Study On The Mechanisms Of Exosomal LncRNA MSTRG.91634.7 Regulates Fibroblast Activation By Targeting PINK1 In The Progression Of Experimental Silicosis

Objective:Silicosis is a fibrotic lung disease caused by the inhalation of free crystalline silica.The pathogenesis of silicosis involves a variety of cells.Its pathogenesis is also extremely complex.The role of exosomal lncRNA in intercellular communication had attracted extensive attention in recent years.However,its role in the pathogenesis of silicosis is still unclear.Therefore,this study aimed to clarify the expression profile of serum exosomal lncRNA in silicosis patients,explored the role of exosomal lncRNA MSTRG.91634.7 in the communication between macrophages and fibroblasts,and explored the effect of lncRNA MSTRG.91634.7 targeting PINK1 on silicosis pathology.We hope that this study could provide a new theoretical basis and potential therapeutic targets for elucidating the pathogenesis of silicosis..Methods:In this study,the exosomes were extracted from the peripheral blood sera of silicosis patients and healthy people,and identified by projection electron microscope,particle size analysis technology,and western blot;The lncRNA of exosomes was sequenced;The differentially expressed lncRNAs were screened by DESeq;Bioinformatics was used to predict and analyze the function of target genes for differentially expressing lncRNA;Macrophages were cultured in vitro and treated with Si O₂.The differentially expressed lncRNA was verified by realtime-PCR.The location of lncRNA MSTRG.91634.7 was analyzed by FISH in cells;LncRNA MSTRG.91634.7 was detected by realtime-PCR;LncRNA MSTRG.91634.7 was verified to binding to PINK1 by RNA Pull-Down;The co-culture system of THP-1 and MRC-5 was established.The lncRNA MSTRG.91634.7,PINK1 and other fibrosis factors were detected in fibroblasts by realtime PCR after Si O₂ stimulated;The exosomes of THP-1culture supernatant were extracted and co-cultured with fibroblasts.The protein expression levels of PINK1 and fibrosis factors were detected by western blot in fibroblasts;The PINK1 silencing model of fibroblasts in vitro was constructed by transfecting lentivirus carrying sh-PINK1.The co-culture system of THP-1 and MRC-5 was established.The expression levels of lncRNA MSTRG.91634.7 and PINK1 were detected by realtime-PCR in fibroblasts after Si O₂ treatment of macrophages;The exosomes of THP-1 culture supernatant were extracted and co-cultured with MRC-5.The protein expression levels of PINK1 and fibrosis factors in fibroblasts were detected by western blot.The above experiments were used to illustrate that the macrophage-derived exosomal lncRNA MSTRG.91634.7 regulates fibroblast activation by targeting PINK1.Male C57BL/6 mice were perfused with silica dust suspension through the unexposed trachea to establish the mouse pulmonary fibrosis model.The mouse pulmonary PINK1overexpression model was established by adeno-associated virus type 6 loaded with ov-PINK1.The mice were randomly divided into six groups according to body weight:control;silica;control+AAV-NC;silica+AAV-NC;control+AAV-ov-Pink1;silica+AAV-ov-Pink1.The silencing effect of PINK1 was confirmed by western blot and realtime-PCR;the pathological conditions of each group were observed by HE staining and surface photos of mouse lung tissue;The expression of IL-1βwas detected by ELISA in alveolar lavage fluid;The m RNA level of IL-1βwas detected realtime PCR in lung tissues.The protein level of inflammatory and fibrosis factors in lung tissue was detected by western blot;The protein level ofα-SMA and COL-3 was detected by immunofluorescence and immunohistochemistry in 56 days lung tissues;The effect of overexpression of PINK1 on collagen deposition was detected by measuring the content of hydroxyproline in mouse lung tissue and Masson staining.The above experiments were used to illustrate that overexpression of PINK1 could reduce pulmonary inflammation and fibrosis caused by silica in mice.Result:1.Basic characteristics of silicosis patients and healthy controls.A total of 3 silicosis patients were recruited in this study,all of which were in phaseⅠ.At the same time,3 dust exposed workers were selected as healthy controls.The age of the patients was 55.3±1.5 years.The control age was 