MiR-322 Negatively Regulates LPS-induced Inflammatory Response

Inflammation is the body’s normal self protection mechanism to eliminate pathogens and resistant pathogen invasion. But excessive inflammatory response will inevitably lead to inflammatory lesions, cause all sorts of autoimmune disease. Therefore,the precise regulation of the inflammatory response is particularly important. micro RNAs(mi RNAs)are non-coding small RNA molecules and the size are 19~24 nucleotides. It is highly conserved during evolution. mi RNAs can degradate the target m RNA or repress its translation through completely or partially binding to the target gene m RNA 3’UTR.mi RNAs mediated post-transcriptional level play an important role in inflammatory response and immune regulation.Mmu-mi R-322, homologous to human mi R-424, is an important member of the mi R-322 family. mi R-322 is differentially expressed in many kinds of disease, and is highly conserved in different tissues and cells. Recent research shows that, mi R-322 play an important role in cell proliferation, cell differentiation and other biological process, but the role of mi R-322 in immune regulation and biological function remains to be further studied. In this research, we analysis the relationship between inflammation and mi R-322;study the role of mi R-322 in macrophages proliferation and differentiation; mi R-322 target gene prediction and reveal the interaction.The purpose is to clarify the regulatory role of mi R-322 in inflammation, and lay the foundation for revealing its immune regulation function.Specific research methods are as follows:q PCR detection the expression of mi R-322: RAW264.7 cells were stimulated with LPS to detect the expression of mi R-322.The results showed that mi R-322 expression was significantly suppressed when exposed to LPS. It revealed that mi R-322 may be involved in the regulation of inflammatory response.Effects of mi R-322 on the inflammatory response: mi R-322 mimics were transfected to RAW264.7 cells which were stimulated with LPS,and then analyzed m RNA expression levels of IL-1β、IL-6、TNF-α by q PCR.The results showed that the m RNA expressions of IL-1β、IL-6、TNF-α were significantly suppressed when transfected with mi R-322. It revealed that mi R-322 negatively regulates LPS-induced inflammatory response.Effects of mi R-322 on macrophages proliferation: mi R-322 mimics were transfectedto RAW264.7 cells which were stimulated with LPS,and then analyzed m RNA expression levels of cyclin D cyclin E P21, P27 by q PCR.The results showed that,when transfected with mi R-322, m RNA expressions of cyclin D、cyclin E were significantly increased, P21、P27 were significantly suppressed. Subsequently, further validation found that overexpression of mi R-322 promotes macrophage cell cycle progression by flow cytometry. It suggested that mi R-322 can promote cell proliferation, alleviate inflammatory damage. Next,we use MTT assay to detecte the viable cells, it also proved the same results.Target genes prediction: Predicted with target gene prediction software(mi Randa,targetscan), we found that mi R-322 can bind to 3’UTR of NF-κB1 m RNA, suggesting that NF-κB1 maybe mi R-322’s target gene.mi R-322 target genes validation: NF-κB1 3’UTR was cloned into the dual luciferase plasmid psi CHECKTM-2, to construct dual luciferase reporter vector and mutant, then transfected to 293 T cells with mi R-322 mimics or inhibitors, detecting the luciferase activity, the results showed that mi R-322 transfection group luciferase activity was significantly reduced, significant difference(P<0.0001), and mi R-322 inhibitors group luciferase activity was significantly increased, the significant difference(P<0.05),indicating that mi R- 322 can interact with the NF-κB1.After these,we use q PCR and western blot to detect NF-κB1’s m RNA and protein expression levels when exposed to mi R-322 mimics or inhibitors, and the results showed that NF-κB1 m RNA and protein levels were significantly reduced when transfected with mi R-322 mimics,while it showed the opposite result when transfected with mi R-322 inhibitors.It indicated that NF-κB1 is mi R-322’s target gene,and it suppresses NF-κB1 expression by degrading its m RNA.Conclusion: mi R-322 negatively regulates LPS-induced inflammatory response through inhibiting the NF-κB1 expression, and mi R-322 can promote cell proliferation,alleviate inflammatory injury.