Effect Of DCLK1 On TGF-tany1-induced Phenotypic Transformation Of Fibroblasts In Pulmonary Fibrosis

Objective: The purpose of this experiment was to study the effect of DCLK1 on the phenotypic transformation of fibroblasts in human pulmonary fibrosis,and to preliminarily explore the molecular mechanism of autophagy and apoptosis of DCLK1 in the phenotypic transformation of human pulmonary fibroblasts,so as to provide new ideas for the exploration of the pathogenesis of pulmonary fibrosis.Methods:(1)First,we used TGF-?1 to induce phenotypic transformation of human lung fibroblasts(HLFs),and then we detected DCLK1 m RNA by q RT-PCR,and expression levels of DCLK1,Fibronectin and ?-SMA were detected by Western Blot.(2)After human lung fibroblast DCLK1 gene Silencd by si-RNA(si-DCLK1),then treated with TGF-?1,we are used Western Blot to detect the expression levels of DCLK1,Fibronectin,?-SMA,and autophagy marker protein LC3-I,LC3-II and p62.Meanwhile,we detected the apoptosis by flow cytometry.(3)With DCLK1 inhibitor(lrrk2-IN-1)applied to human lung fibroblasts,then treated with TGF-?1,we used Western Blot to detect the expression levels of DCLK1,Fibronectin,?-SMA and autophagy marker protein LC3-I,LC3-II and p62,and used flow cytometry to detect the apoptosis.Results :(1)After the induction of phenotypic transformation of HLFs by TGF-?1,we fund that the relative expression levels of DCLK1 m RNA were higher than those in the control group by q RT-PCR,and the expression levels of DCLK1,Fibronectin,?-SMA were significantly higher than those in the control group by Western blot.(2)After silencing the DCLK1 gene(si-DCLK1)in human lung fibroblasts with si-RNA or DCLK1 inhibitor(lrrk2-IN-1)acted on human lung fibroblasts,we found that the expressions of DCLK1,Fibronectin,?-SMA and p62 were decreased compared with the control group.And we found that the expressions of LC3-I and LC3-II about autophagy marker protein were enhanced compared with the control group.Furthermore,the apoptosis was enhanced in the experimental group compared with the control group by flow cytometry.Conclusions:(1)DCLK1 is overexpressed in phenotypic transformation of human lung fibroblasts.(2)In the phenotypic transformation of human lung fibroblasts,after DCLK1 is inhibited or knocked down,the phenotypic transformation process of fibroblasts will be weakened and the extracellular matrix will be reduced;(3)In the phenotypic transformation of human lung fibroblasts,after DCLK1 is inhibited or knocked down,Autophagy and apoptosis were enhanced.