| Silicosis is an occupational disease,which caused by the industrial dust with a large number of free silicon dioxide is inhaled over a long period of time,and the massive nodular fibrosis and diffuse pulmonary interstitial fibrosis are belonging to its pathological features.The regulation mechanism of silicosis is extremely complex and the progress of silicosis is continued even though the dust environment was improved.At present,the effective treatment and sensitive diagnostic are deficient.Those issue bring about great difficulties and challenges for the prevention and treatment measures of silicosis.Therefore,it is always a key and difficult task to explore deeply the prevention and treatment strategy for silicosis fibrosis.MicroRNAs(miRNAs)are a non-coding single-stranded RNA molecule with a length of about 18-24 nucleotides encoded by the endogenous genes.MiRNAs are participates in a variety of physiological and pathological processes including cell proliferation,differentiation and apoptosis,which resulting in the occurrence and development of a variety of diseases with its abnormal expression.In this study,a aspiration silicosis model of rat was established,and firstly,the high-throughput sequencing was used to screen differential expressed miRNAs in lung tissues of silicosis rat,meanwhile,the bioinformatics analysis were carried out on the differential miRNAs.Secondly,validation experiments were conducted on miRNAs with the most significant differences in vivo and in vitro,and their regulatory effects on the expression of collage type ?(Col ?)and alpha smooth muscle actin(?-SMA)were analyzed,this is,the effect of miRNAs on collagen deposition and myofibroblast differentiation.Finally,miR-411-3P was used as the research target to observe the role and the related molecular regulatory mechanism on silicosis through the target genes,including myocardin related transcription factor(MRTF)-A and transforming growth factor(TGF)-?/Smurf2 signaling.The studies were divided into four parts:Part 1 Screening,validation and bioinformatics analysis of differential miRNAs in lung tissues of silicosis ratsObjective:The high-throughput information sequencing technology was used to screen differential miRNAs in the pulmonary tissues of silicosis rats and the differential miRNAs were analyzed by the validation experiments and bioinformatics analysis.Methods:1.Silicosis rat model was established by the system of HOPE-MED8050,wistar rats were random divided into two groups:1)Control 24w group:inhalation of pure gas(SiO2-free);2)silicosis 24w group:inhalation of gas with SiO2(50 1?g/m3).2.The HE staining,VG staining and western blot were used to observe the pathological morphology,collagen deposition and the proteins expression of ?-SMA and Col I in silicosis rat model.3.The differential miRNAs in lung tissues of silicosis rats were screened by the high-throughput information sequencing.4.The target gene prediction,GO and KEGG analysis were performed in the differential miRNAs.5.The real-time fluorescence quantitative PCR(RT-qPCR)was used to verify the expression of several differential miRNAs in the lung tissues of silicosis rats.Results:1.The HE staining results showed that compared with the control 24w group,typical silica nodules appeared in the silicosis 24w group,and its quantity and area perentage were significantly higher than the control 24w group(P<0.05);VG staining results revealed that a large amount of collagen deposition was observed in the silica nodules of the silicosis 24w group,and the collagen content was significantly higher than that of the control 24w group(P<0.05);western blot results indicated that compared with the control 24w group,the proteins expression of ?