The Roles And Underlying Mechanisms Of MALAT1 In The LPS-induced Inflammatory Response

Objective:There is accumulating evidence that lncRNAs are important regulators of physiological and pathological responses. MALAT1, an evolutionary conserved lncRNA in mammalian species, has been found to be associated with tumor development, but its potential importance in immune response is unknown. The purpose of this project is to address the mechanism underlying the regulation of inflammation response by MALAT1 in macrophages, thus enriching our understanding of the role of lncRNA in regulating the inflammatory response.Methods:To explore the role of MALAT1 in inflammation response, THP1-derived macrophages and qPCR are used to analyze the expression of MALAT1 before and after stimulation with TLR4 (Toll-like receptor 4) ligand LPS (Lipopolysaccharide). Whether knockdown of MALAT1 could regulate the expression of proinflammatory cytokines, TNFα、IL-1β and IL-6 of THP-1 macrophages stimulation with LPS are determined using qPCR and ELISA. To address the mechanism underlying the regulation of inflamation response by MALAT1, MALAT1 is knocked down and a reporter gene assay is performed with a NF-KB-dependent luciferase reporter construct to test the impact of MALAT1 on NF-κB activity. To find the the mechanism underlying the regulation of NF-kB activity by MALAT1, qPCR and Western blot are used to explore the influence of MALAT1 on the expression abundance of p50 and p65 and ChIP-qPCR is used to test the role of of MALAT1 in regulating the binding of NF-κB to NF-κB target promoters. RIP assay is used to explore wether MALAT1 interact with NF-κB. To explore weather NF-κB regulates the expression of MALAT1, an inhibitor of NF-κB, PDTC, is used to assess the effect of NF-κB inhibition on MALAT1 transcription. A series of luciferase reporter plasmids containing consecutive non-overlapping deletions spanning the 5’-flanking region of MALAT1 and a luciferase reporter plasmid containing a point mutations on the κB-like motif upstream of the MALAT1 transcription start site are used to determine weather there is a κB site in the promoter of MALAT1. Finally, ChIP is used to check the interaction between NF-κB and MALAT1 promoter.Results:MALAT1 is greatly upregulated in LPS activated macrophages and knockdown of MALAT1 is found to increase LPS-induced expression of inflammatory cytokine, TNFa and IL-6 rather than the production of IL-1β.The reporter gene assay suggests that MALAT1 inhibits the NF-κB activity. However, MALAT1 is found to have no effect on the expression of p50 and p65. Mechanistically, MALAT1 is found to interact with NF-κB subunits p65 and p50 in the nucleus and inhibit NF-κB binding to the promoters of TNFa and IL-6 as revealed by ChIP, RIP and the analysis of subcellular distribution of MALAT1. PDTC, an inhibitor of NF-κB, is found to abolish LPS-induced MALAT1 upregulaton which suggests that LPS-induced MALAT transcription depends on NF-κB. A κB consensus sequence is found in the promoter of MALAT1. Finally, ChIP is performed and the result shows that NF-κB directly binds to the promoter of MALAT1 to activate its transcription.Conclusion:MALAT1 is upregulated in macrophages treated with LPS. And MALAT1 was found to function through interacting with and sequestering NF-κB in the nucleus, leading to decreased binding of the p65/p50 transcription factor to target promoters which decreases the expression of TNFa and IL-6. Besides, LPS-induced MALAT transcription depends on NF-κB. We propose that MALAT1 acts as a negative feedback regulator of NF-κB activity to help tightly control TLR signaling pathways and avoid an overactive inflammatory response.