Transcription factor NF-κB promotes acute lung injury via microRNA-99b-mediated PRDM1 down-regulation

Abstract

Acute lung injury (ALI), is a rapidly progressing heterogenous pulmonary disorder that possesses a high risk of mortality. Accumulating evidence has implicated the activation of the p65 subunit of NF-κB [NF-κB(p65)] activation in the pathological process of ALI. microRNAs (miRNAs), a group of small RNA molecules, have emerged as major governors due to their post-transcriptional regulation of gene expression in a wide array of pathological processes, including ALI. The dysregulation of miRNAs and NF-κB activation has been implicated in human diseases. In the current study, we set out to decipher the convergence of miR-99b and p65 NF-κB activation in ALI pathology. We measured the release of pro-inflammatory cytokines (IL-1β, IL-6, and TNFα) in bronchoalveolar lavage fluid using ELISA. MH-S cells were cultured and their viability were detected with cell counting kit 8 (CCK8) assays. The results showed that miR-99b was up-regulated, while PRDM1 was down-regulated in a lipopolysaccharide (LPS)-induced murine model of ALI. Mechanistic investigations showed that NF-κB(p65) was enriched at the miR-99b promoter region, and further promoted its transcriptional activity. Furthermore, miR-99b targeted PRDM1 by binding to its 3'UTR, causing its down-regulation. This in-creased lung injury, as evidenced by increased wet/dry ratio of mouse lung, myeloperoxidase activity and pro-inflammatory cytokine secretion, and enhanced infiltration of inflammatory cells in lung tissues. Together, our findings indicate that NF-κB(p65) promotion of miR-99b can aggravate ALI in mice by down-regulating the expression of PRDM1. Acute lung injury (ALI), is a rapidly progressing heterogenous pulmonary disorder that possesses a high risk of mortality. Accumulating evidence has implicated the activation of the p65 subunit of NF-κB [NF-κB(p65)] activation in the pathological process of ALI. microRNAs (miRNAs), a group of small RNA molecules, have emerged as major governors due to their post-transcriptional regulation of gene expression in a wide array of pathological processes, including ALI. The dysregulation of miRNAs and NF-κB activation has been implicated in human diseases. In the current study, we set out to decipher the convergence of miR-99b and p65 NF-κB activation in ALI pathology. We measured the release of pro-inflammatory cytokines (IL-1β, IL-6, and TNFα) in bronchoalveolar lavage fluid using ELISA. MH-S cells were cultured and their viability were detected with cell counting kit 8 (CCK8) assays. The results showed that miR-99b was up-regulated, while PRDM1 was down-regulated in a lipopolysaccharide (LPS)-induced murine model of ALI. Mechanistic investigations showed that NF-κB(p65) was enriched at the miR-99b promoter region, and further promoted its transcriptional activity. Furthermore, miR-99b targeted PRDM1 by binding to its 3'UTR, causing its down-regulation. This in-creased lung injury, as evidenced by increased wet/dry ratio of mouse lung, myeloperoxidase activity and pro-inflammatory cytokine secretion, and enhanced infiltration of inflammatory cells in lung tissues. Together, our findings indicate that NF-κB(p65) promotion of miR-99b can aggravate ALI in mice by down-regulating the expression of PRDM1. Acute lung injury (ALI) is a prevalent disease with exceedingly-high rates of morbidity and mortality. ALI can often predispose patients to acute respiratory distress syndrome (ARDS) (1Rubenfeld G.D. Caldwell E. Peabody E. Weaver J. Martin D.P. Neff M. Stern E.J. Hudson L.D. Incidence and outcomes of acute lung injury.N. Engl. J. 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Overexpression of miR-125a in myelodysplastic syndrome CD34+ cells modulates NF-kappaB activation and enhances erythroid differentiation arrest.PLoS ONE. 2014; 9 (24690917)e9340410.1371/journal.pone.0093404Crossref PubMed Scopus (36) Google Scholar), which regulates the expressions of a large variety of genes that are involved in numerous processes like inflammatory and immune responses of the cell, cell growth, and development (20Napetschnig J. Wu H. Molecular basis of NF-kappaB signaling.Annu. Rev. Biophys. 