-SMA and Col I were significantly up regulated in silicosis 24w group(P<0.05).The above tips suggested that the silicosis model was successfully constructed.2.The results of high-throughput information sequencing showed that compared with the control 24w group,there were 70 differential miRNAs in the lung tissues of silicosis 24w group,including 41 miRNAs with up-regulated and 29 miRNAs with down-regulated(P<0.05).KEGG analysis results showed that up-regulated miRNAs were mainly related to cancer,pi3k-akt signaling pathway and MAPK signaling pathway,while down-regulated miRNAs were mainly related to MAPK signaling pathway,tumor proteoglycan signaling,and cgmp-pkg signaling pathway.3.The RT-qPCR results showed that compared with the control 24w group,miR-292-5p,miR-292-3p,miR-295-3p,miR-291a-3p and miR-155-3p were up-regulated in silicosis 24w group,while miR-411-3p,miR-370-3p,miR-409a-5p,miR-433-3p and miR-1193-3p were down-regulated in silicosis 24w group(P<0.05).Part 2 Effect of differential miRNAs on regulating the expression of?-SMA and Col I in rat lung fibroblasts induced by TGF-?1Objective:To investigate the effect of differential miRNAs on regulating the expression of ?-SMA and Col I in rat lung fibroblasts basal leveled and TGF-?1 induced leveled.Methods:1.The rat lung fibroblasts were transfected with the mimics and inhibitors of differential miRNAs and the experiments were performed at the basal level and TGF-?1 induced level,respectively.The cells were divided into groups:1)Control group;2)TGF-?1 group;3)control group with fluorescence;4)mimic-NC group;5)mimic group;6)inhibitor-NC group;7)inhibitor group;8)mimic-NC+TGF-?1 group;9)mimic+TGF-?1 group;10)inhibitor-NC+TGF-?1 group;11)inhibitor+TGF-?1 group.2.The level of miRNA was measured by RT-qPCR.3.The protein levels of ?-SMA and Col I were measured by the western blot.Results:1.The results of RT-qPCR revealed that compared with the control group,the level of miR-155-3p was up-regulated,while the levels of miR-292-5p,miR-411-3p,miR-370-3p,miR-409a-5p and miR-1193-3p were down-regulated in TGF-?1 group(P<0.05).2.Compared with the mimic-NC group,the protein level of ?-SMA was up regulated in mimics of miR-292-5p,miR-291a-3p and miR-155-3p group(P<0.05),while the protein level of ?-SMA was down regulated in mimics of miR-1193-3p,miR-411-3p,miR-409a-5p and miR-433-3p group(P<0.05);and compared with the inhibitor-NC group,the protein level of ?-SMA was down regulated in inhibitor of miR-292-3p,miR-291a-3p and miR-295-3p group(P<0.05),while the protein level of ?-SMA was up regulated in inhibitor of miR-1193-3P,miR-411-3p,miR-409a-5p and miR-433-3p group(P<0.05).Compared with the mimic-NC group,the protein level of Col I was up regulated in mimics of miR-292-5p,miR-155-3p and miR-295-3p group(P<0.05),while the protein level of Col I was down regulated in mimics of miR-411-3p and miR-370-3p group(P<0.05);and compared with the inhibitor-NC group,the protein level of Col I was down regulated in inhibitor of miR-292-5p,miR-155-3p and miR-295-3p group(P<0.05),while the protein level of Col I was up regulated in inhibitor of miR-411-3p group(P<0.05).3.Compared with the control group,the expression of ?-SMA and Col I was obviously up-regulated in TGF-?1 group(P<0.05).4.Compared with the mimic-NC+TGF-?1 group,the protein level of?-SMA was up-regulated in mimic+TGF-?1 of miR-292-5p,miR-292-3p,miR-291a-3p and miR-295-3p group(P<0.05),while the protein level of?-SMA was down regulated in mimic+TGF-?1 of miR-411-3p,miR-370-3p,miR-433-3p and miR-1193-3p group(P<0.05);and compared with the inhibitor-NC+TGF-?1 group,the protein level of ?-SMA was down regulated in inhibitor+TGF-?1 group of miR-292-5p and miR-292-3p group(P<0.05),while the protein level of ?