诱骗寡核苷酸下调MKN-45细胞OP18基因表达及对细胞增殖的影响

Inhibitory effects of E2F decoy ODNs on OP18 gene expression and proliferation in gastric cancer MKN-45 cells

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.

AIM： To investigate the blocking effects of E2F decoy oligodeoxynucleotides （ODNs） in dumbbell shape on the op18 gene transcription and the gastric cancer MKN-45 cell proliferation. METHODS： E2F decoy ODNs were designed, chemically synthesized, annealed and ligated to form dumbbell shape, and then transfected into gastric cancer cell line MKN-45 using lipofectamine^TM 2000 reagent according to the manufacturer＇s instructions. Total RNA was extracted by using TRIzol. The level of op18 mRNA in the transfected tumor cells was detected by RTPCR, The proliferation of the transfected tumor cells was analyzed by MTT assay and the apoptosis of the tumor cells was detected by TUNEL staining. RESULTS： E2F decoy ODNs could be successfully transfected into tumor cells, and RTPCR results indicated that E2F decoy ODNs decreased the expression of op18 gene in the transfected tumor cells. The proliferation of tumor cells was inhibited markedly by E2F decoy ODNs and morphological analysis showed positive staining identified by TUNEL assay. CONCLUSION： E2F decoy ODNs in dumbbell shape may specifically decrease the activity of transcription factor E2F and down-regulate op18 gene expression and inhibit MKN-45 cell proliferation, which demonstrates a potential strategy for cancer gene therapy.