小鼠胚胎诱导型Hsp70基因的RNA干扰有效序列筛选

Ascertainment of Effective RNAi Sequences of Mouse Embryonic Induced Heat Shock Protein70

【目的】应用RNA干扰技术沉默小鼠胚胎成纤维细胞(Mouse embryonic fibroblast,MEF)中诱导型热休克蛋白70(Hsp72)基因的表达,从而筛选出对Hsp72基因具有确实干扰作用的siRNA。【方法】设计并合成4对特异性针对Hsp72基因的siRNA,借助LipofectamineTM2000瞬时转染MEF,41℃热应激处理后,采用RT-PCR和Western blot检测Hsp70 mRNA和Hsp70蛋白表达量。【结果】在热应激组中,siRNA1、siRNA2两组及阳性、阴性和空白3个对照组的Hsp70 mRNA和Hsp70蛋白表达量均极显著高于常温对照组(P<0.01),siRNA3和siRNA4两组的Hsp70 mRNA的表达量极显著低于常温对照组(P<0.01);siRNA3组Hsp70蛋白表达量极显著低于常温对照组(P<0.01),而siRNA4组与常温对照组差异不显著(P>0.05)。与空白对照相比,4对siRNA中siRNA3和siRNA4对MEF中Hsp72 mRNA有极显著(P<0.01)的抑制作用,其对Hsp72 mRNA的抑制率分别为86.2%和75.4%(P<0.01),对Hsp72蛋白表达的抑制率分别为77.3%和57.0%(P<0.01)。【结论】体外合成的四对特异性针对Hsp72基因的siRNA中,靶位点在Hsp72 mRNA二级结构表面位置的siRNA3和siRNA4对Hsp72基因具有显著的抑制作用,靶位点在Hsp72 mRNA二级结构复杂处的siRNA1和siRNA2对Hsp72基因的抑制作用不明显。

[Objective] The RNAi method was used to inhibit the expression of induced Hsp72 in the mouse embryonic fibroblast （MEF） to ascertain an effective siRNA of Hsp72. [Method] Four pairs of siRNA specific for Hsp72 gene were designed for RNAi and were transferred into MEF through the help of Lipofectamine^TM 2000. The expression of Hsp72 mRNA and Hsp72 proteins was detected by RT-PCR and Western blot after heat shock of 41℃. [Result] In the heat shock groups, the expression of Hsp70 mRNA and Hsp70 protein in the siRNA1, siRNA2 and the three control groups was significantly higher （P〈0.01） than that in the normal temperature group, and the expression of Hsp70 mRNA in siRNA3 and siRNA4 was significantly lower （P〈0.01） than that in the normal temperature group. The expression of Hsp70 protein in siRNA3 was significantly lower （P〈0.01） than that in the normal temperature group. While, there was no significant difference （P〉0.05） between siRNA4 and the normal temperature group. The siRNA3 and siRNA4 had a significant （P〈0.01） inhibiting effect on expression of Hsp70 mRNA in MEF compared with the blank control after heat shock. The inhibition rates were 86.2% and 75.4% （P〈0.01） for siRNA3 and siRNA4, respectively, the inhibition rates of siRNA3 and siRNA4 on Hsp72 protein expression were 77.3% and 57.0%, respectively （P〈0.01）. [Conclusion] In the four siRNAs designed specific for Hsp72 gene, siRNA3 and siRNA4 target sites located on the surface of Hsp72 mRNA secondary structure can significantly reduce the expression of Hsp72 gene, but siRNA1 and siRNA2 target sites located on the interior of Hsp72 mRNA secondary structure had an insignificant inhibitory action on Hsp72 gene.