-SMA was up regulated in inhibitor+TGF-?1 of miR-411-3p,miR-370-3p and miR-1193-3p group(P<0.05).Compared with the mimic-NC+TGF-?1 group,the protein level of Col I was up-regulated in mimic+TGF-?1 of miR-292-5p,miR-292-3p,miR-291a-3p and miR-155-3p group(P<0.05),while the protein level of Col I was down regulated in mimic+TGF-?1 of miR-411-3p,miR-370-3p,miR-409a-5p and miR-1193-3p group(P<0.05);and compared with the inhibitor-NC+TGF-?1 group,the protein level of Col I was down regulated in inhibitor+TGF-?1 group of miR-292-5p and miR-291a-3p group(P<0.05),while the protein level of Col I was up regulated in inhibitor+TGF-?1 of miR-411-3p,miR-370-3p and miR-1193-3p group(P<0.05).Part 3 miR-411-3p inhibits the silicosis fibrosis by targeting the MRTF-A/SRF signalingObjective:To observe the role of miR-411-3p in silicosis fibrosis,and investigate the mechanism of miR-411-3p on silicosis though regulating MRTF-A/SRF signaling.Methods:1.Primary of lung fibroblasts was cultured.Cells were divided into 1)control group,2)TGF-?1 group,3)mimic-NC group,4)miR-411-3p mimic group,5)inhibitor-NC group,6)miR-411-3p inhibitor group,7)mimic-NC+TGF-?group,8)miR-411-3p mimic+TGF-?1 group,9)inhibitor-NC+TGF-?1 group,10)miR-411-3p inhibitor+TGF-?1 group.2.Primary of lung fibroblasts was cultured.Cells were divided into 1)siRNA-NC group,2)si-MRTF-A group,3)siRNA-NC+TGF-?1 group,2)si-MRTF-A+TGF-?1 group.2.The bronchial perfused SiO2 was used to constructed a silicosis mouse model,C57BL/6 mouse were random divided into 1)control group,2)Silica group,3)Silica+agomir NC group,4)Silica+agomir miR-411-3p group.3.The HE staining and Sirius red staining were used to observe the pathological morphology and collagen deposition in silicosis rat model.4.The lung function of mice was detected by FinePointe WBP whole-body volume scanning system.5.In situ hybridization was used to detect the expression and localization of miR-411-3p6.CCK-8 was used to detect the activity and scratch assay was carried out to detect the migration capacity of rat lung fibroblasts.7.Immumofluorescence was used to detect the co-expression of MRTF-A and ?-SMA in rat lung tissue of silicosis.8.Dual luciferase reporter assay was used to verify the binding of miR-411-3p to Mrtfa.9.Western blot and RT-qPCR were used to measure the protein and mRNA level of MRTF-A,SRF,?-SMA and Col I.Results:1.The results of in situ hybridization showed that miR-411-3p with red fluorescence expressed on alveolar and bronchial epithelium.And compared with control 24w group,the level of miR-411-3p was significantly decreased in silica nodules of silicosis 24w group;meanwhile,it was also significantly decreased in coal worker's pneumoconiosis lung tissues.In rat lung fibroblasts,compared with control group,the level of miR-411-3p was significantly decreased in TGF-?1 group,and it was localized in the cytoplasm of lung fibroblasts.2.The CCK-8 results showed that the activity of rat lung fibroblasts was obviously inhibited in miR-411-3p mimic group compared with miR-411-3p m-NC group,while the activity of rat lung fibroblasts was obviously improved in miR-411-3p inhibitor group compared with miR-411-3p i-NC group(P<0.05).Compared with control group,the activity of rat lung fibroblasts was obviously increased in TGF-?1 group(P<0.05).Compared with miR-411-3p m-NC+TGF-?1 group,the activity of rat lung fibroblasts was obviously decreased in miR-411-3p mimic+TGF-?1 group,while compared with miR-411-3p i-NC+TGF-?1 group,the activity of rat lung fibroblasts was obviously increased in miR-411-3p inhibitor+TGF-?1 group(P<0.05).3.The scratch assay showed that the migration ability of rat lung fibroblasts obviously increased in TGF-?1 group compared with control group.Compared with miR-411-3p m-NC+TGF-?1 group,the migration ability obviously down regulated in miR-411-3p mimic+TGF-?1 group;while compared with miR-411-3p i-NC+TGF-?1 group,the migration ability obviously up regulated in miR-411-3p inhibitor+TGF-?1 group(P<0.05).4.Westren blot results indicated that the expression of MRTF-A and SRF was obviously up-regulated in silicosis 24w group compared with control 24w group.Both of them were also significantly up-regulated in TGF-?1group compared with control group(P<0.05).5.The immunofluorescence results showed that MRTF-A and ?