利用RNA干涉技术研究Smad2和Smad3在鼠成纤维细胞TGF-β信号通路中的不同作用
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摘要
RNA干扰(RNA interference,RNAi)是由双链RNA(double stranded RNA,dsRNA)介导的一种特异性基因表达沉默,是一种古老但在进化上又高度保守的基因表达调节机制。关于RNAi的基础研究和应用研究迅速成为21世纪初生命科学的热点之一,在人、小鼠及其他哺乳动物中,RNAi已广泛应用于基因功能验证、信号转导和基因治疗等领域的研究。由于哺乳动物缺乏体内siRNA介导的沉默放大效应,利用DNA载体在细胞内稳定转录出siRNAs,并维持长时间抑制效果的方法已被证明是行之有效的。转化生长因子β(TGF-β,transforming growth factors beta),在细胞生长、发育、分化和物质代谢等方面均有极其重要的作用,而Smads蛋白是目前所知的唯一的TGF-β受体胞内激酶底物,在TGF-β信号转导中起着关键的作用。本研究通过构建RNAi的DNA表达载体,特异地、有效地抑制TGF-β/Smads信号通路中通路特异性Smads—Smad2和Smad3基因在NIH/3T3小鼠成纤维细胞中的表达,分别在mRNA水平和蛋白质水平研究Smad2和Smad3的不同作用和相互关系及在TGF-β信号转导过程中所处的地位。具体研究结果如下:
1.确定了外源TGF-β1诱导NIH/3T3细胞中Smads蛋白表达的最佳浓度和时间。通过设置添加TGF-β1处理的不同浓度梯度和时间梯度,利用半定量RT-PCR和Western-blottting分析方法确定诱导Smads蛋白达到最高表达量的浓度和时间分别为5μg/L和3小时,并发现Smad3的表达对TGF-β1的刺激尤为敏感,从0.5小时起一直持续高效表达。
2.构建了靶基因的shRNA DNA表达载体。按照siRNA的设计规则,针对Smad2和Smad3基因分别设计了5条21bp的siRNA序列,合成了10条62-64bp的双链DNA寡核苷酸序列,并将它们分别插入到RNAi-Ready pSIREN-RetroQ-ZsGreen质粒载体中,获得了包括阳性和阴性对照的12个shRNA的DNA表达质粒,并经酶切和测序验证。
3.建立了一套比较有效的RNAi的技术体系。将所获得12个RNAi表达质粒利用脂质体介导的方法分别转染NIH/3T3小鼠成纤维细胞,提取转染细胞的总RNA和总蛋白质,采用半定量RT-PCR和Western-blotting方法分析了siRNA抑制效果,获得了两个特异地、有效地抑制Smad2和Smad3表达的RNAi表达质粒。分析这两个有效的siRNA序列发现,正义链第10位的碱基在设计中具有重要的作用,当第10位为U时,抑制效率最高,其次是C,最后是A/G。
4.分析了Smad2和Smad3在TGF-β信号转导过程中的不同作用。在mRNA水平和蛋白质水平分别检测了Smad2-depleption细胞、Smad3-depleption细胞和Smad2+3-deoleption细胞中Smad2、Smad3和Smad4的表达,发现在Smad2-depleption细胞中Smad3和Smad4的表达提高,而在Smad3-depleption细胞中Smad2的表达没有发生改变,Smad4的表达降低。表明TGF-β信号在NIH/3T3细胞转导过程中,Smad3起到关键的调节作用。
5.建立了混合RNAi表达质粒有效地抑制靶基因表达的方法。分别针对于Smad2和Smad3 mRNA序列不同区段的4个shRNA表达质粒等比例混合,构成Smad2-RNAi池和Smad3-RNAi池,利用脂质体将RNAi混合质粒共同导入NIH/3T3细胞中,经半定量RT-PCR和Western-blotting方法分析,发现RNAi池抑制Smad2或Smad3表达的效果要明显高于单一的RNAi表达质粒。表明针对靶基因mRNA序列不同区段的混合RNAi表达质粒可以提高RNAi的抑制效果,为有效利用RNAi表达载体研究基因的功能提供了一个新的策略。
RNA interference(RNAi) is an old and evolutionarily conserved regulative mechanism of sequence-specific,post-transcriptional gene silencing by the introduction of double stranded RNA(dsRNA) homologous in sequence to the silenced gene to trigger the degradation of homologous mRNA.The fundamental and applied research about RNAi has quickly become the hotspot of life science at beginning of the 21 century.To the human,mouse and the other mammals,RNAi had been wildly used in the field of gene function,signal transduction and gene therapy.Due to the lack of silencing amplification mechanisms in mammalian cells,siRNA-expression vector,which works as a platform to produce a large amount of siRNA for a relatively long period,is a versatile method of application of RNAi.Transforming growth factors beta(TGF-β),plays a key role in cell growth,differentiation,proliferation and substance metabolism.Smads protein, the only substrates of TβR(TGF-βreceptor) kinase,are critical mediators of TGF-βsignaling transducer.In this study,we had constructed the DNA expression vectors of RNAi.The endogenous expression of Smad2 and Smad3 were specifically and effectively suppressed by Smad2- and Smad3 siRNA expression plasmids in NIH/3T3 fibroblast cells. We also investigated the different role of Smad2 and Smad3 and the relationship among Smads in TGF-βsignaling.The main results are as follows:
1.The best concentration and time of Smads expression by TGFβ-induction in NIH/3T3 fibroblast cells were confirmed.The cells were treated with different concentration of TGF-β1 for 4h and cultured for various length of time with 5μg/L TGF-β1-treatment,respectively.At the best concentration(5μg/L) and length of time(3 hours) with TGF-β1-treatment,the expressions of Smads achieve the maxim by RT-PCR and Western-blotting.We also found that the Smad3 would be more sensitive to TGF--, because the Smad3 had been at the high expression status from 0.5h.
