Study Of The Antitumor Potency Of P16 Gene In Osteosarcoma U-2OS Cells

Objective:To construct and identify the eukaryotic expression plasmid pcDNA3-HA-p16 encoding p16 gene, and investigate the the antitumor potency of p16 gene in osteosarcoma U-2OS cells.Methods:1. p16 gene was amplified by RT-PCR.2. The eukaryotic expression plasmid pcDNA3-HA-p16 encoding p16 gene was constructed by molecular biology techniques.3. Applying transfection with liposomes, the exogenous p16 gene was transferred to U-2OS cells.4. The expression of pcDNA3-HA-p16 was detected by immunofluorescence and Western blot in U-2OS cells.5. CCK-8 was used to detect cell proliferation.6. PI staining was applied to analyze cell cycle.7. DNA ladder and PI staining was used to illuminate cell apoptosis.Results:1. The eukaryotic expression plasmid pcDNA3-HA-p16 carrying p16 gene was confirmed by restricted enzyme digestion and DNA sequencing.2. After trasfecting U-2OS cells with liposomes, the recombinant plasmid expressed p16 and distributed mainly in the nucleus.3. p16 notably inhibited cell proliferation in U-2OS cells (P <0.05), and this effect was dose-dependent to a certain extent.4. p16 led to the increase of cell percentage of G1 phase(P <0.05) and the decrease of cell percentage of S phase in U-2OS cells (P <0.05).5. p16 caused to cell apoptosis percentage increased significantly and DNA ladder presented in DNA agarose gel electrophoresis.Conclusion:1. The highly active eukaryotic expression plasmid pcDNA3-HA-p16 was successfully constructed and the recombinant plasmid expressed p16 successfully by trasfecting U-2OS cells with liposomes.2. p16 notably inhibited cell proliferation in U-2OS cells, and this effect was dose-dependent to a certain extent. p16 induced G1 arrest and cell apoptosis significantly. Therefore, p16 markedly exhibits antitumor potency in U-2OS cells.