-SMA were co-expressed in the silicon nodules of silicosis 24w group,compred with control 24w group(P<0.05).6.The western blot results showed that the expression of MRTF-A,?-SMA and Col I were obviously down regulated in Si-MRTF-A group compred with SiRNA-NC group.And both of them were also down regulated in Si-MRTF-A+TGF-?1 group compred with SiRNA-NC+TGF-?1 group(P<0.05)7.The RT-qPCR results showed that the level of miR-411-3p was increased,and the mRNA levels of MRTF-A,SRF,?-SMA and Col I were decreased in miR-411-3P mimic group compred with mimic-NC group(P<0.05).The mRNA levels of MRTF-A,SRF,?-SMA and Col I were slightly changed in miR-411-3P inhibitor group compred with inhibitor-NC group(P>0.05).The western blot results showed that the expression of MRTF-A and SRF were down regulated in miR-411-3P mimic group compred with mimic-NC group,the expression of SRF were up regulated in miR-411-3P inhibitor group compred with inhibitor-NC group(P<0.05).8.The RT-qPCR results showed that the mRNA levels of MRTF-A,?-SMA and Col I were down regulated in miR-411-3p mimic+TGF-?1 group compred with mimic-NC+TGF-?1 group,the mRNA levels of MRTF-A,?-SMA and Col I were up regulated in miR-411-3p inhibitor+TGF-?1 group compred with inhibitor-NC+TGF-?1 group(P<0.05).The western blot results showed that the expression of MRTF-A was obviously down regulated in miR-411-3p mimic+TGF-?1 group compred with mimic-NC+TGF-?1 group.However,it was obviously up regulated in miR-411-3p inhibitor+TGF-?1 group compred with inhibitor-NC+TGF-?1 group(P<0.05).9.The results of the dual-luciferase reporter gene showed that dual-luciferase expression levels in Mrtfa 3'UTR Mut+miR-411-3p mimic group did not change significantly when compared with Mrtfa 3'UTR Mut+miR-NC group(P>0.05).However,the dual-luciferase expression levels were significantly down-regulated in Mrtfa 3'UTR Wt+miR-411-3p mimic group compared with Mrtfa 3'UTR Wt+miR-NC group(P<0.05).10.The HE staining results of mouse lung tissue showed typical silica nodules appeared in the silicosis group,and its quantity and area perentage were significantly higher than the control group,the quantity and area perentage were significantly decreased in Silica+agomir miR-411-3p group compared with Silica+agomir-NC group(P<0.05).The Sirius red staining results showed that collagen deposition was increased in silicosis group compared with control group,the collagen deposition was obviously decreased in Silica+agomir miR-411-3p group compared with Silica+agomir-NC group(P<0.05).11.In situ hybridization results showed that the expression of miR-411-3p was significantly down regulated in lung tissue of silicosis group compared with control group.However,the expression of miR-411-3p was significantly up regulated in Silica+agomir miR-411-3p group compared with Silica+agomir-NC group(P<0.05).12.The results of FinePointe WBP whole-body volume scanning system for lung function of mice shown that the Rpef was obviously down regulated,and the EEP,PAU and Penh were up regulated in silicosis compared with control group;while Rpef was obviously up regulated,and the EEP,PAU and Penh were down regulated in Silica+agomir miR-411-3p group compared with Silica+agomir-NC group(P<0.05).13.Immunohistochemical staining showed the expression of ?