2013; 42 (23495970): 443-46810.1146/annurev-biophys-083012-130338Crossref PubMed Scopus (545) Google Scholar). Moreover, transcriptional repressor PR (PRDI-BF1-RIZ) domain zinc finger protein 1 (PRDM1) has also been identified to be a downstream effector of the NF-κB (21Romagnoli M. Belguise K. Yu Z. Wang X. Landesman-Bollag E. Seldin D.C. Chalbos D. Barille-Nion S. Jezequel P. Seldin M.L. Sonenshein G.E. Epithelial-to-mesenchymal transition induced by TGF-beta1 is mediated by Blimp-1-dependent repression of BMP-5.Cancer Res. 2012; 72 (23054396): 6268-627810.1158/0008-5472.CAN-12-2270Crossref PubMed Scopus (65) Google Scholar). PRDM1 (also referred to as Blimp-1) was originally identified as a post-inductive silencer of interferon beta (IFN-β) gene expression and controls cell fate decisions in multiple tissue contexts (22Elias S. Robertson E.J. Bikoff E.K. Mould A.W. Blimp-1/PRDM1 is a critical regulator of Type III Interferon responses in mammary epithelial cells.Sci. Rep. 2018; 8 (29321612): 23710.1038/s41598-017-18652-9Crossref PubMed Scopus (6) Google Scholar). However, the mechanism underlying the role of miR-99b and NF-κB in ALI remains unclear. In the current study, we performed experiments using ALI mouse models, and found miR-99b expression was increased in the lung tissues. In addition, we observed that NF-κB could increase miR-99b expression and deteriorate ALI of mice induced by LPS. a we the regulated the development of ALI. we set out to the mechanism by which miR-99b the processes of ALI by of LPS-induced MH-S cells and a murine model of LPS-induced ALI. the results of alveolar alveolar alveolar edema and inflammatory cell infiltration in the lung tissues of mice in the ALI group with the control group The ratio of the mice in the ALI group was to the control group activity was in mice ALI was further to the expression of inflammatory IL-6, and in the and the results showed that LPS to IL-6, and in the of mice the expression of miR-99b in lung tissues were detected with and the results showed a in miR-99b expression levels in the lung tissues of mice of the ALI group with the control group a increase in the of macrophages in the lung tissues of mice from the ALI group with the control group findings that miR-99b was in mice with ALI. further the of miR-99b in regulating the of lung injury in mice was by ALI mice with miR-99b results a in the expression levels of miR-99b in the lung tissues of mice in the ALI miR-99b group to the ALI control group of miR-99b in ALI that inflammatory cell infiltration was in the lung tissues of mice in the ALI miR-99b group with the ALI in to in alveolar and pulmonary edema In addition, lung ratio activity and IL-6, and expression levels were observed in the in mice in the ALI miR-99b group with the ALI findings indicate that miR-99b could lung injury in ALI MH-S cell of ALI were by LPS to further study the mechanism of miR-99b in ALI was to the expression of miR-99b in the cells with of and the results showed that miR-99b expression was in the cells of LPS miR-99b was in the and results a in miR-99b expression miR-99b The results of cell counting kit 8 (CCK8) and experiments showed that LPS MH-S cell viability and enhanced and However, with the LPS cell viability was the expression levels of and were in the LPS miR-99b group The expression levels of in the cells were detected using and was found that the expression of in the was with the increase of LPS was to the of LPS on expression and the results that LPS promoted The was to the NF-κB(p65) binding with the miR-99b promoter of a promoted NF-κB(p65) at the miR-99b promoter LPS NF-κB and was used to MH-S and the expression levels of were detected by The results showed a in NF-κB(p65) and expression levels was to the miR-99b expression and was found that miR-99b expression was in the of NF-κB p65 was further by and the expression levels of p65 and miR-99b were detected using and The results a in p65 and miR-99b expression NF-κB was Cell viability of MH-S was observed to be promoted NF-κB while of