Effects of siRNA Silencing TGF-β1 on the Expression of Fibronectin and Type Ⅳ Collagen in the Rat Masengial Cell
|School||Central South University|
|Keywords||shRNA TGF-β1 Mesangial cells SD rats Fibronectin Type IV collagen pEGFP6 plasmid Build Four type I collagen|
Objective: to silence the expression of TGF-β1 by constructing the pEGFP6 vectors expressing shRNA for its mRNA of SD rat and investigate the inhibited expression of fibronectin and typeⅣcollagen so as to search for a new pathway to treat renal fibrosis.Methods: to construct the pGEGP6 vectors expressing shRNAs for two sequences, that is, 538-556 and 895-913, of SD rat TGF-β1 mRNA, transfect the mesangial cells of SD rat and investigate the dynamic expression of TGF-β1, fibronectin （by RT-PCR for mRNA, ELISA for the protein） and typeⅣcollagen in the total cellular protein by ELISA.Results: the DNA sequencing of three constructed pEGFP6 vectors showed that one or both of two sequences of SD rat TGF-β1 mRNA was or were successfully inserted in the relative pEGFP6 vector, respectively. After transfection, only one sequence for TGF-β1 mRNA 538-556 possessed the marked interfering effects on TGF-β1 expression at the mRNA level lasting for 48hrs （RT-PCR results）, at the protein for 48hrs in the supernatant （ELISA results）, or 72hrs in the total cellular protein（Western Blot results）, accordingly, effectively delaying peak concentration of fibronectin stimulated by Lipoinfectamin2000TM up to 48hrs in either mRNA by RT-PCR or the supernatant protein by ELISA, and obviously decreasing the expression of typeⅣcollagen in the total cellular protein by ELISA at 24hrs and 48hrs.Conclusion: the constructed pEGFP6 vectors expressing the shRNA for TGF-β1 mRNA 538-556 sequence of SD rat strongly silenced the expression of TGF-β1, accompanied by down-regulation of fibronectin and typeⅣcollagen. It is possible for them to resist against renal fibrosis. Objective: to construct the pEGFP6 vector expressing small hairpin （shRNA） for the mRNA of TGF-β1 in SD rat in order to silence the expression of TGF-β1 mRNA in the mesangial cellof SD rat after transfection.Methods: Depending upon two specific sequences, that is, 538-556 and 895-913, of TGF-β1 mRNA for SD rat, two interfering sequences were designed in each with 19 nucleotides（nt）. The sense and antisense chain （or vice verse） for each interfering sequence was artificially linked by a loop chain into forming the sense chain （or anti-sense chain） of a small hairpin cDNA with the terminal encodes and relative multiple clone sites （MCS） for the tool enzymes. Then the sense chain and anti-sense chain of the small hairpin cDNA were mixed, thawed at 94℃and annealed at the room temperature into the small hairpin cDNA, which was inserted between BamH I and EcoR I of pEGFP6 Vector U6 by using T4 DNA linkase. The reconstructed pEGFP6 vectors was amplified by transfecting the Bacillus DH5αfor screening and selecting the resistant strain to kanamycin and extracting the plasmids which were identified by the endonuclease digestion for gel electrophoresis and DNA sequencing. Two constructed plasmids identified with the relative interfering sequence by DNA sequencing were further digested by the endonuclease SalI and then Pst, respectively, for two fragments containing two small hairpin cDNAs consisting of the sense and anti-sense chains of two interfering sequences. Then two fragments were linked by T4 DNA linkase to make up a piasmid expressing both of the irterfering sequences. Finally, they were amplified and identified by means of the methods similar to the above.Results: By the endonuclease digestion for gel electrophoresis and DNA sequencing, it was identified that the reconstructed pEGFP6 vectors were inserted with the right one or both of two interfering sequences Conclusion: Two plasmids expressing one of two interfering sequences and one plasmid expressing both of them were successfully recenstructed。 Objective: To transfect the mesangial cellof SD rat by the well constructed pEGFP6 vector expressing shRNA for TGF-β1 mRNA and dynamically investigate changes of TGF-β1.Methods: The mesangial cells was transfected by the TGF-β1-expressing pEGFP6 vector carried by LipofectamineTM 2000 and investigated the expression of TGF-β1 for mRNA （by RT-PCR） and protein in the supernatant （by ELISA） in the periods of 24hrs, 48hrs and 72hrs, respectively, and in the total cellular protein of mesangial cells by Western Blot at 72hrs. By the statistics software SPSS10.0 analysis, the most markedly interfering sequence and periods were found.Results: the shRNA for the SD rat TGF-β1 mRNA sequence （from 538 to 556） obviously silenced the expression of TGF-β1 in the mesangial cellfor mRNA by RT-PCR and protein in the supernatant by ELISA until 48hrs and by western blot even until 72hrs. In contrast, the shRNA for the sequence from 895 and 913 did not show marked specific interfering effect.Conclusion: the shRNA for SD rat TGF-β1 mRNA sequence from 538 to 556 could specifically silence the expression of TGF-β1 mRNA and protein in the mesanigal cellof SD rat. Objective: To investigate the dynamic expression of fibronectin and typeⅣcollagen （Col-Ⅳ） after the expression of TGF-β1 was silenced.Methods: To compare the expression of fibronectin in the supernatant and Col-Ⅳin the total cellular protein by ELISA and RT-PCR for the fibronectin mRNA semiquantitative in the mesangial cells, whose TGF-β1 was markedly silenced by the shRNA-expressing pEGFP6 vector for SD rat TGF-β1, to that in the control groups in the periods of 24hrs, 48hrs and 72hrs, respectively.Results: LipofectamineTM 2000 potently stimulated the mesangial cells to express fibronectin at the level of either mRNA or protein, but fibronectin in the effectively silenced experimental groups was markedly lower than the control groups or the ineffectively silenced experimental group （P＜0.01）, the later whose fibronectin rose to the peak concentration during 24hrs, in contrast to the former in which it was delayed to 48hrs. The measurements of Col-Ⅳin the cellular extract protein showed the changes similar to TGF-β1, that is, the marked lower concentration of Col-Ⅳoccurred in the experimental groups whose TGF-β1 expression was effectively suppressed by shRNA in the period of 24hrs and 48hrs. Even until 72hrs, the former tended to be higher, but without statistics significance.Conclusion: The silence of TGF-β1 mediated by shRNA markedly suppressed the expression of Col-Ⅳand delayed the peak concentration of fibronectin. LipfectamineTM 2000 maybe stimulated the mesangial cells to secret fibronectin by a pathway independent of TGF-β1.