-SMA was up regualted in silicosis group compared with control group,while it was down regulated in Silica+agomir miR-411-3p group compared with Silica+agomir-NC group(P<0.05).14.The results of RT-qPCR revealed that the level of miR-411-3p was down regulated,and the mRNA levels of MRTF-A,SRF,?-SMA and Col I were up regulated in silicosis compared with control group.Compared with Silica+agomir-NC group,the level of miR-411-3p was obviously up regulated and the mRNA levels of MRTF-A,?-SMA and Col I were down regulated in Silica+agomir miR-411-3p group(P<0.05).Western blot results indicated the expression of MRTF-A,SRF,?-SMA and Col I were up regualted in silicosis group compared with control group,while they were down regulated in Silica+agomir miR-411-3p group compared with Silica+agomir-NC group(P<0.05).Part 4 MiR-411-3p inhibits silicosis fibrosis by targeting Smurf2/TGF-?signalingObjective:To discuss the expression of Smurf2/TGF-? signaling in silicosis and investigate the mechanism of miR-411-3p inhibits silicosis by regulating Smurf2/TGF-signaling.Methods:1.Silicosis rat model was established by the system of HOPE-MED8050,wistar rats were random divided into two groups:1)Control 24w group:inhalation of pure gas(SiO2-free);2)silicosis 24w group:inhalation of gas with SiO2(50 ?g/m3).2.Primary of lung fibroblasts was cultured.Cells were divided into 1)control group,2)TGF-?1 group,3)TGF-?1+LY364947 group,4)mimic-NC+TGF-?3 group,5)miR-411-3p mimic+TGF-?1 group,6)inhibitor-NC+TGF-?1 group,7)miR-411-3p inhibitor+TGF-?1 group.3.Primary of lung fibroblasts was cultured.Cells were divided into 1)siRNA-NC group,2)si-Smurf2 group,3)siRNA-Nc+TGF-?1 group,4)si-Smurf2+TGF-?1.4.The bronchial perfused SiO2 was used to constructed a silicosis mouse model,C57BL/6 mouse were random divided into 1)control group,2)Silica group,3)Silica+agomir NC group,4)Silica+agomir miR-411-3p group.5.Immunohistochemical staining was used to measure the expressions of Smurf2,TGF-?R1,TGF-?R2,TGF-?1 and Smad7.6.Dual luciferase reporter assay was used to verify the binding of miR-411-3p to Smurf2.7.The RT-qPCR and Western blot were used to measure the mRNA and protein levels of Smurfl,Smurf2,TGF-?1,TGF-?R1,TGF-?R2,Smad7 and p-Smad2/3.Results:1.The immunohistochemical staining results indicated the expression of Smurf2 was up regulated in silicosis 24w group compared with control 24w group(P<0.05).2.In rat lung tissue the results of RT-qPCR revealed that the mRNA levels of Smurf2,TGF-?1 and TGF-?R1 were obviously up regulated,while the mRNA level of Smad7 was down regualted in silicosis 24w group compared with control 24w group(P<0.05).However,compared with control 24w group,the the mRNA levels of Smurfl changed slightly in silicosis 24w group(P>0.05).Western blot results indicated the expressions of Smurf2,TGF-?1,TGF-?R1,TGF-?3R2 and p-Smad2/3 were up regulated,while Smad7 was down regulated in in silicosis 24w group compared with control 24w group(P<0.05).3.In rat lung fibroblasts the RT-qPCR results indicated that the mRNA levels of Smurf2 and TGF-?R1 were up regualted,while Smad7 was down regualted in TGF-?1 group compared with control group(P<0.05).Western bolt results showed the expression of Smurf2,TGF-?R1,TGF-?R2 and p-Smad2/3 were up regualted,while Smad7 was down regualted in TGF-?1 group compared with control group(P<0.05).4.In rat lung fibroblasts the western bolt results showed that the expression of TGF-?R1,Smurf2 and p-Smad2/3 were down regualted,while Smad7 was upregulated in TGF-?1+LY364947 group compared with TGF-?1 group(P<0.05).5.In rat lung fibroblasts the results of western bolt and RT-qPCR indicated the level of Smurf2 was down regulated in si-Smurf2 1#,2#and 3#group compared with siRNA-NC group,and the level of Smurf2 was the most obvious downregulation in si-Smurf2 2#group(P<0.05).6.In rat lung fibroblasts the results of RT-qPCR indicated the mRNA levels of Smurf2,TGF-?1,TGF-?R1 and ?