Inhibitory Effects Of PIN1 Antisense Gene On Human Osteosarcoma

【Objetive】(1) To evaluate the expression of Pin1 in human osteosarcoma and its correlation with histobiological behaviour;(2) To clone and construct eukaryotic expressing vectors of sense and antisense human PIN1(hPIN1)genes; (3) To evaluate the inhibitory effects of PIN1 antisense gene on the proliferation of human osteosarcoma cells.【Methods】(1) The expression of Pin1 in 42 cases of human osteosarcoma was detected with immunohistochemistry by ABC method; (2) Total RNA was extracted from MG-63 cells ,then the hPIN1 cDNA was amplified by RT-PCR; At the same time the sense and antisense hPIN1 genes were formed by binding BamHⅠand HindⅢin cis and trans-directions; At the end they were cloned into the eukaryotic expressing vector pIRES2-EGFP in cis and trans directions using DNA recombinant technology. The recombinant vectors were further identified by digestion of BamHⅠand HindⅢ; (3) Different doses of antisense PIN1 gene (0 ,20 ,50 ,100 ,200 ,250μl) were transfected into osteosarcoma MG-63 cells. The cells and culture supernatant before and after transfection were collected.The curve of cell growth was made using MTT method.The cell growth cycle and apoptosis were detected by FCM. The expression of PIN1 was detected by Western-blot ; The expression of PIN1 mRNA was detected by reverse transcription polymerase chain reaction (RT- PCR) .【Results】(1) The expression positivity rate of Pin1 was 64.3% in osteosarcoma ; no expression of Pin1 was detected in 10 cases of normal bone and cartilage tissues from patients with traumatic amputation(as control group). The expression of Pin1 was significantly associated with clinical stages ( P < 0. 05), but not with sex,age,tumor locus,Price's grade and soft tissue invassion ( P > 0. 05); (2) Results of sequencing showed that the orientation of the ligations and the reading frame were correct. After digested by BamHⅠand HindⅢ, two fragments which lengthened 5.3kbp and 0.990kbp were formed in sense and antisense eukaryotic expressing vectors. Electrophoretic results were completely coincident with theoretical calculation; (3) MTT and FCM assays indicated that the transfection of antisense PIN1 gene could inhibit proliferation of MG-63 cells and lead to cell apoptosis. Western-blot assays revealed the MG-63 cells transfected with antisense PIN1 gene had weaker expression than those without transfected with antisense PIN1 gene, and band intensity was negatively related with doses. The cells transfected with different doses of gene (0 ,20 ,50 ,100 ,200 ,250μl) had different absorbance rate : 0. 854±0. 136,0. 866±0. 138,0. 732±0. 154,0. 611±0. 121,0. 547±0. 109,0. 398±0. 113,0. 320±0. 151 ,with significant difference assessed by F and q test ( P < 0. 05) . The absorbance rate of PINI mRNA was 0. 983±0. 125,0. 988±0. 127,0. 915±0. 157,0. 786±0. 125,0. 608±0. 124,0. 433±0. 130,0. 410±0. 158 respectively ( P < 0. 05) .【Conclusion】(1) Overexpression of Pin1 in osteosarcoma may play an important role in tumor genesis and tumor progression, and it may provide new target for gene therapy; (2) Human PIN1 sense and antisense genes were successfully cloned and eukaryotic expressing vectors were successfully constructed; (3) The expression of PIN1mRNA in MG-63 cells could be inhibited by antisense PIN1 gene, and then the expression of PIN1 was reduced and the proliferation of human osteosarcoma cells MG-63 was inhibited.