56.3±0.5 years.There was no significant difference between the patients and the control group by t-test.The cumulative exposure time was 26.2±4.3 years for patients and 32.3±7.3 years for controls.The exposure time of controls was longer than that of patients（P<0.05）.2.Identification of differentially expressed lncRNAs in serum exosomes of silicosis.12055 lncRNAs were identified by sequence alignment with the Ensembl database,of which 2685 sequences were consistent with the mature lncRNA records in Ensembl.9370 sequences could not be paired with any records and were identified as predicted lncRNAs.In order to further explore the role of the lncRNAs,we compared their expression between the case group and the control group.The results found that 27lncRNAs were differentially expressed,of which 16 lncRNAs were up-regulated and 11lncRNAs were down regulated.In addition,hierarchical clustering results showed that lncRNAs with the same group of sample allocation or the same regulatory pattern were clustered together.Principal component analysis showed that the overall interpretation rate of the first two components was 26.67%,and the correlation coefficient between any two samples in the same group was greater than 0.5,indicating that the samples in the same group had high interpretation and coherence.3.LncRNA expression level The validation of LncRNA levels.The realtime-PCR method was used to detect the expression of each lncRNA in human macrophages.The results showed that the ones that were consistent with the sequencing results were:MSTRG.91634.7,MSTRG.43085.16（P<0.05）.4.The localization of lncRNA MSTRG.91634.7 in cells and its binding to target gene PINK1.LncRNA MSTRG.91634.7 was detected by realtime-PCR in fibroblasts,macrophages,and epithelial cells.The results showed that the expression was the highest in macrophages.LncRNA MSTRG.91634.7 was detected by fluorescence in situ hybridization.The localization of lncRNA MSTRG.91634.7 in macrophages was mainly expressed in the nucleus.LncRNA MSTRG.91634.7 was tested by RNA Pull-Down.The result showed that lncRNA MSTRG.91634.7 could be combined with PINK1.5.Exosomal lncRNA MSTRG.91634.7 could regulate the level of PINK1 and inhibit the activation of fibroblasts.The transwell co-culture system of THP-1 and MRC-5 was established.THP-1 was by adenovirus was overexpressed lncRNA MSTRG.91634.7 and stimulated with silica.After co-culture for 24 hours,fibroblasts in the upper compartment were collected.LncRNA MSTRG.91634.7,PINK1 and fibrosis factors were detected by realtime PCR.LncRNA MSTRG.91634.7 and Pink1 decreased,and the TGF-β1 and COL-1 m RNA levels increased significantly in the Si O₂ group;The lncRNA MSTRG.91634.7 and Pink1increased significantly,and the TGF-β1 and COL-1 m RNA levels decreased significantly in the Si O₂-ov-lncRNA group（P<0.05）.The expression of COL-1 increased significantly;The level of PINK1 increased significantly,and the TGF-β1 and COL-1 decreased significantly in the Si O₂-ov-lncRNA group（P<0.05）.6.Exosomal lncRNA MSTRG.91634.7 could inhibit Si O₂-stimulated the fibroblasts activation through targeting PINK1.The macrophage-derived exosomes were collected and extracted to co-culture with fibroblasts.LncRNA MSTRG.91634.7 and Pink1 were detected by realtime-PCR in fibroblasts.LncRNA MSTRG.91634.7 levels decreased significantly in the exo _{Si O2} group.LncRNA MSTRG.91634.7 levels increased after over-expressing lncRNA MSTRG.91634.7,interestingly,it is not increased in the exo_{ov-lncRNA-Si O2}+sh-PINK1 group.The results showed that the expression of Pink1 in the Si O₂-exo group was significantly reduced.After overexpressing lncRNA MSTRG.91634.7,pink1 increased significantly in the exo_{ov-lncRNA-Si O2},but not increased in the exo_{ov-lncRNA-Si O2}+sh-PINK1 group.Then,the protein levels of PINK1,TGF-β1 and Fibronectin were detected by western blot.The results showed that PINK1 was significantly reduced,and TGF-β1 and Fibronectin were increased in the exo-_{Si O2}.After overexpressing lncRNA MSTRG.91634.7,PINK1 was increased or TGF-β1 and FN1 were decreased.While PINK1 was not increased or TGF-β1 and FN1 were not decreased in the exo_{ov-lncRNA-Si O2}+sh-PINK1 group.These results indicated that lncRNA MSTRG.91634.7 could inhibit fibroblasts activtation by targeting PINK1 in