Utilization RNA Interference To Identify The Differential Roles Of Smad2 And Smad3 In TGF-β Signal Pathway Of Mouse Fibroblast Cells

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.