RNAi技术体外沉默肌肉环指蛋白1的研究

Silencing muscle RING finger protein 1 by RNAi in vitro Z

目的探讨RNAi技术体外抑制大鼠肌肉环状指蛋白1(MuRF-1)基因表达的效果,筛选出针对MuRF-1基因最有效的siRNA重组质粒。方法根据大鼠MuRF-1基因的mRNA序列,设计4组干扰序列,即siRNA MuRF1-Ⅰ、Ⅱ、Ⅲ、Ⅳ,利用lipofactamine2000转染试剂将siRNA重组质粒转染大鼠成肌细胞系L6,于转染后48 h与72 h,采用实时定量PCR和Western blot或免疫印迹检测其对MuRF-1表达的抑制效果。MuRFl基因siRNA重组质粒瞬时转染后mRNA和蛋白质数据以x±s表示。对照组与四组实验组的比较用单因素方差分析;多个样本均数的两两比较用LSD检验。结果(1)实时定量PCR显示四组干扰质粒MuRF1-Ⅰ、Ⅱ、Ⅲ、Ⅳ对基因MuRF-1的mRNA的抑制率在转染后48 h分别为67﹪、31﹪、11﹪,20﹪,不同干扰序列的抑制效果有差异(F=4.527,P=0.024);72 h分别为79﹪、59﹪、50﹪和61﹪,不同干扰序列的抑制效果有差异(F=19.088,P<0.001),与48 h相比,抑制效应更为明显,但以siRNA MuRF1-Ⅰ的抑制效果最为明显(t=8.201,P<0.001)。(2)使用Western印迹灰度分析显示四组干扰序列对基因MuRF-1的蛋白的抑制率在转染后48 h分别为61﹪、40﹪、9﹪和15﹪,不同干扰序列的抑制效果有差异(F=4.286,P=0.028);72 h分别为70﹪、54﹪、30﹪和46﹪,不同干扰序列的抑制效果有差异(F=3.731,P=0.042);与48 h相比,MuRFl-Ⅰ、MuRFl-Ⅱ抑制效应与对照组相比有显著性差异(tⅠ=3.256,P=0.009;tⅡ=2.512,P=0.03),但MuRFl-Ⅲ和MuRFl-Ⅳ与对照组相比仍无显著性差异(P>0.05)。四对序列中,以siRNAMuRF1-Ⅰ的抑制效果最为明显。蛋白水平的抑制效果与mRNA水平基本一致。结论成功筛选出对MuRF-1基因有效的siRNA重组质粒,即siRNA MuRF1-Ⅰ,为通过RNAi技术进一步研究其功能以及基因靶向治疗奠定了基础。

Objective To investigate the efficiency of RNAi technique in inhibiting MuRF-1 gene expression in rat muscle. Methods Four siRNA recombinant plasmids were designed according to the rat MuRF-1 gene mRNA sequence （siRNA MuRF-1- Ⅰ, Ⅱ, Ⅲ and Ⅳ）. Lipofactamine 2000 transfection reagent was used to transfect the plasmids into rat myoblasts L6 and MuRF-1 expression was evaluated after 48 h and 72 h respectively, using real-time quantitative PCR, Western blot amd immunoblot. Results （1） Real-time quantitative PCR showed plasmids MuRF-1- Ⅰ, Ⅱ,Ⅲ, and Ⅳ inhibited MuRF-1 mRNA by 67%, 31%, 11%,and 20 % respectively after 48 h, the inhibitory effects of the four plasimds were significant different （F = 4.527, P = 0.024）; At 72 h, the inhibition rates were 79%, 59%, 50%and 61% for the 4 plasimds and difference was significant among the 4 plasimds （F = 19.088, P 〈 0.001） Compared with 48h, the inhibitory effect was greater at 72 h, especially for siRNA MuRF-1- Ⅰ （t = 8.201, P 〈 0.001 ）. （2） Western blot analysis revealed the protein inhibition rates after transfection for 48h were 61%, 40%, 9% and 15% respectively The inhibitory effect of different plasmids was significantly different （F = 4.286, P = 0.028）. At 72h, the inhibition rates were 70 %, 54 %, 30 %, and 46 %, respectively （F = 3.731, P = 0.042）. At 72 h, the inhibition was greater for MuRF1 - Ⅰ and MuRF1 - Ⅱ compared to 48 h （tⅠ= 3.256, P = 0.009; tⅡ= 2.512, P = 0.03）. MuRF1-Ⅲ and MuRF1-Ⅳ did not inhibit the gene expression as compared with the control group （ P 〉 0.05 ）. Among the four plasmids, the inhibitory effect of siRNA MuRF-1- Ⅰ was greatest. The inhibitory effect on mRNA and protein levels were similar. Conclusion siRNA MuRF- 1- Ⅰ may be used for further study of gene function and gene therapy.