[Knockdown of cyclin A2 expression by small interfering RNA in MG-63 cells].

Abstract

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.

CONCLUSION:

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.

• Authors: Ye Liu, Jia-Yi Ding, Wei-Liang Shen, Xing Zhao, Shun-Wu Fan
• Journal: Zhonghua zhong liu za zhi [Chinese journal of oncology] (Zhonghua Zhong Liu Za Zhi) Vol. 29 Issue 9 Pg. 670-5 (Sep 2007) ISSN: 0253-3766 [Print] China
• PMID: 18246796 (Publication Type: English Abstract, Journal Article)

Chemical References

• Cyclin A2
• Cyclin B1
• Proliferating Cell Nuclear Antigen
• RNA, Messenger
• RNA, Small Interfering

Topics

• Bone Neoplasms (metabolism, pathology)
• Cell Cycle
• Cell Line, Tumor
• Cell Proliferation
• Cyclin A2 (genetics, metabolism)
• Cyclin B1 (metabolism)
• Fibroblasts (cytology, metabolism)
• Gene Knockdown Techniques
• Humans
• Osteosarcoma (metabolism, pathology)
• Proliferating Cell Nuclear Antigen (metabolism)
• RNA Interference
• RNA, Messenger
• RNA, Small Interfering
• Skin (cytology)
• Transfection