Screening Of ShRNA Targeting TGF-β1and Inhibiting Effects Of Silencing TGF-β1Expression On The Renal Tubular Epithelial To Mesenchymal Transition

Background Renal tubular epithelial to mesenchymal transition plays important role in renal interstitial fibrosis. TGF-β1is recognized as the important regulatory factor in renal tubular epithelial cell transdifferentiation. RNA interference triggers the degradation of homologous mRNA, suppressing the expression of the target gene. Therefore, it is important for preventing and curing tubulointerstitial fibrosis to screen of shRNA targeting the silencing of TGF-β1.Objectives To screen shRNA silencing TGF-β1expression, evaluate the inhibiting effects of silencing TGF-β1expression on the renal tubular epithelial HKC epithelial to mesenchymal transition. Our work hope to lay the foundation of studying the downstream molecules participating in renal tubulointerstitial fibrosis induced by TGF-β1, so as to provide a scientific basis for the feasibility of preventing and curing tubulointerstitial fibrosis by silencing the expression of TGF-β1via RNA interference.Methods1. According to the general design principles of siRNA, the siRNA was designed targeting TGF-β1mRNA covering the cDNA full-length of TGF-β1gene through manually and siDirect software. All of the siRNA targeting TGF-β1mRNA were homology analyzed, and screen5pairs of siRNA. Based on the above mentioned5pairs of siRNA,5pairs of shRNA targeting TGF-β1mRNA were obtained.2. The eukaryotic expression recombinant plasmid (p-Genesil-shRNAl, p-Genesil-shRNA2, p-Genesil-shRNA3, p-Genesil-shRNA4and p-Genesil-shRNA5) targeting TGF-β1and unrelated shRNA (p-Genesil-1-shRNA-vect) were constructed by gene recombination technology. The plasmids were identified by enzyme digestion and gene sequencing respectively.3. Taken the renal tubular epithelial HKC cells,the above mentioned eukaryotic expression recombinant plasmids were transfected into HKC cells which have activated by Angiotensin II or high glucose via liposomes, then named p-Genesil-1-vect cells, p-Genesil-shRNAl cells, p-Genesil-shRNA2cells, p-Genesil-shRNA3cells, p-Genesil-shRNA4cells and p-Genesil-shRNA5cells. The shRNA silencing TGF-β1expression was screened through detecting the changes of expression of TGF-β1by western blotting. Compared with p-Genesil-1-vect cells, detecting the changes of expression of a-SMA、type I collagen、keratin and vimentin in p-Genesil-shRNA4cells, to observe the inhibiting effects of silencing TGF-β1expression on the renal tubular epithelial to mesenchymal transition.Results1. Construction and identification of the eukaryotic expression recombinant plasmid.The results of enzyme digestion showed that the length of digestion fragments were consistent with expected lenth respectively. The results of gene sequencing showed that, compared with designed sequences, gene coding sequences of shRNA were exactly consistent, no mutation, no insertion, no loss and the directions were correct.2. Screen the shRNA silencing TGF-β1expression.The results of Western blotting showed that:compared with HKC cells, the expression of TGF-β1in HKC cells stimulated by high glucose or Ang Ⅱ and p-Genesil-1-vect cells was significantly increased (P<0.05or0.01). However, the expression of TGF-β1in HKC cells stimulated by high glucose or Ang Ⅱ and p-Genesil-1-vect cells was no significant difference (P>0.05). Compared with p-Genesil-1-vect cells stimulated by high glucose or Ang II, the expression of TGF-β1in p-Genesil-shRNA cells were significantly depressed (P<0.05or0.01). The expression of TGF-β1in p-Genesil-shRNA4cells was reduced significantly. 3. Inhibiting effects of silencing TGF-β1expression on the renal tubular epithelial to mesenchymal transition.The results of Western blotting showed that:compared with p-Genesil-1-vect cells, the expression of a-SMA、type I Collagen and Vimentin in p-Genesil-shRNA4cells were significantly depressed (P<0.01). However, the expression of Keratin was significantly increased (P<0.01).Conclusions1. The one pair of shRNA which have silenced TGF-β1expression and have not been reported up to now was screened.2. The silencing of TGF-β1expression can inhibit the renal tubular epithelial HKC epithelial to mesenchymal transition.