The Role Of Per2 Gene In The Proliferation, Invision And Apoptosis Of Osteosarcoma Cells And The Relative Mechanisms

Background:Osteosarcoma is the most common primary bone tumor that mainly affects children and adolescents. Early metastasis usually occurs, and its prognosis is poor. 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%. As many other malignant tumors, the etiology of osteosarcoma is mostly unclear. Recently, various chemotherapies and new diagnostic techniques have been developed. These developments have dramatically improved the 5-year survival rate for osteosarcoma 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, especially gene therapy.Circadian rhythms are endogenous self-sustained, near 24-h biological cycles. Organisms from bacteria to human have evolved endogenous molecular clocks. In mammals, the master circadian pacemaker is located in the suprachiasmatic nuclei (SCN). This clock is in constant contact with the environment through daily light-dark cycle. The master circadian clock coordinates peripheral circadian clocks within virtually every cell in the body. This coordination is accomplished directly through autonomic nervous system innervation and indirectly through daily rhythmic synthesis and release of an array of hypothalamic, pituitary and dispersed endocrine hormones. Disruption of circadian organization has significant effects on human health, causing sleep disorders, gastrointestinal and cardiovascular illnesses and depression. It is also associated with an increased incidence of several epithelial cancers. In mouse models, transplanted tumors grow twice as fast in SCN-lesioned mice than in sham-lesioned animals. Circadian disruptions caused by changing the light/dark cycle, as in rotating shift work, or by constant light exposure increases cancer risk in humans and cancer growth rate in mice. Reciprocally, cancer bearing affects the quality of circadian organization of individual cancer patients. Those patients suffering from cancer associated circadian disruption die sooner from their disease, have more disrupted sleep, suffer increased daytime fatigue and have poorer quality of life than those who maintain their usual circadian organization more fully.To date, at least nine known core clock genes are found in mammalian, including periodl (Per1), period2 (Per2), period3 (Per3), CLOCK, cryptochrome1(CRY1), cryptochrome2 (CRY2), BMAL1, casein kinasel epsilon (CSNK1E), and timeless (TIM). Per2 is a core circadian clock gene. PER2 protein,which involves a series of cell pathways to regulate the biological activities of the body, is expressed differently in different tissues. Dysregulation of hPer2 gene has been found in many types of poor-prognosis human cancers, including breast cancer, hepatoma, colorectal cancer, and pancreatic cancer. Genetic studies also showed that mice with dysfunctional circadian rhythms are prone to many kinds of cancer developing. Mice deficient in mPer2 showed significant higher tumor incidence. Moreover, functional studies found that overexpression of Per2 inhibited cancer cell growth in both culture system and xenograft mouse model.However, there is scarce information available in the literature as to whether and how circadian gene hPer2 on osteosarcoma cells. To confirm the influence of Per2 gene to osteosaecoma cells, we transfected the eukaryotic expression vector pcDNA3.1-hPer2 and Per2 small interference RNA(siRNA) into MNNG/HOS oeteosarcoma cells respectively. After transfection, we analysed the variations of cell proliferation, invation and apoptosis. Furthermore, to investigate the mechanism of hPer2 to osteosarcoma cells, we checked the effects of hPer2 overexpression&downregulation on proliferation/apoptosis-related genes and Akt signal pathway.Objective:1. To determine the variations of PER2 protein expression after transfection of hPer2 plasmid and siRNA.2. To investigate the effect of hPer2 overexpression/downregulation on the proliferation,invation and apoptosis of osteosarcoma cells.3. To determine the effect of hPer2 on Akt signaling pathway, Bcl-2, p21, p27 genes in osteosarcoma cells, and clarify the role of hPer2 in the anti-osteosarcoma effects.Materials and methods:1. hPer2 plasmid and siRNA were transfected into HOS cells by Lipofectamine Reagent. The overexpression&downregulation of hPer2 in HOS cells were confirmed by immunofluorescence technique and Western blot.2. The HOS cells were divided into five groups:blank control group(without any treatment), pcDNA3.1-hPer2 group, pcDNA3.1 group, siRNA group, unrelated interference RNA group.3. (1) Cell proliferation analysis. CCK-8 assay was performed to detect the osteosarcoma cell viability of each group.