Inhibition Of The Expression Of Proliferation Cell Nuclear Antigen In Humen Osteosarcoma Cell By Antisense Oligodeoxynucleotide In Vitro

Malignant tumor is a polygenic disease, It has been shown that the formation and development are not only associated with dysfunction of gene expression and regulation, including abnormal secretion of growth factor and abnormal signal pathway, but also involved attenuation of apoptosis. It is their successfully escaping from apoptosis by various mechanisms that make tumor cells gain immortalization, formation and progression. Nevertheless, resistance to apoptosis of tumor cells may lead a way to resistant to chemical therapy or radiation therapy. The duplication, transcription and translation were changed by these factors, which would result in the abnormal differentiation and infinite proliferation of tumor cells. Thus, it is significant for us to demonstrate the pathogenesis of malignant tumor by studying the expression of the related gene in the tumor cell proliferation and apoptosis, and it has realistically meaning to promote the healing rate and develop new therapies of tumor.Osteosarcomas are the most commen malignant tumor that primarily arise from skeletal system, except plasma cell myeloma, and occur most frequently during childhood and adolescence (15~25 years old), and happen to the male more than the female. There is comparatively large disparity in each country's census of the incidence rate, which is about 1~0.1 per million people, and the precise statistics data of the incidence and mortality rate in our country is still lacking. Osteosarcomas have highly malignancy and bad prognosis, which take place metastasis early, and recur after treatment frequently, and do great harm to the patient. By the development of new adjunctive chemical therapy, surgical technic, bony remodeling method and improved staging methods, now more and more patients have been treated with limb-sparing surgery and can expect their disease to be cured. However, many patients still can't take advantage from exiting treatment methods, whose survival rate in 5 year from 50% to 65%. Recur and metastasis still are deadly result, and survival rate will lower (30%) if metastasis occur. Now, the total therapeutic efficacy of the osteosarcoma sufferer is still poor, and how to improve the healing rate and explore new therapy methods are the clinical focal points right along.Antisense gene therapy is based on the principle of Watson-Crick base pair, synthesis minor antisense oligodeoxynucleotide chain that can combine specifically with target gene was imported into tumor cells, then block the target gene expression from transcription and translation level. Because of its effectiveness, simplicity and no vector, antisense gene therapy has been one of hot points of tumor gene therapy in recent years. As a helper factor of DNA polymeraseδ, proliferation cell nuclear antigen (PCNA), is absolutely necessary to DNA synthesis, and take part in the regulation of cell cycle, and is positively correlated with various regulatory protein of liver nucleic acid metabolism. Previous studies have revealed that PCNA plays an important role of the malignancy and prognosis of osteosarcoma. Some researches about antisense genetheray target PCNA have be reported, but up to now, the data of the effect of antisense oligodeoxynucleotide(ASODN) on the expression of proliferation cell nuclear antigen(PCNA) in osteosarcoma cell line MG-63 still is not available. We choose PCNA as target gene, and design this study:Objective To investigate the inhibitory effect of antisense oligodeoxynucleotide (ASODN) on the expression of proliferation cell nuclear antigen(PCNA) in osteosarcoma cell in vitro.Methods Phosphorothioate PCNA-ASODN and sense oligodeoxynucleotide were transfected to MG-63 cell by dosper liposomal. The expression of PCNA mRNA and protein were examined by RT-PCR and Western blot method, respectively. The inhibition of the cell proliferation was estimated by MTT method and colony forming test.Results The number of the colonies of ASODN group was significantly smaller than the other groups (p<0.01). PCNA-ASODN could significantly reduce the expressions of PCNA mRNA and protein. The expressions of PCNA mRNA in ASODN groups were 31.36% of the control, the expression of protein at the same time was 40.00 % of the control, and the inhibition rate at 12h, 24h, 48h, 72h were 56.25%, 65.64%, 55.08%, 39.79%, respectively.Conclusion PCNA-ASODN could significantly reduce the expressions of PCNA mRNA and protein and inhibit the proliferatiom of the MG-63 cell.