| Pulmonary fibrosis is a chronic,progressive,and usually lethal fibrotic lung disease.The disease is characterized by formation of(myo)fibroblast foci and exaggerated accumulation of extracellular matrix in the lung parenchyma.However,there is no efficacious treatments so far.Thus,it is urgent to unravel the pathogenesis of pulmonary fibrosis to develop effective treatment targeting for this fatal disease.Wnt/β-catenin pathway is crucial in development of pulmonary fibrosis.The aberrant up-regulation of Wnt signaling could induce proliferation of lung epithelial cell,(myo)fibroblast activation and thus excessive collagen synthesis.Furthermore,the specific inhibitors of Wnt/β-catenin pathway are able to markedly inhibit the activated Wnt/β-catenin signaling and finally prevent the development of pulmonary fibrosis.MicroRNAs are a class of 20-to 25-nucleotides single-stranded,non-coding RNAs that suppress translation or promote cleavage of target mRNAs via partial complement to untranslated regions(UTRs).Dysregulation of miRNAs has been found to be implicated in the development of pulmonary fibrosis.There has been accumulating evidence suggesting that Wnt/β-catenin pathway is partly regulated by specific microRNAs in organ development and disease.However,the relationship between miRNAs and the Wnt/β-catenin signaling in the development of pulmonary fibrosis is still undergoing further exploration.miR-154 was reported to be up-regulated in the lung tissues of IPF patients.DKK2 is one direct target gene of miR-154 as determined by luciferase in our previous studies.DKK2 is also characterized by a secretory protein of the Wnt pathway antagonist families.Thereby,the regulative role of miR-154 on Wnt pathway in the development of pulmonary fibrosis needs further study.Objectives:(1)To explore the expression of miR-154 in BLM-induced pulmonary fibrosis mice models.(2)To demonstrate the expression and role of Wnt/β-catenin signaling pathway in mouse pulmonary fibrosis models.(3)To investigate the impact of miR-154 on the development of pulmonary fibrosis model and the expression of Wnt/β-catenin signaling pathway.(4)To verify the profibrotic role of miR-154 is mediated through the Wnt/β-catenin signaling pathway in vivo.Methods:(1)Mice were randomly divided into 2 groups,Saline and BLM groups.The mice model of pulmonary fibrosis is established through intratracheal injection BLM(3mg/kg).Mice were killed on day 21 after BLM instillation.Protein levels ofα-SMA were examined by Western Blot.Histological changes and collagen contents in lung tissues were examined through H&E stain and Masson trichrome stain.miR-154expression was examined by qRT-PCR,(2)The ICG-001 intervention model is established on the base of pulmonary fibrosis mice model.Mice were randomly divided into 3 groups,Saline+Vehicle,BLM+Vehicle,and BLM+ICG-001 groups.The intervention method of ICG-001 is that the mice received daily intraperitoneal injection of ICG-001(5mg/kg)on the day before BLM pretreatment to the day 20 after BLM pretreatment.Mice were sacrificed on day 21 after BLM instillation.Protein levels ofβ-catenin,α-SMA and COL1 were examined by Western Blot.Histological changes and collagen contents in lung tissues were examined through H&E stain and Masson trichrome stain.(3)The miR-154 intervention model is established through lentiviral vector on the base of pulmonary fibrosis mice model.Mice were randomly divided into 5groups,Blank,anti-miR CON,miR-154 CON,anti-miR-154,and miR-154 groups.The intervention method of lentivirus is that the mice were intratracheally instilled with lentivirus at a dose of 3×10~7 TU/mouse.On the day21 after lentivirus instillation,one mouse of each group was sacrificed and cryo-sections of lung tissues were prepared to check the efficiency of lentivirus in mice lungs.The rest mice of each group subsequently received intratracheal instillation of 3mg/kg BLM.Mice were killed on day 21 after BLM instillation.The expression of miR-154 were examined by qRT-PCR.Protein levels of DKK2,β-catenin,α-SMA and COL1 were examined by Western.Histological changes and collagen contents in lung tissues were examined through H&E stain and Masson trichrome stain.(4)The co-intervention model of over-expressed mi R-154 and ICG-001 is established on the base of pulmonary fibrosis mice model.Mice were randomly divided into 4 groups,miR-154 CON+Vehicle,miR-154+Vehicle,miR-154CON+ICG-001 and miR-154+ICG-001 groups.Mice were killed on day 21 after BLM instillation.Protein levels ofβ-catenin,α-SMA and COL1 were examined by Western Blot.Histological changes and collagen contents in lung tissues were examined through H&E stain and Masson trichrome stain.Results:(1)H&E stain showed that the structures of the alveolar were severely damaged with inflammatory cell infiltration and(myo)fibroblastic foci on day 21 after BLM instillation.BLM instillation also induced a significantly increase of collagen deposition as measured by Masson’s trichrome stain.The mice in the BLM group showed a greatly up-regulation in the the expression ofα-SMA compare with mice in saline group,measured by Western Blot.Experimental mice pulmonary fibrosis model was successfully established via intratracheal instillation with BLM(3mg/kg).The expression of miR-154 in lungs from BLM-induced pulmonary fibrosis mice models is increased as examined by qRT-PCR.(2)Protein levels ofβ-catenin were abnormally up-regulated in mouse model of pulmonary fibrosis,and this activation was effectively blockage by ICG-001 as measured by Western Blot.Protein expression ofα-SMA and COL1 after BLM pretreatment was significantly increased,which was markedly down-regulated by ICG-001 compared with Vehicle controls.Furthermore,the severity of lung fibrosis was attenuated by ICG-001 compared with Vehicle controls as examined by histological analysis and Masson’s trichrome stain.(3)Mice lung were successfully infected by lentivirus as observed in the cryo-section under a fluorescence microscope.Expression of miR-154 was effectively mediated by lentiviral vector.Severity of lung fibrosis induced by BLM was attenuated by down-regulation while promoted by up-regulation of miR-154expression as assessed by histological evaluation.Protein expression of a-SMA and COL1 was increased in the miR-154 group and decreased in the anti-miR-154 group compared with corresponding controls.Dkk2 is a direct target gene of miR-154 and also an antagonist proteins of Wnt signaling,Protein levels of Dkk2 andβ-catenin were correspondingly changed in the mi R-154 group and anti-miR-154 group,measured by Western Blot.(4)The inhibitors against Wnt/β-catenin attenuated the elevated expression ofβ-catenin,α-SMA and COL1 induced by the over-expression of miR-154 as measured by Western Blot.Furthermore,Histological analysis showed that the aggravating damage in structures of the alveolar caused by increased miR-154 was blocked by ICG-001.Conclusions:(1)The expression of miR-154 is increased in BLM-induced pulmonary fibrosis mice models.(2)The up-regulated Wnt/β-catenin signaling pathway plays an important role in mouse pulmonary fibrosis models.(3)miR-154 could up-regulate Wnt/β-catenin pathway and performs a profibrotic role in BLM-induced pulmonary fibrosis mice models.(4)The profibrotic role of miR-154 in pulmonary fibrosis is mediated through the Wnt/β-catenin signaling pathway. |
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