ABSTRACT
In this study, we aim to explore the effects of short hairpin RNAs (shRNAs) targeting human telomerase reverse transcriptase (hTERT) on the proliferation and apoptosis of osteosarcoma cells. After the synthesis of shRNA that target hTERT, osteosarcoma cells were assigned into three experimental groups-shRNA group, scramble group and blank group. The transcription and expressions of the hTERT gene in transfected cells were measured with quantitative real-time polymerase chain reaction and western blotting. Cell proliferation in each group was detected by Cell Counting Kit-8 assay. Cell cycle and rates of apoptosis were measured by flow cytometry. Expressions of apoptosis-related proteins, caspase-9 and caspase-3, were detected by western blotting. Telomerase activity was measured by PCR enzyme-linked immunosorbent assay. Results show that both the mRNA and protein expressions of hTERT were significantly lowered after the transfection of hTERT-shRNA. The proliferation capacity of transfected osteosarcoma MG-63, SaOS2 and U2OS cells in the shRNA group was lower than that in the blank group. We also found changes and differences in the amount of cells throughout the cell cycle. All cells in the G0/G1 phase increased in numbers, whereas the number of cells in the S phase were reduced, with elevated apoptosis rates. Expressions of apoptosis-related proteins, caspase-9 and caspase-3, were increased and telomerase activity was decreased in the transfected shRNA group (all P<0.05). Our results showed that shRNA targeting of the hTERT gene was able to inhibit cell proliferation and promote apoptosis of osteosarcoma cells by reducing the telomerase activity.
Subject(s)
Osteosarcoma/genetics , RNA, Small Interfering/genetics , Telomerase/genetics , Apoptosis/genetics , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Bone Neoplasms/therapy , Cell Line, Tumor , Cell Proliferation/genetics , Genetic Therapy , Humans , Osteosarcoma/pathology , Osteosarcoma/therapy , RNA, Small Interfering/administration & dosage , TransfectionABSTRACT
OBJECTIVE: To observe the effect of transplantation of marrow mesenchymal stem cells (BMSCs) transfected with insulin-like growth factor-1 (IGF-1) on fracture healing of rats with diabetes and discuss the gene therapy for diabetic fracture. METHODS: 60 8-week-old male Wistar rats weighing 180-200 g were divided into the control group and experimental group at random. All of them suffered from right tibia fracture following the model of diabetes induced by streptozotocin. BMSCs were transfected with Ad- IGF-1 and BMSCs of the appropriate group were transplanted to part of the fracture area. 6 rats were selected from each group at 1, 2, 3, 5 and 7 weeks after the surgery. Local bone callus was stained with hematoxylin-eosin (H-E) and IGF-1 in the bone callus and serum was tested. RESULTS: Osteoid tissues formed at 3 weeks in the experimental group; mature lamellar bone formed at 7 weeks in the experimental group; fibrous bone callus was observed in the control group. IGF-1 in bone callus of the experimental group is increasing and significantly different from that of the control group (p < 0.05). Concentrations of IGF-1 in the serum of the two groups were increasing gradually from the first week. The control group reached its peak in the 5th week. The experimental group reached a high concentration in the 5th week and maintained a high concentration in the 7th week. The differences at various times between the two groups have statistical significance (p < 0.05). CONCLUSION: Transplantation of BMSCs transfected with IGF-1 gene can promote fracture healing of rats with diabetes (Tab. 4, Fig. 1, Ref. 20).