(2) Invasion assay.Cells of each group,in which expression of Per2 was confirmed by Western blot, were seeded in serum-free medium onto the upper chamber, while complete medium was added in the lower compartment as a chemoattractant. After 24h of incubation, abilities of invasion were quantified by counting the number of invaded cells from Transwell chamber.(3) Apoptosis analysis. Apoptosis was detected by the TUNEL assay, and the experiment was performed according to the instruction of the TUNEL kit. TUNEL positive cells (DAB+) were believed to be apoptotic cells.4. Akt signal pathway and Bcl-2, p21, p27 gene assay. Cells of each group were collected. Total proteins were extracted by RIPA. Western blot was performed to investigate the protein expression of Akt, Bcl-2, p21, p27.5. Statistical analysis. SPSS for Windows v.16.0 was used for statistical analysis. Data are described as mean ± SEM. Comparisons between two groups involved in Student’s t-test and comparisons among groups involved one-way ANOVA followed by LSD’s test (with equal variances assumed) or Dunnett’sT3 test (with equal variances not assumed). Probabilities of 0.05 or less were considered to be statistically significant.Results:1. siRNA successfully downregulate per2 expression, the HOS proliferation, invation was increased, the apoptosis was decreased Immunofluorescence assay confirmed that hPer2 siRNA had been successfully transfected into MNNG/HOS cells and efficiently expressed.Western blotting analysis showed that PER2 protein displayed significantly downregulation.48h after transfection, CCK-8 assay showed that Per2 downregulation significantly accelerated proliferation of HOS cells (p<0.01). There was no statistical difference between blank control group and non-related RNA group (p>0.05).24h after transfection, Transwell assay showed that Per2 downregulation significantly enhanced invision of HOS cells (p<0.01). There was no statistical difference between blank control group and non-related RNA group (p>0.05).48h after transfection, TUNEL assay showed that the number of apoptotic cells in non-related RNA group increased significantly than blank control group(p<0.01).Cell apoptosis in Per2 siRNA group was inhibited significantly than non-related RNA group (p>0.05)2. Overexpression of hper2 successfully upregulate per2 expression, the HOS proliferation, invasion was decreased, the apoptosis was increased Consistent with above results. Immunofluorescence assay confirmed that hPer2 plasmid had been successfully transfected into MNNG/HOS cells and efficiently expressed.Western blotting analysis showed that PER2 protein displayed significantly overexpression.48h after transfection, CCK-8 assay showed that Per2 overexpression significantly inhibited proliferation of HOS cells (p<0.01). There was no statistical difference between blank control group and non-related RNA group (p>0.05).24h after transfection, Transwell assay showed that Per2 overexpression significantly inhibited invision of HOS cells (p<0.01). There was no statistical difference between blank control group and non-related RNA group (p>0.05).48h after transfection, TUNEL assay showed that the number of apoptotic cells in both pcDNA3.1-hPer2 and pcDNA3.1 groups increased significantly than blank control group (p<0.05 and p<0.01).Cell apoptosis in pcDNA3.1-hPer2 group was promoted significantly than pcDNA3.1 group (p>0.01)3. Effects of Per2 on Akt protein expression.48h after transfection, Western blot assay showed that Per2 overexpression could inhibit expression of Akt.4.48h after transfection, Western blot assay showed that Per2 overexpression could inhibit expression of Bcl-2.5. Per2 overexpression promoted expression of p27.6. Per2 overexpression promoted expression of p21.7. Per2 downregulation promoted the phosphorylation of Akt.8. Per2 downregulation promoted the expression of Bcl-2.9. Per2 downregulation inhibited the expression of p21.10. Downregulation of Per2 had no significantly effect on expression of p27.Conclusion:1. Per2 plasmid and siRNA can successfully transfect into MNNG/HOS osteosarcoma cells.2. Overexpression can inhibit the proliferation and invasion of MNNG/HOS osteosarcoma cells and can promote its apoptosis. Consistent with the overexpression data, downregulation of Per2 can enhance the proliferation and invasion of MNNG/HOS osteosarcoma cells and can inhibit its apoptosis.3. Per2 overexpression inhibits Akt signaling pathway. The Akt pathway plays an important role in cell proliferation and apoptosis. Per2 may affect proliferation and apoptosis of osteosarcoma cells through Akt signal pathway.4. Per2 may regulate the apoptosis of osteosarcoma cells through Bcl-2.5. Per2 may regulate the proliferation of osteosarcoma cells through p27 and p21.