人工合成小干扰RNA抑制涎腺腺样囊性癌DNA甲基转移酶1的表达

Synthesized small interfering RNA inhibits the expression of DNA methyltransferase 1 in salivary adenoid cystic carcinoma

目的通过筛选针对涎腺腺样囊性癌(adenoid cystic carcinoma,ACC)DNA 甲基转移酶1(DNA methyltransferase 1,DNMT-1)基因有效的小于扰 RNA(small interfering RNA,siRNA)序列和分析 RNA 干扰(RNA interference,RNAi)作用的时效、量效关系,为建立 ACC 稳定抑制 DNMT-1的表达载体,深入了解 DNMT 在 ACC 抑癌基因甲基化过程中的作用。方法设计并人工合成4对siRNA,脂质体介导分别转染 ACC 细胞系 ACC-2、ACC-3和 ACC-M 细胞,分别于转染后24、48和72 h,采用 PCR、RT-PCR 和 Western blot 方法检测各细胞系 DNMT-1的 mRNA 和蛋白表达,筛选有效的siRNA 干扰序列,同时设置 siRNA 不同浓度转染组,分析 RNAi 作用的量效关系。以 Cy3标记甘油醛-3-磷酸脱氢酶 siRNA 作为阳性对照组。结果 2对 siRNA 对3株 ACC 细胞系细胞 DNMT-1的 mRNA表达产生了抑制作用,它们分别使 ACC-2、ACC-3、ACC-M DNMT-1表达抑制67%、86%、92%和76%、76%、86%,抑制作用出现的时间为转染后24～48 h,维持24～48 h,与 siRNA 浓度成正比,Westernblot 检测符合 mRNA 的检测结果。结论得到了2对针对 ACC DNMT-1基因有效的 siRNA 干扰序列。它们能显著抑制涎腺 ACC 细胞系 DNMT-1的 mRNA 和蛋白表达,该抑制作用具有时间依赖性和浓度相关性。

Objective To select the effective siRNA which could inhibit the expression of DNA methyltransferase 1 （DNMT-1） in adenoid cystic carcinoma （ACC） and discuss the time-, and dosedependant effect of RNA interference（RNAi）. Methods Four pairs of siRNA were designed, synthesized and transfected through oligofectamine reagent into ACC cell lines ACC-2, ACC-3 and ACC-M. 24, 48 and 72 h after transfection, total RNA and protein were harvested respectively, mRNA and protein expression level of DNMT-1 were detected by real time PCR, RT-PCR and Western blot, and then the effective siRNA was subsequently selected. ACC-3 as also transfected by different concentration of siRNA and the dosedependant effect of RNAi was discussed. Cy3 labelled GAPDH siRNA was used as a positive control. Results Two of 4 pairs of siRNA inhibited the mRNA expression of DNMT-1 in three ACC cell lines and the expression of DNMT-1 was downregulated by 67%, 86%, 92% and 76%, 76% , 86% respectively. The gene inhibition was detected at 24 h or 48 h after transfection, maintained only 1-2 days and showed direct relationship to the concentration of siRNA. The change of protein expression level of DNMT-1 was consistent to the changes of mRNA. Conclusions The effective siRNA which could inhibited the expression of DNMT-1 of ACC were achieved. The inhibition effect of RNAi was time-dependant and dose-dependent.