Apoptosis Of Osteosarcoma Cells Induced By Targeting Expression Of Long Double-stranded RNA

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. It is a highly aggressive neoplasm typically composed of spindle cells producing osteoid. Osteosarcoma is mainly treated by surgical excision or chemotherapy. The introduction of multi-agent chemotherapy has dramatically improved the outcome for the patients and the majority of modern series report 3-year disease-free survival of 60%-70%. Because the prognosis of unresectable, recurrent and chemotherapy-resistant cases still remains quite poor, new therapies for the tumor are being developed.Gene therapy is one of the most promising strategies developed to cure malignancies, but few clinical trials of gene therapy for osteosarcoma have been done to date. It had been reported that targeting osteosarcoma cells by HSV-1 tk gene driven by human osteocalcin promoter or hTERT promoter produced good outcomes. However, low transcriptional activities of osteocalcin promoter could be detected in lung, brain and other tissues. And re-activation of telomerase .could be found in stem cells, germ cells and activiated lymphocytes. All these data demonstrated that the expression of therapeutic genes could not be strictly limited to the bone tumor tissues even using tissue-specific or tumor-specific promoters and may result in unexpected toxicities to normal tissues. Therefore novel strategies should be developed to further improve the safety and efficiency of current gene therapy methods.In this project, we designed a novel osteosarcoma gene therapy method based on that long dsRNA could induce apoptosis of many types of cells. A long dsRNA expression vector with the sense and antisense strands of a non-coding gene of certain length without any homology to human genome driven by human osteocalcin promoter and hTERT promoter respectively was constructed. And only in osteosarcoma cells, both strands of the long dsRNA could be expressed to activate the PKR-dependent signal transduction pathway and 2'5'-oligoadenylate synthetase/RNase L pathway to induce tumor cells apoptosis. In nonmalignant cells, only one strand of long dsRNA is transcribed and takes no effects on the normal cellular functions because it is non-coding and with no homology to human genome. In our opinion, the strategy of targeting expression of long dsRNA is superior to any other gene therapy methods with potential clinical application.Objective: To establish a novel, efficient and highly safe strategy of osteosarcoma gene therapy, evaluate its specificity in inducing apoptosis of osteosarcoma cells and elucidate the mechanisms involved in the process.Methods: Parti: The knockdown effects of shRNA driven by human osteocalcin promoter without 5'UTR and hTERT core promoter with a reconstructed transcriptional start site on the expression of exogenous luciferase and endogenous P53 were evaluated. Partll: A shuttle plasmid pDC311-dsRNA containing sense and antisense expression cassettes of a designed gene with no homology to human genome, driven by modified human osteocalcin promoter and hTERT promoter respectively, was constructed. A recombinant replication-defective adenovirus Ad-dsRNA was prepared by co-tansfecting HEK293 cells with pDC311-dsRNA and a adenovirus genomic plasmid pBHGlox(delta)El,3Cre. After being confirmed by PCR using specific primers, Ad-dsRNA was purified and titered. The survival rates and apoptotic cells rates of MCF-7(a hTERT⁺ human breast adenocarcinoma cell line), A549(a hTERT⁺ human alveolar cell carcinoma cell line), U2OS(a hTERT" osteosarcoma cell line), MG63(a hTERT⁺ osteosarcoma cell line) and HOS(a hTERT⁺ osteosarcoma cell line) post-infected with Ad-dsRNA were evaluated by MTT assays and flow cytometry respectively. The cleavage of PARP, phosphorylation of PKR and eIF2α induced by Ad-dsRNA infection was detected by Western blot. A luminescent assay was used to detect the activities of casapse-3 and caspase-8of MG63 post-infection.Results: Part I: Using human genomic DNA as template, a 830bp-length human osteocalcin promoter without 5'UTR was obtained. A plasmid vector pGL3-OCP-shLuc carrying shRNA coding sequence driven by the promoter and a self-designed mini-polyA as transcriptional terminal sequence was constructed. Osteoblast-specific knockdown effect on the expression of luciferase was seen only in MG63 cells but not in HEK293 cells, and the knockdown rate was about 69.8%. Using pGL3-basic-hTERT harboring hTERT core promoter as template, a 290bp-length modified hTERT promoter with a reconstructed transcriptional start site was amplified by PCR. Specific knockdown effects on exogenous luciferase gene and endogenous P53 gene could only be obtained in hTERT+ osteosarcoma cells when the shRNA coding sequences were driven by the modified hTERT promoter. Part II: A shuttle plasmid pDC311-dsRNA with both sense and antisense expression cassette driven by the modified human osteocalcin promoter and hTERT promoter was constructed and verified by restrictive digestion analysis. Ad-dsRNA was prepared by co-tansfecting HEK293 cells with pDC311-dsRNA and pBHGlox(delta)El,3Cre and confirmed by a 724bp fragment successfully amplified by PCR using specific primers. The titer of the purified Ad-dsRNA was determined to be 1.8×10¹⁰ pfu/ml and 2.2×10¹⁰ pfu/ml by end-point dilution assay and plaque assay respectively. MTT assay demonstrated that infection with Ad-dsRNA had no obvious effects on the survival rates of MCF7, A549 and U2OS, but with the increase of MOI, the. survival rates of MG63 and HOS decreased significantly. Flow cytometry analysis demonstrated that the apoptotic cells rates of MG63 and HOS increased with the increase of MOI, reached 73.3% and 80.8% at MOI of 100:1, but had no impact on the apoptotic cells rates of MCF7, A549 and U2OS. Western blot analysis revealed that the cleavage of PARP could be induced in MG63 and HOS cells by infection with Ad-dsRNA, but not in U2OS cells. And with the increase of MOI, the cleaved PARP elevated. All these data indicated that Ad-dsRNA could only induce the hTERT+ osteosarcoma cells to undergo apoptosis, and have no cytotoxic effects on the hTERT" cells and hTERT⁺ non-bone derived cells. Phosphorylation of PKR and eIF2a were induced in MG63 cells by Ad-dsRNA infection but not in U2OS cells using anti-phospho-PKR(Thr451) and anti-phospho-eIF2a(Ser51)...