| [FOREWORD]:Osteosarcoma is one of the most common adolescent primary bone cancer,the prognosis is poor.At present,the clinical treatment of osteosarcoma is integrated surgery and chemotherapy,so that the patients’ five-year survival rate has been significantly improved than previous.But the serious side effects caused by high-dose chemotherapy and multidrug resistance and other issues make progress of osteosarcoma treatment limited.At present,the use of live pathogens,attenuated pathogens or pathogens purified product for cancer treatment is one of the hot research.Microbial pathogens can cause degeneration of malignant tumors in humans and animals,but it’s viable because it can produce significant toxicity and limits its application in human cancer therapy.Therefore,at present people tend to look for pure metabolites with anti-tumor effects of pathogens for cancer treatment.Azurin is one of recently discovered biological anti-tumor proteins.Recent studies have confirmed that azurin in vitro and in tumor-bearing nude mice selectively induce apoptosis on some melanoma and human breast tumor cells,people also found that when azurin induces apoptosis in vivo,non-toxic side effects was found.For osteosarcoma prone to drug resistance characteristics,we used bacterial redox protein azurin treated U20S osteosarcoma cells,and we found significantly apoptosis on it.The synthesis of azurin is expensive,we prepared through genetic engineering of bacteria strains expressing azurin,through exploring expression conditions,to achieve the most optimal results.And we confirmed its apoptosis-inducing activity with recombinant proteins azurin,and we explored the p53 gene in osteosarcoma cells,the impact on the azurin-induced apoptosis.[PURPOSE]:1.We first use a recombinant azurin cDNA insert into DH5a strains,and we confirmed the expression of the protein azurin.Then we explored the expression conditions by different temperature,concentration of ITPG,and time,we tried to find out the best conditions for maximized expression.2.p53 has an important function in azurin-induced apoptosis,we try to study if the wild-type and mutant p53 affect the use of the anti-tumor effect of azurin on osteosarcoma cell lines.3.By using RNA interference technology,we tried to explore how the azurin play anti-tumor effect with p53 gene more accurate and efficient.[METHODS]:1.We use a pqe30/azurin plasmid,which we have already and contains the azurin’s cDNA,to transfect the DH5a stain with pGEM T plasmid tranfection system.By changing the bacterial incubation temperature,time and concentration of inducer ITPG explore optimal expression conditions;2.In order to study changes in the expression levels of p53 azurin different role,we use U20S cell lines contains wild-type p53 and MG-63 cell line contains mutant p53 to study the impact of azurin in different cells containing p53 gene.We treated these two cell lines the azurin under the concentration that we know can have effection at different times,then we detectd by MTT cell viability,cell morphology was observed through a microscope to observe the changes in cell ultrastructure by electron microscopy,cell cycle analysis by flow cytometry changes detected by RT-PCR the expression of apoptosis-related genes and the expression level by western blot detection of apoptosis proteins;3.We choose U2OS cell line with a wild-type p53,using the p53 siRNA construct to interfere with the expression of p53 in cells,then treated with azurin.We tested cell cycle,cell morphology,apoptosis-related protein expression again to locate the role of azurin target points.[RESULTS]:1.When the external conditions were remaining unchanged,the pqe30/azurin plasmid transfected DH5 α strain will have the highest expression efficiency at 37℃,ITPG concentration 0.8ug/ml for 5 hours;2.As the MG-63 cell line has mutant p53,and U2OS has wild-type,so there are significant differences between two groups with azurin.U2OS cell line was found significantly apoptosis induced by azurin,apoptosis-related cell morphology,ultrastructural changes were observed,and there were significant changes in cell cycle,apoptosis-related protein levels were significantly increased,especially Bax and Bcl-2,which are associated with p53 related apoptosis,had significant change.In MG-63 cells,these changes were not so obvious’.It means p53 may be an important role in azurin induce apoptosis on osteosarcoma.However,due to the azurin is a protein,its role in the cell is more complex,requiring more precise studies to confirm.So,once again we designed another experiment.3.We use the p53 siRNA + azurin treat U20S cells,and because p53 siRNA can only inhibit the expression of p53,we found that after using azurin it had more cell survival,lower rate of apoptosis,lower caspase-3 activity,it can increase the level of Bcl-2 and Bax were lower than the control group,which did not inhibit p53 with a negative siRNA group.In the TUNEL assay,p53 siRNA + azurin treated cells have a lower positive rate than the control group.It means p53 siRNA can inhibit apoptosis induced by azurin.Due to the use of p53 siRNA for transient transfection techniques,its function will decline over time.Therefore,we observed 72 hours,and we found the apoptosis-related reactions occur again.So it tell us azurin can play its role after binding with p53,it can act on the upstream and cause the downstream changes directly by the relevant product.This research found p53 has a similarity of oncogene during its cell cycle regulation,and azurin can reverse this function.[CONCLUSION]In this study,we found the azurin’s precise target is located on p5 3 gene by using RNA interference technology,it confirmed that it does have an effect by binding to p5 3,explained the reasons for azurin that it has weak effection on inducing apoptosis in some cell lines,and we found p53 has an oncogene likely function in wild type osteosarcoma cells,it gives us a new pioneering road to further tumorigenesis and explore gene therapy. |
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