| Objective: To elucidate the expression of CXXC5 in the mouse lung fibroblast(MLFs).To investigate the effect of CXXC5 overexpression and silence expression on the differentiation of MLFs into myofibroblasts.To investigate the effect of CXXC5 overexpression and silence expression on the differentiation of TGF-β1 stimulated MLFs into myofibroblasts and Wnt/β-catenin signaling pathway,and to explore the role and mechanism.of CXXC5 in pulmonary fibrosis.Methods:1.Construction of adenoviral vectors containing CXXC5 with green fluorescent protein(GFP)(Ad-CXXC5)and specificity Sh RNA adenoviral vector(Ad-CXXC5-sh RNA).2.Mouse lung fibroblasts were cultured in vitro and divided into 8 groups: cell group,Ad-GFP infection group,Ad-CXXC5 infection group,Ad-sh RNA-CXXC5 infection group,TGF-β1group,TGF-β1-Ad-GFP infection group,TGF-β1-Ad-CXXC5 infection group,TGF-β1-Ad-sh RNA-CXXC5 infection group.3.According to groups,Lung fibroblasts were infected with Ad-GFP,Ad-CXXC5,and Ad-CXXC5-Sh RNA,then stimulated with TGF-β1,normal group received no treatment,we collected cells at 48 h.4.Then we detected the cell proliferation and apoptosis by CCK8 assay and flow cytometry;detected the m RNA levels of CXXC5,β-catenin,pro Colla I,pro Colla III,and α-SMA by RT-q PCR;Western blot was used to detect CXXC5,β-catenin,、Dvl.、E-cadherin、Vimentin、Fibronectin、α-SMA。Results:1.The optimal infection efficiency(MOI)of Ad-GFP,Ad-CXXC5 and Ad-sh RNA-CXXC5 was 100.2.the proliferation rate of CXXC5 overexpression group decreased and the apoptosis increased.The CXXC5 silencing expression group increased cell proliferation rate and decreased apoptosis.the proliferation of each component fibroblasts stimulated by TGFβ increased and the apoptosis decreased.The comparison between TGFβ treatment groups showed that overexpression of CXXC5 could attenuate the effect of TGFβ on proliferation and apoptosis of fibroblasts.The down-regulation of CXXC5 expression could be combined with TGFβ to promote cell proliferation and inhibit apoptosis.3.RT-q PCR test results show:(1)Compared with Ad-GFP group,CXXC5 m RNA expression was increased in Ad-CXXC5 group,and CXXC5 m RNA expression was decreased in Ad-sh RNA-CXXC5 group.Compared with the cell group,the expression of CXXC5 m RNA was decreased in TGF-β1 group.(2)Compared with Ad-GFP group,the expression of pro Colla I m RNA,pro Colla III m RNA,and α-SMA m RNA was decreased in Ad-CXXC5 group and increased in Ad-sh RNA-CXXC5 group.Compared with the cell group,the expression of pro Colla I m RNA,pro Colla III m RNA,and α-SMA m RNA was increased in TGF-β1 group.Compared with TGF-β1-Ad-GFP,TGF-β1-Ad-sh RNA-CXXC5 group CXXC5 down-regulated synergistically with TGF-β1to promote the expression of pro Colla I m RNA,pro Colla III m RNA and α-SMA m RNA,while TGF-β1-Ad-CXXC5 Overexpression of Group CXXC5 Impairs the Above Effects of TGF-β1.(3)Compared with Ad-GFP group,β-Catenin m RNA was significantly decreased in Ad-CXXC5 group,and β-Catenin m RNA expression was significantly increased in Ad-sh RNA-CXXC5 group.Compared with the cell group,the expression ofβ-Catenin m RNA was increased in TGF-β1 group.Compared with TGF-β1-Ad-GFP group,β-Catenin m RNA was significantly decreased in TGF-β1-Ad-CXXC5 group,andβ-Catenin m RNA expression was significantly increased in TGF-β1-Ad-sh RNA-CXXC5 group.4,Western blot results show:(1)Compared with Ad-GFP group,the expression of CXXC5 protein was increased in Ad-CXXC5 group,and the expression of CXXC5 protein was decreased in Ad-sh RNA-CXXC5 group.Compared with the cell group,CXXC5 protein expression was decreased in the TGF-β1 group.(2)Compared with the Ad-GFP group,the expression of Vimentin,Fibronectin and α-SMA protein was decreased in Ad-CXXC5 group and increased in Ad-sh RNA-CXXC5 group.Compared with the cell group,the expression of Vimentin,Fibronectin and α-SMA protein increased in TGF-β1 group.Compared with TGF-β1-Ad-GFP,down-regulation of CXXC5 in TGF-β1-Ad-sh RNA-CXXC5 group promoted the expression of Vimentin,Fibronectin,and α-SMA proteins in cooperation with TGF-β1,while in TGF-β1-Ad-CXXC5 group CXXC5 Overexpression can reduce the above effects of TGF-β1.(3)Compared with Ad-GFP group,the amount of β-Catenin and Dvl protein was significantly decreased in Ad-CXXC5 group,and the expression of β-Catenin and Dvl protein was significantly increased in Ad-sh RNA-CXXC5 group.Compared with the cell group,the expression ofβ-Catenin and Dvl protein increased in TGF-β1 group.Compared with TGF-β1-Ad-GFP,the down-regulation of CXXC5 expression in TGF-β1-Ad-sh RNA-CXXC5 group promoted the expression of β-Catenin and Dvl proteins in cooperation with TGF-β1.The TGF-β1-Ad-CXXC5 group showed that overexpression of CXXC5 attenuated the above effects of TGF-β1.5.The results of ELISA showed that compared with Ad-GFP group,the expression of Coll I,Colla III was decreased in Ad-CXXC5 group,and the expression of Ad-sh RNA-CXXC5 group was increased.Compared with the cell group,the expression of Coll I and Colla III increased in the TGF-β1 group.Compared with TGF-β1-Ad-GFP,TGF-β1-Ad-sh RNA-CXXC5 group CXXC5 down-regulated synergistic TGF-β1promoted the expression of the cell Collal I,Colla III,while the TGF-β1-Ad-CXXC5 group CXXC5 over-expression reduced TGF-The above effect of β1.Conclusion:1.Mouse lung fibroblasts express CXXC5.2.After TGF-β1 stimulation of mouse lung fibroblasts,the expression of CXXC5 was decreased and the Wnt/β-catenin signaling pathway was activated.3.Over-expression of CXXC5 inhibited fibroblast proliferation and promoted apoptosis.Silencing of CXXC5 promotes the proliferation of fibroblasts and inhibits their apoptosis.4.CXXC5 overexpression inhibits the activation of Wnt/β-catenin signaling pathway,inhibits the transformation of fibroblasts into myofibroblasts,inhibits the production of extracellular matrix,and inhibits pulmonary fibrosis. |
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