fibroblasts（P<0.05）.7.Overexpression of PINK1 could reduce the lung inflammation induced by silica in mice.The inflammatory factor IL-1βin the alveolar lavage fluid of mice in each group for7 days was measured by ELISA.The m RNA transcription level and protein expression level of inflammatory factors in mice lung homogenate were measured by realtime-PCR and Western blot.ELISA results showed that IL-1βin the alveolar lavage fluid of mice increased significantly in the silicosis group（P<0.05）;Compared with the silica group,the IL-1βin the alveolar lavage fluid of silica-ov-Pink1 group was significantly reduced（P<0.05）.Realtime-PCR results showed that the m RNA levels of IL-1βand NLRP3 in the silica group increased significantly（P<0.05）;Compared with the silica group,the m RNA levels of IL-1βand NLRP3 were significantly decreased in the lung tissue in the silica-ov-Pink1 group（P<0.05）.Western blot results were consistent with realtime-PCR results.8.Overexpression of PINK1 can reduce the lung inflammation induced by silica dust exposure in mice.Paraffin sections of lung tissues of mice in each group at the early stage of silica dust exposure were stained with HE.The lung weight to body weight ratio of mice was recorded.The results of HE staining showed that the early stage of silica exposure was lower,compared with mice perfused with the control group.There were obvious infiltration of inflammatory cells,obvious thickening of alveolar wall cells,and shedding of epithelial cells in the lung tissue of mice.Overexpression of PINK1 could significantly reduce the pulmonary inflammatory changes caused by silica exposure.The lung photos of mice in each group showed that the lung tissue of mice in the silica group was significantly damaged,and overexpression of PINK1 could reduce the pulmonary inflammation caused by silica in the early stage of silica exposure.The lung weight to body weight ratio of mice in each group showed thatthe lung weight to body weight ratio of mice in the silica group increased significantly,compared with the control group,while the lung weight to body weight ratio of mice decreased significantly after overexpressing PINK1（P<0.05）.The results showed that the number of macrophages,neutrophils and lymphocytes in alveolar lavage fluid increased significantly in the silica group,while they increased significantly after overexpressing PINK1（P<0.05）.9.Overexpression of PINK1 could reduce the lung fibrosis induced by silica dust exposure in mice.The m RNA level and protein level of lung fibrosis-related fators in mice lung tissue were measured by realtime-PCR and Western blot.Realtime-PCR results showed that TGF-β1,COL-3 and FN1 in lung tissue of silica group mice were significantly increased,while after overexpression of PINK1,m RNA levels of TGF-β1,COL-3 and FN1 decreased significantly（P<0.05）.The protein level of COL-1 and TGF-β1 were significantly increased,while the protein level of COL-1 and TGF-β1 decreased significantly in the silica-ov-Pink1 group（P<0.05）.Immunofluorescence and immunohistochemistry were used to detect the secretion ofα-SMA and COL-3 in the lung tissue of mice in the late period of silica exposure,which supports the results of realtime-PCR and Western blot.Masson staining and hydroxyproline in mice lung tissue.Masson staining showed that collagen deposition in lung tissue of the silica group was more serious during the late period of silica exposure.Compared with silica group mice,collagen deposition in lung tissue of the silica-ov-Pink1 group was significantly reduced.Hydroxyproline showed that in the late period of silica exposure,the content of hydroxyproline in lung tissue of the silica group increased significantly（P<0.05）;when PINK1 was overexpressed,the content of hydroxyproline in lung tissue of mice decreased significantly（P<0.05）.The lung photos of mice in each group showed that the lung tissue injury of mice in the silica group was obvious.Overexpression of PINK1 could reduce the lung fibrosis caused by silica.Conclusion:1.Exosomal lncRNA MSTRG.91634.7 was low expressed in serum exosomes of silicosis patients.2.Exosomal lncRNA MSTRG.91634.7 inhibited fibroblast activation by targeting PINK1.3.Overexpression of PINK1 could reduce pulmonary inflammation and fibrosis caused by silica in mice.