Effect Of Human Circadian Gene Period2 On The Proliferation Of Osteosarcoma Cells

Background Osteosarcoma is the most common primary bone tumor that mainly affects children and adolescents. Twenty percent of osteosarcoma patients succumb to this disease as a result of tumor metastasis or an unresectable tumor. The remaining 80% of patients generally present with small metastases when diagnosed; a number of these patients develop pulmonary metastasis within a year and the 5-year survival rate is only 15%.The etiology of osteosarcoma is mostly unclear because of the difficulties in understanding the molecular mechanism of tumor development in the complex structure and numerous genomic rearrangements of bone cancer cells. Recently, various chemotherapies and new diagnostic techniques have been developed. These developments have dramatically improved the 5-year survival rate for osteo-sarcoma patients up to 55-70%. Complete radical surgery remains a preferable choice of osteosarcoma treatment, whereas adjuvant chemotherapy is given before surgery. If surgical excision is not possible, the addition of radiation therapy might be beneficial to control the local tumor. Still, a number of patients with osteosarcoma have a risk of local relapse after chemotherapy. For this reason, it is very necessary to explore novel and alternative strategies for osteosarcoma treatment. In order to improve the long-term survival rate of this type of cancer, it is crucial for research efforts to identify new targets and techniques, especially for gene therapy.The physiological and behavioral activities of many organisms are controled by the daily light/dark cycles of the Earth. These temporal activities are often referred to as the circadian rhythm, which has the biological molecular basis, namely the circadian gene. Recent studies have demonstrated that the circadian genes regulate other molecular and biochemical processes beyond their established role in the mammalian circadian clock, a growing body of research suggests that the role of the circadian clock could be a fundamental regulator for tumor suppression in humans. Several lines of evidence generated from cancer studies indicate that period2 (Per2), as one of the key genes, has been shown to play critical roles in growth control and tumor development, and frequently dysregulated in several poor outcomes, aggressive, metastatic human cancers, including breast cancer, hepatocellular carcinoma, colorectal carcinoma, pancreatic cancer, etc. In the present study, we sought to construct the recombinant pEGFP-N1-hPer2 plasmid with pEGFP-N1 vector carrying fluorescent protein expressed gene then transfected into MG63 cells in order to ob- serve the biological behavior of the cells. These data might provide scientific information for prognosis prediction and targeted therapy for osteosarcoma.Objective (1) Total mRNA was extracted from human osteosarcoma MG63 cells, hPer2 gene was obtained by RT-PCR and cloned into pEGFP-Nl vector. (2) The pEGFP-N1-hPer2 eukaryotic expression vector was constructed and transfected into cultured MG63 cells using Lipofectamine 2000. The over-expression of hPer2 in MG63 cells was verified by qRT-PCR and Western blotting, respectively. (3) Investigated the effects of hPer2 protein over-expression on MG63 cells viability, cycle, apoptosis and invasive ability.Results (1) Corrected construction of pEGFP-N1-hPer2 was identified by double enzyme digestion and DNA sequencing. hPer2 gene expressed by the transfected cells was testified by qRT-PCR and Western blot. (2) the pEGFP-N1-hPer2 eukaryotic expression vector was constructed and transfected into cultured MG63 cells successfully. (3) The viability, proliferation, and invasive abilities were suppressed, and the apoptosis was enhanced in MG63 cells after transfected with pEGFP-N1-hPer2 eukaryotic expression vector.Conclusion The recombinant pEGFP-N1-hPer2 plasmid had been constructed successfully and expressed effectively in MG63 cells. Furthermore, results also showed that the viability, proliferation, and invasive abilities were suppressed, and the apoptosis was enhanced in MG63 cells by over-expression of hPer2. This preliminary study provides ground work for further research on the roles of circadian gene hPer2 in osteosarcoma cells MG63, and would offer promise for the development of novel therapeutic strategies in the treatment of osteosarcoma.