[Knockdown of cyclin A2 expression by small interfering RNA in MG-63 cells].
OBJECTIVE
To study the inhibitory effect of small interference RNA (siRNA) targeting cyclin A2 gene on the growth of osteosarcoma MG-63 and human normal skin fibroblast HSF cells and to explore whether cyclin A2 siRNAs could become a useful tool in the treatment of osteosarcoma.


METHODS
Three pairs of siRNAs targeting cyclin A2 mRNA and a pair of nonsense siRNA were designed according to the current criteria. SiRNAs were chemically synthesized and purified. The siRNAs were transfected into MG-63 cells and HSF cells via oligofectamine. The cells transfected with nonsense siRNA served as negative control group and those only treated with PBS as blank control group. Quantitative fluorescence RT-PCR, Western-blot, MTT assay, reverse transcriptase (RT)-PCR, flow cytometry and clone forming test were employed to evaluate the efficacy of RNA interference. At the same time, the mRNA expression of PCNA and cyclin B1 in siRNA-treated MG-63 cells were examined.


RESULTS
Although all three siRNAs could reduce the cyclin A2 expression, siRNA, appeared to be the most effective. After 48 h treatment with siRNA1, cyclin A2 mRNA and protein expression in MG-63 cells was significantly reduced by nearly 80% as compared with that of the blank control group, whereas the negative and blank control groups had similar expression levels. MG-63 cells treated with siRNA1 were arrested at G0/G1 phase by 80.1% and the proliferation of these tumor cells was suppressed 48 h after transfection. Furthermore, MG-63 cells showed a decreased colony forming ability after siRNA1 treatment. In addition, the cyclin A2-depleted MG-63 cells showed decreased levels of PCNA and cyclin B1. In contrast, although cyclin A2 expression in HSF reduced by nearly 60% after treatment by siRNA1 for 48h, these cells exhibited only a slight change in cell cycling, and neither clear inhibition of proliferation nor impaired colony forming ability was observed.


CONCLUSIONS
Cyclin A2 is critical for proliferation of MG-63 cells. Cyclin A2-siRNAs can induce obvious inhibition of cyclin A2 mRNA and protein expression in MG-63 and HSF cells, which consequently down-regulate the proliferation of MG-63 cells. There is little effect on the proliferation of siRNA-treated HSF cells. Those results indicate that siRNAs against cyclin A2 may become a potential antiproliferative tool in future antitumor therapy.
Liu
Ye
Y
Department of Orthopaedics, Sir Run Run shaw Hospital, Zhejiang University, Hangzhou 310016, China.
Ding
Jia-Yi
JY
Shen
Wei-Liang
WL
Zhao
Xing
X
Fan
Shun-Wu
SW
D001859:Bone Neoplasms / Q000378:metabolism / Q000473:pathology*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D056751:Cyclin A2 / Q000235:genetics / Q000378:metabolism*; D056744:Cyclin B1 / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D012516:Osteosarcoma / Q000378:metabolism / Q000473:pathology*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012867:Skin / Q000166:cytology; D014162:Transfection
D056751:Cyclin A2; D056744:Cyclin B1; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D034741:RNA, Small Interfering