[Inhibition of osteosarcoma cell proliferation by a short hairpin RNA targeting proliferation cell nuclear antigen].
OBJECTIVE
To construct a plasmid vector expressing the short hairpin RNA (shRNA) targeting proliferation cell nuclear antigen (PCNA), and to investigate its effect in vitro on the expression of PCNA, proliferation and apoptosis of human osteosarcoma cells.


METHODS
A plasmid vector expressing the short hairpin RNA targeting at PCNA was constructed and transfected into human osteosarcoma cell line MG-63 by dosper liposomal method. PCNA mRNA and protein expressions were examined using RT-PCR and immunohistochemical staining, respectively. Inhibition of the cell proliferation was studied by MTT method and colony forming assay. DNA synthesis was analyzed by (3)H-TdR incorporation and the cell cycle was determined by flow cytometry. The apoptotic cells were stained with acridine orange.


RESULTS
Expression of PCNA mRNA after the transfection was markedly inhibited by 80.51% with a PI value of 25.68% of that of the control group. PCNA shRNA inhibited MG-63 cell growth detected by using MTT method, with an inhibition rate of 61.78% at 48 h. DNA synthesis rate also decreased in the (3)H-TdR incorporation test. Flow cytometry analysis showed an increase of the percentage of G(0)/G(1) phase cells, along with a decrease of cell population in the S phase. The apoptosis rate of cells transfected with the plasmid vector was 16.54%.


CONCLUSIONS
PCNA shRNA significantly suppresses the expression of PCNA at both mRNA and protein levels, corresponding to an inhibition of the proliferation of MG-63 cell and an increase of the cellular apoptosis.
Zhang
Qi-liang
QL
Department of Orthopedics, Xiehe Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. zhangqiliang2004@yahoo.com.cn
Yang
Shu-hua
SH
Liu
Hong-yun
HY
Wu
Cui-huan
CH
D017209:Apoptosis; D001859:Bone Neoplasms / Q000378:metabolism / Q000473:pathology*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005822:Genetic Vectors; D006801:Humans; D012516:Osteosarcoma / Q000378:metabolism / Q000473:pathology*; D010957:Plasmids; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection
D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D034741:RNA, Small Interfering