| Objective:This study was designed to establish a stable pulmonary fibrosis model in mouse,and investigate the expression profile of IncRNA in the paraquat induced pulmonary fibrosis.Screen pulmonary fibrosis related lncRNA.Study on such lncRNAs potential rules in the procession of pulmonary fibrosis.Assess the value of pulmonary fibrosis related IncRNA in A549 cells.Methods1.In vivo(1)Preparation for pulmonary fibrosis modelFemales BALB/c mice were administered 10mg/kg paraquat dissolved in sterile saline via intraperitoneal injection(once per day for 5 days).The normal control group were administered an equal volume of saline at the same time.(2)Establishment,identification,and evaluation of mice modelsMice were examined daily,body weight and survival rate were recorded daily.Lung tissues were harvested on the 12th day following the first treatment.Histological changes were detected by HE and Masson stain in lung of every group mice.Immunohistochemistry show the expression of a-SMA in lung tissue.Quantitative real-time reverse transcription PCR(qRT-PCR)examine the expression of fibrosis related genes.2.Microarray analysis(1)Analysis of IncRNA microarray.Arraystar Mouse IncRNA Microarray v2.0 was designed for the global profiling of mouse IncRNAs.Using standard enrichment computation method,GO and KEGG pathway analysis were performed to determine the role of these closest genes which IncRNAs preferentially located next to in GO categories or biological pathways.By using UCSC genome browser(http://genome.ucsc.edu/)and other database,we get the sequence of pulmonary fibrosis related IncRNAs and their associated protein-coding genes.(2)Verification of IncRNAs expression in lung of fibrosisQuantitative real-time reverse transcription PCR(qRT-PCR)were used to test and verify the microarray data infibrosis lung tissues.Furthermore,we measured IncRNA uc.77 and 2700086A05Rik expression levels in each group.Also the potential genes ZEB2 and HOXA3 of IncRNA uc.77 and 2700086A05Rik are further investigated.3.Plasmid construction and cell transfectionLncRNAs(uc.77 and O5RiK)were PCR amplified and cloned into pEX-3 expression vectors.For overexpression of IncRNAs,A549 were transfected with plasmid and Lipofectamine 2000.Cells were harvested 2 days after transfection for the analysis of IncRNA and gene expression.We also tested the effects of uc.77 and 05Rik increase on the expression of well-known EMT markers E-Cadherin and so on.Results1.In vivo(1)The clinical evaluation in each group miceWe observed a decrease of mobility and a loss of body weight following paraquat treatment.In control group,no significant respiratory symptom was observed,also no death occur during the watch.(2)Histopathology change in each group miceResults revealed a well-defined pulmonary structure with integral air-blood barriers and low collagen levels in the control group.In contrast,in paraquat-treated mice pulmonary tissue structure was distorted with severe damage to the air-blood barrier.Significant thickening of the pulmonary inter-alveolar septum and increased deposition of collagen fibers were noticed.(3)Immunohistochemistry analysisIHC staining with ?-SMA,a marker for the myofibroblast,revealed visible fibroblast accumulation after paraquat injection.(4)Quantitative real-time reverse transcription PCR(qRT-PCR)analysisE-cadherin,a-SMA,COL-1,COL-3,CTGF,MMP-3,Fibronectin were differentially expressed in the paraquat group.2.Microarray analysis(1)Analysis of IncRNA microarray.Lung tissues from control or treatment group were subjected to microarray expression analysis of IncRNA and messenger RNA(mRNA).Scatter plot is adopted to visualize and assess the IncRNA or mRNA expression variation between control samples and paraquat-treated fibrotic samples.Unbiased hierarchical clustering revealed a distinct IncRNA expression pattern in paraquat-treated samples.we identified 513 up-regulated and 204 down-regulated IncRNAs in the paraquat-induced fibrotic lung tissues compared with normal tissues.GO analysis revealed that the most statistically significantly enriched functional terms in IncRNAs down-regulated in paraquat-treated lung tissues include gene expression,regulation of apoptosis and cell differentiation.The top enriched functional terms in up-regulated lncRNAs include cell differentiation,tissue development and MAPKKK cascade.Up to 19 pathways were enriched in up-regulated lncRNAs including pyrimidine metabolism and p53 signaling pathway,while cell adhesion molecules and peroxisome were among the 34 pathways significantly associated with down-regulated lncRNAs.(2)Verification of IncRNAs expression in lung of fibrosisRT-qPCR was used to validate our microarray data,including seven up-regulated lncRNAs and seven down-regulated lncRNAs.The variation trend was in accordance with the microarray results.Two up-regulated lncRNAs:uc.77 and 2700086A05Rik(05Rik),were found to overlap with Zeb2 and Hoxa3 genes,respectively.Also,Zeb2 expression was up-regulated in paraquat-treated pulmonary tissues,while Hoxa3 expression was down-regulated.3.Plasmid construction and cell transfectionWe overexpressed uc.77 and 05Rik in human lung epithelial A549 cellsand observed that increased uc.77 led to an enhanced expression of Zeb2,while 05Rik overexpression suppressed Hoxa3 transcription.We also tested the effects of uc.77 and 05Rik increase on the expression of well-known EMT markers.E-Cadherin,EpCAM and KRT5,marker for epithelial cells,were down-regulated upon uc.77 and 05Rik overexpression.And conspicuous changes were also observed in the expression of a-SMA,vimentin?MMP-2?MMP-9,markers for mesenchymal cells.Conclusion:1.The mice administered 10mg/kg paraquat via intraperitoneal injection can be a available model for research of pulmonary fibrosis.The establishment of pulmonary fibrosis depend on histopathology observation.2.A set of IncRNAs that were differentially expressed between normal and fibrotic lung tissues in a paraquat-induced pulmonary fibrosis mouse model.3.we predicted and used quantitative real time-PCR analysis to confirm that two lncRNAs up-regulated in paraquat-treated lung tissues,uc.77 and 2700086A05Rik(05RiK),acted through zeb2 and Hoxa3 genes,respectively.4.Consistent with Zeb2 and Hoxa3 as key regulators of EMT,we found that transfecting human lung epithelial cells with uc.77 or 05Rik was sufficient to regulate a set of genes' expression.5.The novel concept that IncRNAs may control EMT during lung fibrosis may open new avenues for the diagnosis and treatment of IPF. |
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