Growth Inhibition Of MG-63 Cells By CyclinA2 Gene-specific Small Interfering RNA

Osteosarcoma is the most common primary malignant bone tumor of children and always lead to a very poor prognosis. It accounts for about 60% of malignant bone tumors diagnosed in the first two decades of life. Modern treatment programmes for this disease are typically multimodal, with surgery combined with both pre- and postoperative chemotherapy (neoadjuvant chemotherapy). Despite advances in anticancer therapy, survival has become plateaued nowadays largely due to the high frequency of metastases, chemotherapy resistance and other related reasons. So, there is a clear need to explore a more effective and less toxic treatment to eradicate osteosarcoma.Disruption of components of cell proliferation pathway can lead to the loss of growth control that underlies a tumor's development. Indeed, aberrant control of Cdks is a central feature in the molecular pathology of cancer. In osteosarcoma, it is also true.Cdks(cyclin-dependent kinase) play a crucial role in regulating cell cycle processes. As its name suggests, Cdk requires cyclin binding for its activity. Cdk and cyclin compose a complex in which Cdk is the catalytic subunit and cyclin is the regulatory subunit. The cyclin protein levels oscillate in a cell cycle-dependent manner and thus modulate the kinase activity of the Cdks.CyclinA2 is particularly interesting among the cyclin family because it can activate two different cyclin dependent kinases and functions in both S phase andmitosis phase. cyclinA2-Cdk is believed to be important for initiation of DNA replication and to restrict the initiation to only once per cell cycle. More and more evidence showed that deregulated expression of cyclinA2 could be involved in malignant transformation and possibly in tumorigenesis. In particular, many oncogene direct or indirectly cooperates with cyclinA2 to malignantly transform normal cells. Previous Study have proved Transgenic mice overexpressing the c-Fos oncoprotein develop osteosarcomas that are associated with deregulated expression of cyclinA2 protein level. Overexpression and amplification of cyclinA2 is detected in various primary tumors including osteosarcoma and this overexpression is always associated with poor prognosis. Taken together, these observations indicated that downregulation or depletion of cyclinA2 in osteosarcoma cells could be an attractive strategy for antiproliferative therapy.RNA interference (RNAi) is a widespread mechanism in eukaryotes for post-transcriptional gene silencing. It is initiated by the presence in the cell of double-stranded RNA (dsRNA), which is cleaved into -20-bp fragments by the cytoplasmic enzyme Dicer. Single strands from these fragments become incorporated into an RNA-induced silencing complex (RISC) that can induce cleavage of any mRNA capable of pairing with the RNA strand of the RISC. These -20-bp dsRNA fragments are called small interfering RNAs (siRNAs). RNAi overcome many drawbacks of antisense technology is considered as the most potent and promising gene silence technology discovered so far. In the mean time, although the original studies of siRNA silencing suggested high specificity, off-target and other toxic effects have been reported recently in cell culture experiments. These problem have to be solved before this technology is used for specific gene silence.In this study, three pairs of siRNAs targeting the cyclinA2 mRNA were designed according the current criteria. The siRNAs were chemically synthesized and purified. These siRNAs were transfected into osteosarcoma cell line MG-63 and normal human skin fibroblast ( HSF ) cells respectively in order to choose one which is the most effective. Then the selected cyclinA2-siRNA was used to silence the cyclinA2 gene expression in MG-63 and HSF cells. MTT assay, reverse transcriptase (RT)-PCR, flow cytometry and clony-forming test were employed to evaluate thebiological activity of MG-63 and HSF cells after cyclinA2 inhibition, and to explore whether cyclinA2 could be served as a new target gene for the treatment of osteosarcoma.Part ISuppressive effects of cyclinA2 gene-specific small interfering RNA in MG-63 and HSF cellsObjective To study the inhibitory effect of 3 pair of siRNAs targeting the cyclinA2 gene in MG-63 and HSF cells and to choose the most effective one. Methods Three pairs of siRNA_1-3 targeting the cyclinA2 mRNA and a pair of nonsense siRNA were designed according the current criteria. The siRNAs were chemically synthesized and purified. In all experiments except the analysis of dose kinetics, cells were transfected with siRNAs at a concentration of 50nM. Blank control cells were transfected with PBS to substitute siRNA. Negative control cells were transfected with nonsense siRNA. Real-Time Quantitative PCR and Western-blot were employed to evaluate the efficacy of RNA interference. Result1. The cyclinA2 mRNA level in MG-63 cells after siRNAs treatment was analyzed at different time point by real-time RCR. The mRNA level of cyclinA2 standardized to the level of GAPDH was decreased by all three cyclinA2-siRNAs(P < 0.05) , in which siRNA1 appeared to be more effective than the two others (P < 0.05) . There was no further decrease of cyclinA2 mRNA after 48h.Transfection with PBS or with the control siRNA had almost no effect ( P >0.05).2. 