-SMA were down regulated,while Smad7 was up regualated in si-Smurf2 group compared with siRNA-NC group(P<0.05).Western bolt results showed the expression of Smurf2,TGF-?1,TGF-?R1 and TGF-?R2 were down regualted,while Smad7 was up regualted in si-Smurf2 group compared with siRNA-NC group(P<0.05).7.In rat lung fibroblasts induced by TGF-?1 the results of RT-qPCR indicated the mRNA levels of Smurf2,TGF-?1,TGF-?R1,?-SMA and Col I were downregualted,while Smad7 was up regualted in si-Smurf2+TGF-?1 group compared with siRNA-NC+TGF-?1 group(P<0.05).Western bolt results showed the expression of Smurf2,TGF-?1,TGF-?R1,TGF-?R2,p-Smad2/3,?-SMA and Col I were down regulated,while Smad7 was up regualted in si-Smurf2+TGF-?1 group compared with siRNA-NC+TGF-?1 group(P<0.05).8.The results of the dual-luciferase reporter gene showed that dual-luciferase expression levels in Smurf2 3'UTR Mut+miR-411-3p mimic group changed slightly compared with Smurf2 3'UTR Mut+miR-NC group(P>0.05).However,the dual-luciferase expression levels were significantly down-regulated in Smurf2 3'UTR Wt+miR-411-3p mimic group compared with Smurf2 3'UTR Wt+miR-NC group(P<0.05).9.In rat lung fibroblasts induced by TGF-?1 the results of RT-qPCR indicated the mRNA levels of Smurf2,TGF-?1 and TGF-?R1 were down regulated,while Smad7 was up regualted in miR-411-3p mimic+TGF-?1 group compared with m-NC+TGF-?1 group;compared with i-NC+TGF-?1 group,the mRNA levels of Smurf2,TGF-?1 and TGF-?R1 were up regualted,while Smad7 was down regualted in miR-411-3p inhibitor+TGF-?1 group(P<0.05).Western blot results indicated the expression of Smurf2,TGF-?1,TGF-?R1,TGF-?R2 and Smad2/3 were down regualted,while Smad7 was up regualted miR-411-3p mimic+TGF-?1 group compared with m-NC+TGF-?1;compared with i-NC+TGF-?1,the expression of Smurf2,TGF-?1,TGF-?R1,TGF-?R2 and p-Smad2/3 were up regulated,while Smad7 was down regualted in miR-411-3p inhibitor+TGF-?1 group(P<0.05).10.In mouse lung tissue the RT-qPCR results indicated that the mRNA level of Smurf2,TGF-?1 and TGF-?R1 were up regualted,while Smad7 was down regualted in silicosis group compared with control group;and the mRNA level of Smurf2,TGF-?1 and TGF-?R1 were down regualted,while Smad7 was up regualted in Silica+agomir miR-411-3p group compared with Silica+agomir-NC group(P<0.05).11.The immunohistochemical staining results indicated the expression of Smurf2,TGF-?1,TGF-?R1 and TGF-?R2 were increased,while Smad7 was decreased in silicosis group compared with control group;and Smurf2,TGF-?1,TGF-?R1 and TGF-?R2 were decreased,while Smad7 was increased in Silica+agomir miR-411-3p group compared with Silica+agomir-NC group.12.In mouse lung tissue the western blot results indicated that the expression of Smurf2,TGF-?1,TGF-?R1,TGF-?R2 and p-Smad2/3 were up regualted,while Smad7 was down regualted in silicosis group compared with control group;and the expression of Smurf2,TGF-?1,TGF-?R1,TGF-?R2 and p-Smad2/3 were down regualted,while Smad7 was up regualted in Silica+agomir miR-411-3p group compared with Silica+agomir-NC group(P<0.05).Conclusions:1.Through high-throughput sequencing technology,70 differentially expressed miRNAs were screened from the silicosis rats model constructed the system of HOPE-MED8050,among which some differential miRNAs regulated collagen deposition and myofibroblast differentiation.2.miR-411-3p attenuate silicosis fibrosis by regulating target genes Mrtfa and Smurf2. |
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