA/FAT10, Ad-siRNA/LacZ or PBS, cell apoptosis ratio wasdetected by flow cytometry. Apoptosis cells were determined by AnnexinV and propidiumiodide (PI). The results demonstrated that the apoptosis rate of Hep3B cells obviouslyincreased compared with control groups. The results suggested that Ad-siRNA/FAT10hadpotential to induce the apoptosis of HCC cells. 3.Ad-siRNA/FAT10inhibits tumor growth and improves the lifespan of nude miceTo determine whether the Ad-siRNA/FAT10could serve as a therapeutic agent againstHCC growth, we established a tumor model in nude mice bearing Hep3B xenografts. Twoweeks after inoculation of Hep3B, the nude mice were received intratumor injection ofAd-siRNA/FAT10, Ad-siRNA/LacZ or PBS. In addition, tumor volume and lifespan ofnude mice were observed. The result demonstrated that the tumor growth was obviouslyinhibited after treatment with Ad-siRNA/FAT10compared with the Ad-siRNA/LacZ or PBSgroup. Furthermore, the death only occurred later in40days after tumor challenge in theAd-siRNA/FAT10group. However, all control mice died within40days. In addition, themean lifespan of nude mice in the Ad-siRNA/FAT10group was prolonged remarkably, withapproximately half of mice surviving beyond60days. Taken together, these data indicatedthat targeting FAT10by Ad-siRNA could exert a robust antitumor effect in vivo whichcould inhibit tumor growth and prolong the lifetime of tumor bearing nude mice.ConclusionsIn this study, we employed the adenovirus delivered small interfering RNA (siRNA)technique to study the effects of knock down of FAT10on HCC cell growth in vitro and invivo.Our results demonstrated that Ad-siRNA/FAT1could supress the FAT10expression ofHCC cell, which(1) inhibited HCC cell growth and colony formation in vitro;(2) inhibited S-phase entry and induced the apoptosis of HCC cells in vitro;(3) inhibited tumor growth and improved the lifespan of nude mice.Therefore, our findings suggested that knock down of FAT10by adenovirus-deliveredsiRNA might be a promising therapeutic strategy in the treatment of HCC in clinic. |
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