2.The shRNA expression plasmids of targeted genes were constructed.We designed 5 shRNAs against Smad2 and Smad3 mRNA on the base of siRNA rules,respectively. Individual dsDNA oligonucleotide encoding an appropriate shRNA was inserted into a pSIREN-RetroQ-ZsGreen vector,in which the interfering RNAs were driven by the human U6 promoter followed by CMV IE promoter-driven ZsGreen.We obtained 12 recombinant plasmids,including negative control and positive control.These plasmids were confirmed by enzymic digestion and sequencing.
3.The effectively technology about RNAi was established.The 12 recombinant plasmids were transfected into NIH/3T3 fibroblast cells by Lipofectamin~(TM) 2000 Reagent. The total RNA and protein of transfected cells were isolated.By analysis with RT-PCR and Western-blotting,we obtained two effective shRNA expression plasmids,which could specifically and effectively inhibit the expression of Samd2 and Smad3.We found the position 10 was very important to designing by analysis the sequences of the two functional siRNA.We believed that position 10 of the sense strand with U rather than A, G or to a lesser extent,C for satisfactory inhibition of gene expression.
4.The different role of Smad2 and Smad3 in TGF-βsignal transducer was discussed. We detected the expression of Smad2,Smad3 and Smad4 in Smad2-depletion cells, Smad3-depletion cells and Smad2+3-depletion cells at the level of mRNA and protein. We observed that the expression of Smad3 and Smad4 were increased in Smad2-depletion cells,but,the expression of Smad2 wasn't changed and the mRNA expression of Smad4 was decreased in Smad3-depletion cells.We suggested that the Smad3 could be the critical mediator in TGF-βsignal pathway.
5.The method of mixture RNAi expression plasmids for effectively suppression expression of targeted gene was established.Four shRNA-expression plasmids,which homologize in sequence to the different position of the targeted gene,were mixed according to the same concentration and made up of the pool of Smad2-RNAi or Smad3-RNAi.The pool of Smad2-RNAi or Smad3-RNAi was trandfected into NIH/3T3 fibroblast cells by Lipofectamin~(TM) 2000 Reagent.By RT-PCR and Western-blotting,we found the inhibition the expression of Smad2 or Smad3 with the pool RNAi would be better than that of the one shRNA-expression plasmid.This result indicated that the mixture of RNAi expression plasmid,which act on the different region of the targeted mRNA,could increase the inhibitory ability of siRNA.This method provide for studying gene function with the technology of RNAi a new strategy.
1.确定了外源TGF-β1诱导NIH/3T3细胞中Smads蛋白表达的最佳浓度和时间。通过设置添加TGF-β1处理的不同浓度梯度和时间梯度,利用半定量RT-PCR和Western-blottting分析方法确定诱导Smads蛋白达到最高表达量的浓度和时间分别为5μg/L和3小时,并发现Smad3的表达对TGF-β1的刺激尤为敏感,从0.5小时起一直持续高效表达。
2.构建了靶基因的shRNA DNA表达载体。按照siRNA的设计规则,针对Smad2和Smad3基因分别设计了5条21bp的siRNA序列,合成了10条62-64bp的双链DNA寡核苷酸序列,并将它们分别插入到RNAi-Ready pSIREN-RetroQ-ZsGreen质粒载体中,获得了包括阳性和阴性对照的12个shRNA的DNA表达质粒,并经酶切和测序验证。
3.建立了一套比较有效的RNAi的技术体系。将所获得12个RNAi表达质粒利用脂质体介导的方法分别转染NIH/3T3小鼠成纤维细胞,提取转染细胞的总RNA和总蛋白质,采用半定量RT-PCR和Western-blotting方法分析了siRNA抑制效果,获得了两个特异地、有效地抑制Smad2和Smad3表达的RNAi表达质粒。分析这两个有效的siRNA序列发现,正义链第10位的碱基在设计中具有重要的作用,当第10位为U时,抑制效率最高,其次是C,最后是A/G。
4.分析了Smad2和Smad3在TGF-β信号转导过程中的不同作用。在mRNA水平和蛋白质水平分别检测了Smad2-depleption细胞、Smad3-depleption细胞和Smad2+3-deoleption细胞中Smad2、Smad3和Smad4的表达,发现在Smad2-depleption细胞中Smad3和Smad4的表达提高,而在Smad3-depleption细胞中Smad2的表达没有发生改变,Smad4的表达降低。表明TGF-β信号在NIH/3T3细胞转导过程中,Smad3起到关键的调节作用。
5.建立了混合RNAi表达质粒有效地抑制靶基因表达的方法。分别针对于Smad2和Smad3 mRNA序列不同区段的4个shRNA表达质粒等比例混合,构成Smad2-RNAi池和Smad3-RNAi池,利用脂质体将RNAi混合质粒共同导入NIH/3T3细胞中,经半定量RT-PCR和Western-blotting方法分析,发现RNAi池抑制Smad2或Smad3表达的效果要明显高于单一的RNAi表达质粒。表明针对靶基因mRNA序列不同区段的混合RNAi表达质粒可以提高RNAi的抑制效果,为有效利用RNAi表达载体研究基因的功能提供了一个新的策略。
