The Effect Of RNA Interference In Mediated Transforming Growth Factor-beta Type Receptor Gene Silence On Human Embryonic Lung Fibroblasts

Objective To construct a lentiviral vector for RNA interference ofTGFβRⅠ gene and to study its effect on TGFβRⅠ expression and underthe action of TGF-β1, to observe its effect on cell proliferation, the cell cycle,collagen synthesis, α-SMA and TNF-α expression in the human embryoniclung fibroblasts.Methods (1) According to the Genebank information of humanTGFβRⅠ, four RNA interfering sequences and a negative sequence weredesigned and corresponding complementary DNA containing both sense andantisense Oligo DNA was synthesized, and then they were respectivelyinserted to the pMagic4.0/EGFP vector that had been digested byendonuclease. Agarose gel electrophoresis and sequencing were used toselect and indentify the positive clones. The recombinant plasmids incombination with the helper plasmids were transfected into293T cells toconstruct the lentiviral vectors and then measure the viral titer.(2)Four Recombinant lentivirus vectors and a negative control vector were used to transfect the human embryonic lung fibroblasts cell lineMRC-5. After48and72hours of transfection, the expression level ofTGFβRⅠ mRNA and protein were detected by Real-time PCR and Westernblot respectively. We selected the lentivirus vector with the highestinterfering efficiency to the following experiments.(3) MRC-5cells were respectively transfected with the RNAi lentivirusvector and the negative control lentivirus vector. After48hours oftransfection, the cells were divided into four groups: normal group, cellscultured with DMEM; TGF-β1group, cells cultured with TGF-β1(5ng/ml);negative control group, cells tansfected negative control vector cultured withTGF-β1(5ng/ml); RNAi group, cells tansfected TGFβRⅠsiRNA lentivirusvector cultured with TGF-β1(5ng/ml). The proliferation activity of MRC-5cells in different groups were determined by MTT assay, the cell cycle wasanalyzed by flow cytometry, the collegan expression was observed by siriusred staining, the level of α-SMA was detected by RT-PCR and thesupernatant TNF-α level was measured by ELISA.Results (1) PCR analysis and DNA sequencing demonstrated that theRNAi sequences targeting TGFβRⅠ gene were successfully inserted intothe lentiviral vectors.(2) Green fluorescence was appeared on the cellssurface in each transfection group observed by inverted fluorescencemicroscope. When multiplicity of infection (MOI) was30and cultured withPolybrene (5ng/ml), the efficiency of infecting virus of MRC-5was about 93%. Compared with normal group, the expression of TGFβRⅠmRNA wassignificantly lower in the RNAi groups and the TGFβRⅠmRNA inhibitionrate was26.8%、80.8%、70.7%and79.7%(P<0.05). The TGFβRⅠ-siRNA2had highest interfering efficiency. Western blot analysis showed that thelower expression of TGFβR Ⅰ protein was consistent with TGFβR ⅠmRNA.(3) Compared with other groups, MTT analysis showed that theproliferation of MRC-5was significantly inhibited in RNAi group(P<0.05). Flow cytometry analysis showed that the number of cells in Sphase decreased while the number of cells in G0/G1phase increased in RNAigroup (P<0.05). The cell proliferation and cell cycle of MRC-5in the restgroups were no statistical differences (P>0.05). Collegan expression wasobserved by sirius red staining. The study showed that Collegan expressionlevel was significantly higher in TGF-β1group than that in normal group(P<0.05).Compared with TGF-β1group, RNAi group can significantlyrestrained collagen synthesis (P<0.05). RT-PCR analysis showed that theα-SMA mRNA expression level was significantly higher in TGF-β1groupthan that in normal group (P<0.05). RNAi group was found to significantlydown-regulate the expression of α-SMA mRNA relative to TGF-β1group(p<0.05). ELISA showed that the supernatant TNF-α level in TGF-β1groupwas higher than that in normal group (p<0.05). Compared with TGF-β1group and negative group, TNF-α level in RNAi group was much lower(p<0.05). Conclusions (1) Lentivirus-mediated RNAi targeting TGFβRⅠ genecan effectively suppress the expression of TGFβR Ⅰ in MRC-5cells.(2)After the downregulation of TGFβR Ⅰ expression, proliferation ofMRC-5was inhibited and the number of cells in S phase decreased while thenumber of cells in G0/G1phase increased.(3) Lentivirus-mediated RNAitargeting TGFβRⅠ gene can inhibit the activation of MRC-5stimulated byTGF-β1, and effectively suppress its effect on cell proliferation, the cellcycle, collagen synthesis, α-SMA and TNF-α expression in the humanembryonic lung fibroblasts.