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目的筛选高效dsRNA/mdr1,以备研究RNA干扰逆转肝细胞癌多药耐药之用。方法首先根据siRNA设计原则,以多药耐药基因(mdr1)为靶基因,设计并选择4~5条siRNA/mdr1,经BLAST后体外转录法合成dsRNA/mdr1,用Oligofectamine试剂分别转染HepG2/mdr1,然后从mRNA、蛋白(P-gp)表达水平和细胞功能变化评价HepG2/mdr1耐药性被逆转的程度,比较各个dsRNA/mdr1的逆转效率,筛选出有效的siRNA/mdr1。结果成功合成5条dsRNA/mdr1(其中1条为阴性对照),dsRNA/mdr1-4mRNA表达(18.73±1.33)%、蛋白表达变化(79.1±1.6)%~(16.8±0.4)%与其他各组细胞比较,有显著性差异;细胞内柔红霉素(DNR)累积量也较其他组明显增加(平均荧光强度79.58,阳性率84.25%,P<0.05)。结论体外转录法结合脂质体转染适用于筛选高效siRNA,肯定了siRNA干扰序列能够有效阻抑mdr1基因编码蛋白p170的功能。
Objective To screen highly efficient dsRNA / mdr1 in order to study the effect of RNA interference on reversing the multidrug resistance of hepatocellular carcinoma. Methods According to the principle of siRNA design, 4 to 5 siRNA / mdr1 were designed and selected based on the multidrug resistance gene (mdr1). The dsRNA / mdr1 was synthesized by BLAST in vitro and HepG2 / mdr1, and then evaluated the extent of reversal of HepG2 / mdr1 resistance by the expression of mRNA and protein (P-gp) and the change of cell function. Then, the efficiency of reversal of dsRNA / mdr1 was compared and the effective siRNA / mdr1 was screened out. Results Five dsRNA / mdr1 (1 negative control) was successfully synthesized. The expression of dsRNA / mdr1-4 mRNA was (18.73 ± 1.33)% and the protein expression was (79.1 ± 1.6)% ~ (16.8 ± 0.4)% Compared with other groups, the intracellular accumulation of daunorubicin (DNR) was significantly increased (mean fluorescence intensity 79.58, positive rate 84.25%, P <0.05). Conclusion In vitro transcription combined with liposome transfection is suitable for the screening of high efficient siRNA, confirming that siRNA interference sequence can effectively suppress the function of mdr1 gene encoding protein p170.