RNA interference(RNAi) is an old and evolutionarily conserved regulative mechanism of sequence-specific,post-transcriptional gene silencing by the introduction of double stranded RNA(dsRNA) homologous in sequence to the silenced gene to trigger the degradation of homologous mRNA.The fundamental and applied research about RNAi has quickly become the hotspot of life science at beginning of the 21 century.To the human,mouse and the other mammals,RNAi had been wildly used in the field of gene function,signal transduction and gene therapy.Due to the lack of silencing amplification mechanisms in mammalian cells,siRNA-expression vector,which works as a platform to produce a large amount of siRNA for a relatively long period,is a versatile method of application of RNAi.Transforming growth factors beta(TGF-β),plays a key role in cell growth,differentiation,proliferation and substance metabolism.Smads protein, the only substrates of TβR(TGF-βreceptor) kinase,are critical mediators of TGF-βsignaling transducer.In this study,we had constructed the DNA expression vectors of RNAi.The endogenous expression of Smad2 and Smad3 were specifically and effectively suppressed by Smad2- and Smad3 siRNA expression plasmids in NIH/3T3 fibroblast cells. We also investigated the different role of Smad2 and Smad3 and the relationship among Smads in TGF-βsignaling.The main results are as follows:
1.The best concentration and time of Smads expression by TGFβ-induction in NIH/3T3 fibroblast cells were confirmed.The cells were treated with different concentration of TGF-β1 for 4h and cultured for various length of time with 5μg/L TGF-β1-treatment,respectively.At the best concentration(5μg/L) and length of time(3 hours) with TGF-β1-treatment,the expressions of Smads achieve the maxim by RT-PCR and Western-blotting.We also found that the Smad3 would be more sensitive to TGF--, because the Smad3 had been at the high expression status from 0.5h.
2.The shRNA expression plasmids of targeted genes were constructed.We designed 5 shRNAs against Smad2 and Smad3 mRNA on the base of siRNA rules,respectively. Individual dsDNA oligonucleotide encoding an appropriate shRNA was inserted into a pSIREN-RetroQ-ZsGreen vector,in which the interfering RNAs were driven by the human U6 promoter followed by CMV IE promoter-driven ZsGreen.We obtained 12 recombinant plasmids,including negative control and positive control.These plasmids were confirmed by enzymic digestion and sequencing.
3.The effectively technology about RNAi was established.The 12 recombinant plasmids were transfected into NIH/3T3 fibroblast cells by Lipofectamin~(TM) 2000 Reagent. The total RNA and protein of transfected cells were isolated.By analysis with RT-PCR and Western-blotting,we obtained two effective shRNA expression plasmids,which could specifically and effectively inhibit the expression of Samd2 and Smad3.We found the position 10 was very important to designing by analysis the sequences of the two functional siRNA.We believed that position 10 of the sense strand with U rather than A, G or to a lesser extent,C for satisfactory inhibition of gene expression.
4.The different role of Smad2 and Smad3 in TGF-βsignal transducer was discussed. We detected the expression of Smad2,Smad3 and Smad4 in Smad2-depletion cells, Smad3-depletion cells and Smad2+3-depletion cells at the level of mRNA and protein. We observed that the expression of Smad3 and Smad4 were increased in Smad2-depletion cells,but,the expression of Smad2 wasn't changed and the mRNA expression of Smad4 was decreased in Smad3-depletion cells.We suggested that the Smad3 could be the critical mediator in TGF-βsignal pathway.
5.The method of mixture RNAi expression plasmids for effectively suppression expression of targeted gene was established.Four shRNA-expression plasmids,which homologize in sequence to the different position of the targeted gene,were mixed according to the same concentration and made up of the pool of Smad2-RNAi or Smad3-RNAi.The pool of Smad2-RNAi or Smad3-RNAi was trandfected into NIH/3T3 fibroblast cells by Lipofectamin~(TM) 2000 Reagent.By RT-PCR and Western-blotting,we found the inhibition the expression of Smad2 or Smad3 with the pool RNAi would be better than that of the one shRNA-expression plasmid.This result indicated that the mixture of RNAi expression plasmid,which act on the different region of the targeted mRNA,could increase the inhibitory ability of siRNA.This method provide for studying gene function with the technology of RNAi a new strategy.
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