48h after treatment with 1nM, 10nM, 50nM and 100nM siRNA₁, cyclinA2 mRNA level in MG-63 cells was analyzed by real-time RCR. Compared with blank control, cyclinA2 mRNA level was almost no change after incubation with lnM of siRNA₁. Then the expression was rapidly reduced by 54.3% after incubation with 10nM of siRNA₁ (P < 0.05) . At 50nM, the reduced level furtherreached to 80.6% (p < 0.01) . Increasing the concentration of siRNA₁ from 50 to 100nM did not result in a obvious decrease in mRNA expression ( p > 0.05).3. After transfection MG-63 cells with different concentrations of siRNA₁ for 48h, the expression of cyclinA2 protein was negatively correlated to the concentration of siRNA₁ from 1nM to 100nM with slightly inhibition by 1nM and maximum inhibition by 100nM siRNA₁. The control siRNA transfection or treated with PBS did not affected the protein level of cyclinA2.4. The inhibitory effect of cyclinA2-siRNAs on HSF after 48h treatment was examined. Consistent with the finding in MG-63, siRNA₁ was still the most efficient one in knocking down cyclinA2 mRNA in HSF. It downregulate 58.1% cyclinA2 mRNA expression compare with 39% and 40.2% by siRNA2 and siRNA3 respectively (p < 0.05) . The inhibition of cyclinA2 protein expression was also seen in HSF cells after 50nM siRNA₁ treatment for 48h.Conclusion1. All 3 cyclinA2-siRNAs could suppress the expression of cyclinA2, in which siRNA1 is the most effective one.2. siRNA₁ took on a dose dependent manner under 50nM and the optimical inhibiting time is 48h after transfection.3. Using chemically synthesized siRNA and low concentration are effective ways to avoid nonspecific effects and off-target effects of siRNA.Part IIGrowth inhibition of MG-63 cells by suppressing cyclinA2 expressionObjective To study the impact of cyclinA2 depletion on the growth of MG-63 and HSF cells and to explore whether cyclinA2 could become a gene target in the treatment of osteosarcoma.Methods MG-63 cells were transfected with siRNA₁ at a concentration of 10nM and 50nM respectively. HSF cells were transfected with siRNA₁ at a concentration of 50nM alone. Blank control cells were transfected with PBS to substitute siRNA₁. Negative control cells were transfected with 50nM nonsense siRNA. Cells were harvested 48h after siRNA treatment. MTT assay, reverse transcription (RT)-PCR, flow cytometry and plate clony-forming test were employed to evaluate the biological activity of two kinds of cells after RNA interference. At the same time, the mRNA expression of PCNA and cyclinB1 in 50nM siRNA₁-treated MG-63 cells were examined. Result1. For MG-63 cells, 10nM and 50nM group could obviously inhibit cell proliferation after 24h 48h 72h transfection with siRNA₁ (compared with blank controls p < 0.05). For HSF cells, not inhibition of cell proliferation by 50nM siRNA₁ was seen after the same treatment. No inhibitory effect was detected with negative control siRNA in two kinds of cells (p > 0.05).2. For MG-63 cells, after incubation for 48h with 10nM and 50nM siRNA₁ respectively, the cell cycle analysis revealed most of MG-63 cells were arrested in G0/G1 phase (compared with blank controls p < 0.05). For HSF cells, After treatment for 48h with 50nM siRNA₁, the cell cycle analysis revealed 74.3% ±6.7%, 75.1%±5.6% and 80.4%±6.8% G0/G1 phase cells in blank, control and siRNA₁ group respectively. This suggests that most of HSF cells gathered in G0/G1 phase even without siRNA₁ treatment. siRNA₁ group exhibited no increase in G0/G1 population compared with blank controls ( p > 0.05 ). Compared with blank controls, there was no G0/G1 arrest in two kinds of cells treated with negative control siRNA (p > 0.05).3. After 50nM siRNA₁ treatment for 48h, the mRNA expression of cyclinA2, PCNA and cyclinB1 in MG-63 cells, which were standardized to the amount of β-actin, were reduced by 76.5%, 72.4% and 80.2% respectively. The relative mRNA level of each siRNA₁ group was dramatically decreased in comparison with that of blank control of each group (p < 0.01 ).4. Plate colony-forming assays were performed to examine whether MG-63 and HSF cells changed their growth ability after siRNA₁ treatment. For MG-63 cells, after cultured for 14 days, The result showed that the cloning efficiency of 10nM and 50nM group were decreased compared with that of blank control group(p < 0.05 ). While HSF cells showed no change in colony-forming ability after 50nM siRNA₁ treatment (p > 0.05 ).Conclusion1. Downregulation of cyclinA2 expression is an effective approach to inhibit biological activity in osteosarcoma cell MG-63. At the same time, it also results in decreased expression of PCNA and cyclinB1 in MG-63 cells.2. Downregulation of cyclinA2 expression has almost no effect on proliferation of HSF cells.3. CyclinA2 is critical for proliferation of MG-63 cells, which means that it can serve as an ideal target for gene therapy.