miR-99b the viability The results that NF-κB(p65) was enriched at the miR-99b promoter region, and promoted its transcriptional activity, thus inflammatory to the The downstream genes of miR-99b were using and microRNA of the miRNAs were found at the ALI mRNA expression set was from the which of and lung injury and a set of were PRDM1 was the gene with the in the previous has that of PRDM1 in cells the expression levels of and while of cytokines and the of including the increase of E. H. L. Wang L. Wang J. Yang X. Liu W. J.M. H. L. Kim H. of and of by the Rep. 29 Full Text Full Text PDF PubMed Scopus Google Scholar). was that the PRDM1 gene was involved in the regulation of and was further The results further that targeted the PRDM1 gene and the PRDM1 expression in the set was much in the lung injury to The results of gene that of miR-99b the activity of while on the activity of in cells The results of and showed that the mRNA and protein expression levels of PRDM1 were in miR-99b MH-S cells PRDM1 expression were also detected in MH-S cells p65 or in with miR-99b the results of which showed that the PRDM1 levels were by with the and with the PRDM1 levels were by miR-99b further that the PRDM1 gene be involved in the regulation of lung injury via expression was performed to the PRDM1 expression in ALI mouse lung and a in the PRDM1 expression was observed with the control group results showed a of PRDM1 expression in MH-S cells LPS PRDM1 was by PRDM1 overexpression increased cell viability of MH-S cells LPS findings that miR-99b targeted PRDM1 and its expression to increase the of LPS induced cell further PRDM1 regulates ALI in the of lung injury was by into ALI The expression of PRDM1 in lung tissues were by of ALI mice an increase in PRDM1 expression in the lung tissues of results the protein expressions of PRDM1 to that of was further to lung tissue and was observed that inflammatory cell infiltration and edema were in mice of the ALI group with the ALI group Moreover, the ALI group ratio of lung activity and expression levels of inflammatory IL-6, and in with the ALI In a PRDM1 lung injury in ALI findings showed with the ALI the expression levels of miR-99b were and of PRDM1 were enhanced in the ALI of NF-κB(p65) to an miR-99b expression with an PRDM1 miR-99b expression levels were while of PRDM1 were in the ALI miR-99b group with the ALI group findings were with results In addition, an in the infiltration of inflammatory edema of lung tissues ratio of lung activity in the and the secretion of pro-inflammatory in the lung tissues of the ALI group with the ALI ALI results with of the ALI the infiltration of inflammatory cells in lung tissues was increased the ratio of lung was activity in was and the of inflammatory IL-6, and in lung tissues were increased in the ALI miR-99b group in to the ALI findings indicate that NF-κB(p65) promoted the of ALI in mice via miR-99b to the PRDM1. ALI with and morbidity rates M.A. in acute lung into the of acute lung 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). In we NF-κB(p65) promotion of miR-99b can the processes of ALI in LPS-induced MH-S cells and a murine model of LPS-induced ALI by down-regulating the expression of PRDM1. our findings that miR-99b was in the LPS-induced mouse ALI up-regulated levels of have been identified in of acute respiratory distress syndrome C. X. M. Wang Y. Liu L. and mRNA expression in acute respiratory distress Med. 2014; 7 PubMed Scopus Google Scholar). We found that lung injury was increased in the ALI mouse as evidenced by up-regulated activity and the of IL-6, and while the infiltration of inflammatory cells was in the lung the could be by the of miR-99b which is much in with the previous Moreover, has been documented that of in the expression levels of cytokines IL-6, and thus the of X. Wu L. X. W. Y. Wang M. K. Wu J. Y. X. X. F. S. of miRNAs PubMed Scopus Google Scholar). we the miR-99b and and found that miR-99b with the of PRDM1 its miRNAs possess the ability to mRNA degradation or translation by with the of D. microRNAs as Biol. 7 PubMed Scopus Google Scholar). More in with our miR-125a miR-125a on in has been to be to to PRDM1 M. S. D. S. M. of cell differentiation in Immunol. 