Conditionally Replicative Adenovirus Armed With Short Interference RNA Against Ki67 Gene For Renal Cancer Therapy

Renal cell carcinoma (RCC) is the most common malignant disease of theadult kidney. Approximately 30%-40% of all patients afflicted with metastasisdisease at the time of diagnosis and the five-year survival rates are less than 10%.The tumor is considered to be relatively resistant to radiotherapy and chemotherapyand immunotherapy. It is therefore necessary to explore a new therapeutic agent toimprove the patients' responses.RNA interference (RNAi) provides a new approach for suppression of geneexpression. RNAi is a sequence-specific, post-transcriptional gene silencingmechanism by the introduction of small interfering RNA (siRNA) homologous insequence to the silenced gene. SiRNA can mediate strong and specific suppressionof gene expression. Many examples demonstrate the usefulness of siRNA as aneffective method for inhibiting the expression of oncogenes. RNAi with siRNAopens new avenues for cancer gene therapy. But gene therapy in clinical trial has notbroken through in essential to this day yet. To further improve the anticancer efficacyof gene therapy, gene-virus therapy of cancer put forward.Gene-virus therapy strategy is to clone antitumoral gene into conditionallyreplicative adenovirus (CRAds) which replicate selectively in cancer cells and result in cancer-specific cytotoxicity, but not in normal cells. Accompanied with thereplication of virus, the expression of antitumoral gene is greatly increased. At thesame time, the CRAds itself has potent antitumoral efficacy. One of strategy toconstruct CRAds is to delete adenoviral E1B 55Kda gene gene which is necessaryfor efficient viral replication in normal cells. In this paper we construct CRAdsexpressing siRNA targeting Ki67 gene (ZD-Ki67) and evaluate its antitumoralactivity on human renal carcinoma cells.Partâ… Selection siRNAs sequence and construction its expression plasmidtargeted Ki67 gene and evaluate their effects on the cell proliferationand apoptosis of human renal carcinoma cellsFirst, the siRNA which targeted the region containing the 364～382 bases ofKi67 complementary DNA was synthesized and transfected into human renalcarcinoma 786-0 cells. The Ki67 expressions of different levels were detected byRT-PCR and Western blot and immunohistochemical technique respectively. Theproliferation and apoptosis of 786-0 cells were detected by MTT assay and TUNELassay respectively. RT-PCR and Western blot analysis showed that the Ki67expression levels of 786-0 cells treated with Ki67-siRNA were reduced significantly.The Ki67 positive expression rate of 786-0 cells treated with Ki67-siRNA wasreduced markedly. The proliferation of 786-0 cells treated with Ki67-siRNA wasinhibited and the apoptosis of 786-0 cells treated with Ki67-siRNA increased. Theseresults indicate that siRNA against Ki67 gene can inhibit the proliferation andinduce apoptosis by blocking Ki67 expression of human renal carcinoma 786-0cells.Then we constructed Ki67 siRNA expression plasmid. According to Ki67 siRNA sequence above, two complementary template oligonucleotides encodinghairpin RNAs targeting Ki67 gene were designed and synthesized and inserted intoplasmid pSilencer 3.1 to construct the recombinant plasmid (pSilencer-Ki67). ThepSilencer-Ki67 was identified by restrictive enzyme digestion and sequence analysis.The pSilencer-Ki67 were transfected into human renal carcinoma 786-0 cells and itstumor xenografts. It is found that using pSilencer-Ki67, we were able to knock downKi67 gene expression, resulting in inhibition of proliferation and induction ofapoptosis of 786-0 cells and its tumor xenografts. Successful cloning of Ki67 siRNAexpression plasmid helps to construct CRAds armed with short interference RNAagainst Ki67 gene.Partâ…¡Construction of conditionally replieative adenovirus (CRAds)expressing siRNA targeting Ki67 geneAlthough gene silencing by transfection of synthetic siRNA and pSilencer-Ki67plasmid is effective in human renal carcinoma cells, both regimens reduce Ki67expression for only a short period. To circumvent the problem, We constructed theconditionally replicative adenovirus(CRAds) expressing Ki67-siRNA. Firstly, Theannealed Ki67-siRNA template DNA sequences were inserted into pCA13 plasmidto construct the recombinant plasmid (pCA13-Ki67). When pCA13-Ki67 wasidentified, it was cut with Bglâ…¡to get the expression cassette of Ki67-siRNA. Theexpression cassette was cloned to pZD55 