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目的:探讨转录因子E2F哑铃形诱骗寡核苷酸(Decoy ODNs)对MKN-45细胞中op18基因转录调控的影响及对细胞增殖的抑制作用.方法:设计合成针对op18启动子上E2F的结合位点序列的哑铃形Decoy ODNs,然后将合成的序列进行退火、连接,使其形成哑铃形的结构,再应用阳离子脂质体LipofectamineTM2000将哑铃形Decoy ODNs转染MKN-45细胞中.TRIzol法提取细胞总RNA,用RT-PCR方法检测细胞中op18 mRNA表达水平的变化.通过MTT实验监测细胞的生长增殖状况并绘制生长曲线,最后用TUNEL法观察细胞的凋亡的情况.结果:哑铃形Decoy ODNs被成功转染MKN-45细胞,并成功提取了细胞总RNA.RT-PCR检测发现哑铃形Decoy ODNs转染后的MKN-45细胞中op18 mRNA表达水平明显低于空白对照组,MTT实验所作的生长曲线显示转染细胞增殖速度与空白对照组相比较明显减慢,TUNEL凋亡染色可见凋亡细胞.结论:哑铃形Decoy ODNs能特异性抑制E2F转录因子对op18基因的转录调控,进而抑制op18基因表达及MKN-45细胞增殖.
Objective: To investigate the effect of transcription factor E2F Decoy ODNs on the transcriptional regulation of op18 gene in MKN-45 cells and its inhibitory effect on cell proliferation. METHODS: Design and synthesis of binding sites for E2F on op18 promoter. Dot-shaped dumbbell-shaped Decoy ODNs were spotted, and then the synthesized sequences were annealed and connected to form a dumbbell-shaped structure. The dumbbell-shaped Decoy ODNs were then transfected into MKN-45 cells using cationic liposome LipofectamineTM 2000. TRIzol extraction was performed on the cells. Total RNA was detected by RT-PCR method to detect the change of the expression level of op18 mRNA in the cells. MTT assay was used to monitor the growth and proliferation of cells and draw the growth curve. Finally, the cell apoptosis was observed by TUNEL method. Results: Dumbbell Decoy ODNs MKN-45 cells were successfully transfected and total cellular RNA was successfully extracted. RT-PCR showed that the expression of op18 mRNA in MKN-45 cells transfected with Dumbbell Decoy ODNs was significantly lower than that in the blank control group. The growth curve showed that the proliferation rate of transfected cells was significantly slower compared with the blank control group, apoptotic cells were seen by TUNEL apoptosis staining. Conclusion: Dumbbell Decoy ODNs can specifically inhibit E2F transcription Op18 transcriptional regulation of the gene, thereby inhibiting expression and MKN-45 cell proliferation op18.