2010; PubMed Scopus Google Scholar). Moreover, down-regulated levels of PRDM1 have been found in lung expressions promoted cell in and lung in vivo Z. Wang H. Wei Y. F. Liu Z. Zhang Z. of PRDM1 and lung Biol. 2017; 39 Scopus Google Scholar). Furthermore, of PRDM1 is known to the release of and by binding to multiple in human cells M.A. M. B. Yu J. Wei S. J. E. M.A. controls effector cytokine in human Immunol. 2010; PubMed Scopus Google Scholar). a we that miR-99b targeted PRDM1 expression to the of LPS-induced cell ALI is by inflammatory cell pro-inflammatory cytokine (3Grommes J. Soehnlein O. 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Li Zhou modulates cell 2016; PubMed Scopus (28) Google Scholar). In addition, study that NF-κB be with PRDM1 during the process of differentiation C. F. S. C. A. A. S. M. A. O. M. expression is to during Immunol. Res. PubMed Scopus Google Scholar). results the of NF-κB on ALI via PRDM1 In the current study that NF-κB(p65) promoted the miR-99b expression by the miR-99b promoter miR-99b was in ALI mouse lung tissues and targeted causing its the of ALI findings that miR-99b be in the of PRDM1 in ALI. miR-99b could as a ALI to our findings and further to the of experiments were by the and of of mice were as the control mice with ALI with LPS ALI mice with miR-99b ALI miR-99b mice with miR-99b ALI overexpression ALI mice with ALI mice with 1 ALI mice with 1 ALI RNA NF-κB mice with 1 ALI mice with 1 of which is a NF-κB ALI mice with 1 of and and ALI miR-99b mice with 1 of and miR-99b a mice were with a of the mice were by LPS as a in the with and lung tissues were 6 LPS MH-S cell and cells were from the cell of of were cultured in with and and the cells were cultured in with in a at and were to LPS MH-S cells at the of were in a cell The were to the The were by and the and were into the The were as LPS LPS LPS miR-99b LPS NF-κB LPS NF-κB LPS LPS LPS miR-99b miR-99b LPS and LPS mouse lung tissues from were into the 7 with and 1 with in the mouse lung tissues were observed an mouse lung tissues from were into and The kit was The NF-κB(p65) and PRDM1 were to the and at with the were with the the were with with 1 and were as were and and the or The mouse lung were from The of lung tissues was measured and using an The tissues were in an at were in the The of lung tissues was measured and and the was was to lung tissue The was and the was using to activity. activity of lung tissues was as the The expression of the protein were detected in the lung tissues using the kit In the cells were with with and with the on a by the of to the cells the cells were with the at The the was to the cells and at 1 was performed on the and the was which the expression of the were observed a RNA was from the cells and tissue with the of The RNA and were detected using For was using miRNA and was with The was performed using as a with to kit The was out using a and mRNA levels were as the the The are in and the the gene expression of the group and the control used in a protein was from the cells or tissue using a The were with the of to a the NF-κB(p65) and PRDM1 were to the and The the was with with the at and with the was used protein MH-S cells were with LPS and 24 to LPS and were to the MH-S with a of was to and of was measured at using an lung tissues were and the was mouse was The levels of IL-6, and in the were using an complex to the The kit was from the of the kit the cells were with and by the NF-κB(p65) ratio of was used of the complex was from with protein The of complex was and the was was performed to The of miR-99b promoter were as and of PRDM1 and the binding were into were cultured in a with gene and in with miR-99b or to the of the activity of and was measured using a kit activity was by activity. were performed using the were as was using an and multiple were on of by with multiple was The during the current study are from the on We like to our to the their on was by the and of Acute lung injury acute respiratory distress syndrome

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