[Growth inhibition of MG-63 cells by cyclin A2 gene-specific small interfering RNA].
OBJECTIVE
To study the impact of small interference RNA (siRNA) targeting cyclin A2 gene on the growth of MG-63 and HSF cells and to explore whether cyclin A2 siRNAs could become a useful tool in the treatment of osteosarcoma.


METHODS
One pair of siRNA targeting the cyclin A2 mRNA and a pair of nonsense siRNA were designed according the current criteria. SiRNA was chemically synthesized and purified. The siRNA was transfected into osteosarcoma cell line MG-63 and normal human skin fibroblast (HSF) cells via oligofectamine. Cells transfected with nonsense siRNA served as the negative control and those only treated with PBS as the blank control group. Quantitative fluorescence RT-PCR, Western-blot, MTT assay, reverse transcriptase (RT)-PCR, flow cytometry and colony-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
1 nmol/L, 10 nmol/L, 50 nmol/L and 100 nmol/L cyclin A2-siRNA can reduced cyclin A2 mRNA and protein expression respectively by 9.43%, 56.35%, 79.17% and 84.30% as compared with that of the blank control group, whereas the negative and blank control groups had similar expression levels. After 48 h treatment with 10 nmol/L siRNA, MG-63 cells were arrested in G0/G1 phase and the proliferation of this tumor cell was suppressed by 39.06% 48 h after transfection. Furthermore, the treated MG-63 cells showed less colony-forming ability. Increasing the siRNA concentration to 50 nmol/L can further inhibit the proliferation of MG-63 cells by 54.94%. In addition, the cyclin A2-depleted MG-63 cells showed decreased levels of PCNA and cyclin B1. In contrast, although cyclin A2 mRNA and protein expression in HSF reduced 58.13% 48 h after treatment by 50 nmol/L siRNA, these cells exhibited only a slight change in cell cycle, and no clear inhibition of proliferation and impaired plate colony-forming ability was observed.


CONCLUSIONS
Cyclin A2 gene maybe served as a potential target for tumor therapy. RNA interference induces obvious inhibition of cyclin A2 mRNA and protein expression in MG-63 and HSF cells, which consequently downregulate the proliferation of MG-63 cells. There is few inhibitory effect on the proliferation by siRNAs for HSF cells. These results indicate that siRNAs against cyclin A2 could become a potential antiproliferative tool in future antitumor therapy.
Liu
Ye
Y
Department of Orthopaedics, Sir Run Shaw Hospital, Zhejiang University, Hangzhou 310016, China.
Fan
Shun-Wu
SW
Ding
Jia-Yi
JY
Zhao
Xing
X
Shen
Wei-Liang
WL
D015153:Blotting, Western; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019925:Cyclin A / Q000235:genetics* / Q000378:metabolism; D056751:Cyclin A2; D019926:Cyclin B / Q000235:genetics / Q000378:metabolism; D056744:Cyclin B1; D005434:Flow Cytometry; D016193:G1 Phase / Q000235:genetics / Q000502:physiology; D006801:Humans; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D016192:Resting Phase, Cell Cycle / Q000235:genetics / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
C503721:CCNA2 protein, human; C534699:CCNB1 protein, human; D019925:Cyclin A; D056751:Cyclin A2; D019926:Cyclin B; D056744:Cyclin B1; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D034741:RNA, Small Interfering