目的 观察FAM64A基因对宫颈癌细胞周期调控的影响,探讨其作为分子靶点在肿瘤治疗中的潜在价值.方法 阿霉素(ADR)处理HeLa细胞,半定量RT-PCR方法检测DNA损伤后FAM64A表达水平的变化；胸腺嘧啶脱氧核苷/诺考达唑阻断法将细胞阻断在M期,并依次释放入G1、S、G2、M期,半定量RT-PCR方法检测FAM64A在不同细胞周期中的表达情况；化学合成法合成特异性的短链siRNA,脂质体转染法将siRNA分别导入HeLa细胞,RT-PCR法检测其对FAM64A表达的抑制效果；选取特异性的FAM64A siRNA,转染入HeLa细胞,采用流式细胞仪分析FAM64A表达被抑制后对HeLa细胞周期的影响.结果 FAM64A的表达水平在ADR处理后期约24 h呈现明显下调,到48 h时表达量几乎为零.FAM64A的表达水平在G2/M期表达升高,G1、S期表达降低.半定量RT-PCR结果表明,合成的特异性短链FAM64A siRNA能高效、特异性地抑制HeLa细胞中FAM64A的表达.流式细胞分析结果表明,FAM64A表达被抑制后,细胞周期被阻滞在G1期.结论 FAM64A基因参与了宫颈癌HeLa细胞周期的表达和调控,是一个潜在的肿瘤治疗靶点.%Objective To investigate the effects of FAM64A on the cell cycle regulation of cervical cancer cell line (HeLa cells) and the therapeutic potential of FAM64A against cancer.Methods ADR was used to treat HeLa cells and RT-PCR was used to check the expression changes of FAM64A after DNA damage.Thymidine/Nocodazole block method was used to block the cell cycle in M-phase,and then gradually the cell cycle came into G1,S,G2 and M phases with time going by,and RT-PCR was used to detect the expression changes of FAM64A in different cell cycle phases.Three small interfering RNAs (siRNA) were constructed and used to specifically deplete FAM64A in HeLa cells.RT-PCR was used to detect the inhibitory effect of siRNA against FAM64A.The effects of FAM64A depletion on the cell cycle of HeLa cells were determined by flow cytometry.Results FAM64A expression level was significantly decreased at 24 h after ADR treatment and almost zero at 48 h.FAM64A showed a higher expression level during G2/M phase and lower expression levels during G1 and S phases.Synthetic siRNA duplexes against FAM64A were transfected into HeLa cells,which subsequently resulted in a significant inhibition of FAM64A expression.Flow cytometric analysis showed that the expression of FAM64A was inhibited,and the cell cycle arrested in G1 phase.Conclusion The FAM64A gene is involved in the expression and regulation of cell cycle of HeLa cells,and it is also a potential target for cancer therapy.
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