which had been cut with Bglâ…¡anddephosphated to form pZD-Ki67 plasmid. The plasmid pCA13-Ki67 and pBHGE3were transfected in 293 cells together to construct the recombinedreplication-deficient adenovirus. The plasmid pZD-Ki67 and pBHGE3 weretransfected in 293 cells together to construct the CRAds. The viral plaques of the recombined replication-deficient adenovirus and CRAds appeared 9-12 days afterinfection. The DNA extracted from the recombined replication-deficient adenovirusand CRAds was verified by PCR. Viruses were plaque purified and propagated onHEK293 cells and purified by ultracentrifugation with CsCl according to standardtechniques and functional PFU titers were determined by plaque assay on 293 cells.It was confirmed by the analysis of restriction enzyme digestion andnucleotides sequencing that Ki67-shRNA template DNA sequences were cloned intopCA13 successful and the pZD-Ki67 was formed successfully. The analysis of theDNA extracted from ZD-Ki67 by PCR indicated the ZD-Ki67 containedKi67-siRNA gene and without wild adenovirus, while Ad-Ki67 containingKi67-siRNA gene and without E1 gene. Functional PFU titers of Ad-Ki67 andZD-Ki67 were 4×10¹⁰ and 1×10¹¹ PFU/ml, respectively.These results indicate that the CRAds expressing Ki67-siRNA (ZD-Ki67) hadbeen constructed successfully. The CRAds expressing Ki67-shRNA may be used forfurther investigation of Gene-Viro therapy of renal cancer.Partâ…¢Conditionally replicative adenovirus armed with siRNA against Ki67gene for renal cancer therapy in cell culture and in nude mousemodelWe studied the tumor-selective replication of ZD-Ki67 first. Renal carcinoma786-0 cells and normal renal tube HK-2 cells were infected with ZD-EGFP andAd-EGFP respectively at an MOI of 1. The expression of EGFP was assessed byphotomicrographic examination at 120 hours after infection. It is found thatZD-EGFP as gene-virus vector increased the expression of EGFP gene in renalcarcinoma cells, but not in normal cells. Western blot assay of E1A expression indicated 786-0 and ACHN cells infected with ZD-Ki67 and ZD-EGFP can expressE1A, but the cells infected with Ad-Ki67 could not.To investigate its antitumoral efficacy of ZD-Ki67, renal carcinoma cells (786-0,ACHN) were infected with ZD-Ki67, ZD-EGFP and Ad-Ki67. 7 days later, cellswere stained with crystal violet to assay tumor-selective cytotoxicity. Cellproliferation was assayed by MTT method. TUNEL assay was used to measure theapoptosis of carcinoma ceils. Ki67 expression levels were detected by RT-PCR andWestern blot and immunocytochemistry analysis. Results of crystal violet stain andMTT demonstrated the antitumor effect of ZD-Ki67 was more potent than bothZD-EGFP and Ad-Ki67. ZD-Ki67 treatment of renal carcinoma cells resulted inincreased apoptotic cell death than ZD-EGFP and Ad-Ki67 treatment. Significantreductions in Ki67 expression were observed in lysates from cells infected withZD-Ki67. Similar significant reductions in the proportion of 786-0 and ACHN cellsmanifesting immunoreactivity for Ki67 were seen after incubated infected withZD-Ki67. Collectively, these results indicate significant knockdown of Ki67expression by ZD-Ki67 treatment.ZD-Ki67, ZD-EGFP and Ad-Ki67 were directly injected into BALB/C nudemice bearing subcutaneous 786-0 cells xenografts daily for 3 days at 7×10⁸PFU/day, respectively. Tumor size was measured every week. Both Ki67 protein andE1A protein expression of tumor xenografis were detected by immunohistochemicaltechnique and Laser Scanning Confocal Microscope (LSCM). The apoptosis oftumor xenografis was measured by in TUNEL technique. Results demonstrated thatthe tumor volume of ZD-Ki67 treatment group was significantly less than those ofZD-EGFP and Ad-Ki67 treatment groups. The Ki67 positive expression rates inZD-Ki67 treatment group were significantly lower than those in in ZD-EGFP andAd-Ki67 treatment groups. ZD-Ki67 treatment of renal carcinoma cells resulted in increased apoptotic cell death than ZD-EGFP and Ad-Ki67 treatment.Immunofluorescence for E1A protein indicated that ZD-Ki67 group and ZD-EGFPgroups could express E1A protein by Laser Scanning Confocal Microscope. Theconditionally replicative adenovirus ZD-Ki67 can replicate in tumor xenografts andinhibit tumor xenografts growth.From these findings we conclude that antitumor effect of Gene-Viro Therapywas better than virotherapy alone. Conditionally replicative adenovirus armed withsiRNA against Ki67 gene may prove to be a useful novel tool for renal cancertherapy.