The Mechanism About Roles Of MiR-497 In The Development Of IPF

Idiopahic pulmonary fibrosis(IPF)is a chronic,progressive and fatal fibrotic lung disease characterized by profound changes in stem cell differentiation,epithelial cell phenotype and fibroblast proliferation.However,the underlying mechanism remain unknown.Recently,microRNAs(miRNAs),a small noncoding RNAs that regulate gene expression,has been reported to play a key role in many physiological processes,including tissue development and differentiation,cellular proliferation,and tissue repair.It was reported that a lot of miRNAs could regulate cell differentiation and play a key role in the development of IPF.In our previous study,we screened differentially expressed miRNAs by miRNA chip in the myofibroblast differentiation process of LR-MSCs.In our study,we found miR-497-5p was upregulated both in myofibroblast differentiation of lung resident mesenchymal stem cells(LR-MSCs)and lung tissues of pulmonary fibrosis model.In addition,as determined by luciferase assays and Western blotting,reversion-inducing cysteine-rich protein with kazal motifs(RECK)was identified to be one of target genes of miR-497-5p,and it could regulate the expression of MMP-2,MMP-9 and the activation of latent transforming growth factor-?1(TGF-?1).To test the potential therapeutic significance of this miRNA,we modulated miR-497-5p expression in LR-MSCs and animal models.The results proved that miR-497-5p was a profibrotic factor and inhibiting miR-497-5p expression could profoundly attenuate LR-MSCs differentiate into myofibroblasts and retard fibrogenesis.Our research supported that controlling pulmonary fibrogenesis via inhibition of miR-497-5p expression could serve as a potential therapeutic strategy for IPF.Part I Exploration of the mechanism of miR-497 on the differentiation of LR-MSCsObjectiveWe verify that miR-497 could target the 3'UTR of RECK,and explored the role of miR-497 in the fibroblast differentiation of LR-MSCs.Methods1.The 3'UTRs of mouse RECK encompassing the conserved miR-497 response elements were cloned into the GV306 vector.These luciferase reporter were transiently transfected,together with miR-497-mimic and control-mimic,into 293T cells which were a model system for the validation of miRNA target genes.2.To investigate the regulatory effects of miR-497 on the differentiation of LR-MSCs,LR-MSCs were transfected with miR-497 or miR-497-inhibitor before incubated with or without TGF-?1.3.Expression of miR-497,RECK,a-SMA,Collagen were determined by RT-PCR.4.Expression of RECK,?-SMA,Collagen,fibronectin,MMP-9,TGF-?1 vimentin were determined by Western blot.5.Expression of ?-SMA,Collagen was measured by immunofluorescence.Results1.miR-497 is high regulated in the fibroblast differentiation of LR-MSCs.2.miR-497 could directly target the 3'UTR of RECK,and regulated the mRNA and protein level of RECK.3.Result of RT-PCR,Western blot,Immunocytochemistry showed that upregulated the expression of miR-497 could induce the expression of a-SMA,collagen and some other fibrotic related factors,which could induce LR-MSCs differentiate into fibroblasts finally.In contrast,knock down miR-497 could restrain the profibrotic effect of TGF-?1 on LR-MSCs.ConclusionIn our study,we proved that miR-497 could induce LR-MSCs differentiate into fibroblasts by regulating the expression of RECK,which could restrain the expression of MMP-9 and the release of activated TGF-?1.Part II Explore the effect and mechanism of miR-497 on mouse pulmonary fibrosisObjectiveMouse pulmonary fibrosis model was established to explore the function and molecular mechanism of miR-497 in the development of pulmonary fibrosis.Methods1.C57BL/6J mouse were intratracheal injected with miR-497 regulator for 7 days,then,each group was intratracheal injected with bleomycin or saline.14 days later,mouse was sacrificed and relevant indexes were investigated.2.Lung tissues were detected by H&E staining and the masson staining to determine histology structure integrity and the extent of collagen deposition.3.RT-PCR was used to determine the expression of miR-497,RECK,?-SMA,Collagen.4.The protein level of RECK,?a-SMA,Collagen,fibronectin,MMP-9,MMP-2,TGF-?1and vimentin were measure by Western blot.5.Expression of ?-SMA,Collagen was measured by immunofluorescence.6.Immunocytochemistry was used to determine the expression of a-SMA,MMP-2,MMP-9,and investigated the location and expression of ABCG-2,which was a marker of LR-MSCs.Results1.The results of HE and masson staining showed that miR-497 could induce alveolar collapse and collagen deposition increase,which contribute to pulmonary fibrogenesis.In contrast,suppress the expression of miR-497 could effectively attenuate bleomycin induced pulmonary fibrosis and promote alveolar structure remending,and decrease the deposition of collagen.2.The RT-PCR results showed that miR-497 could suppress the expression of RECK,and induce the expression of a-SMA and collagen.Moreover,suppressing the expression of miR-497 also could restrain bleomycin induced the high level of a-SMA and collagen,and sustain the expression of RECK.3.The immunofluorescence results showed that miR-497 could induce the expression of fibrotic marker,such as a-SMA and collagen.Suppressing miR-497 also could attenuate the profibrotic effect of bleomycin.4.The results of Western blot and immunocytochemistry suggested that miR-497 could increase the level of a-SMA,Collagen,MMP2/9,vimentin,and contribute to release activated TGF-b1 which characterize the development of pulmonary fibrosis.5.The results of immunocytochemistry also showed that miR-497 could decrease the expression of ABCG-2,which was a marker of LR-MSCs.In contrast,suppress the expression miR-497 could sustain the phenotype of LR-MSCs in lung tissues.ConclusionIn the development of pulmonary fibrosis,miR-497 plays a profibrotic role in the process.Upregulated miR-497 suppressed the expression of RECK,which was an inhibitor of MMP-2 and MMP-9.Thus,the high level of MMP2/9 caused dysregulated EMC degradation,which resulted the release and activation of TGF-b1,and aggravated the development of pulmonary fibrosis.In contrast,suppress the expression of miR-497 could sustain the phenotype of LR-MSCs,and restrain LR-MSCs differentiate into fibroblasts,which could contribute to alveolar structure restoration,and effectively attenuate pulmonary fibrogenesis.