The Role And Mechanism Of YTHDF2-meditated M6A Modification Of IL-6R MRNA Inhibiting The Release Of HMGB1 In Sepsis

BackgroundSepsis,defined as a lethal organ dysfunction caused by the "non-homeostatic" response of the host,is usually manifested as severe systemic infection,blood coagulation dysfunction,tissue damage,and multiple organ failure.It is the predominant complication of severe burns and remains the leading cause of mortality.Treatment strategies for sepsis contain infection control(such as antibiotic administration,surgical removal of infectious sources),maintaining hemodynamic stability(such as fluid administration,anti-shock treatment),and organ support therapies(such as mechanical ventilation,renal replacement therapy).Although the treatment strategies for sepsis have been extensively studied,to date,symptomatic supportive care has been the most common treatment for sepsis,and no specific drugs for sepsis have emerged in clinics.Therefore,the treatment strategies for sepsis still need to be further studied.The pathogenesis of sepsis is complex,involving multiple pathological processes such as dysregulated inflammatory response,mitochondrial damage,immune dysfunction,and autophagy,of which dysregulated inflammatory response is critical,and occurs throughout the whole process of the occurrence and development of sepsis.However,the pathogenesis of sepsis remains unclear,so elucidating the molecular mechanisms that regulate the inflammatory response in sepsis will undoubtedly help to provide novel targets for the effective prevention and treatment of sepsis.In recent years,apart from the classic inflammatory factors like IL-6,TNF-α,and C-reactive protein and so on,it has been found that serum levels of high mobility group box-1 protein(HMGB1)in patients with sepsis are elevated,and are closely related to the lethality of sepsis.HMGB1 is a nuclear protein present in almost all cells.As an evolutionarily conserved protein,HMGB1 maintains the stability of DNA structure and regulates the activity of transcription,and low level of HMGB1 can be found in cytoplasm and serum under physiological conditions.Additionally,HMGB1 functions as a secreted protein,whose passive or initiative release from cells can result in inflammatory response under stress conditions such as trauma,burns,and infection.The classic inflammatory response occurs relatively early,with the release of TNF-α or IL-1 reaching a peak level within a few hours in sepsis.In contrast,the release of HMGB1 is significantly later,reaching its peak level in 16 to 32 hours after the sepsis onset.As a late inflammatory mediator,HMGB1 can promote the release of early inflammatory factors such as TNF-α,IL-1,IL-6 and IL-8,and these inflammatory factors can in turn promote the release of HMGB1,thereby continuously amplifying inflammatory signals,and leading to dysregulated inflammatory response in sepsis.Targeting HMGB1 undoubtedly has a wider therapeutic window compared to regulating early inflammatory factors for treating sepsis.However,only the extracellular HMGB1 can function as a proinflammatory molecule.Therefore,inhibiting the release of HMGB1 is expected to become an effective strategy for the treatment of sepsis.The inflammatory signaling pathway JAK2/STAT1 has been reported to be a key pathway mediating the release of HMGB1 and play a vital role in the regulation of inflammatory response in sepsis.Therefore,effective inhibition of the activation of JAK2/STAT1 pathway can impede the release of HMGB1,contributing to the prevention and treatment of sepsis.It has been reported that IL-6R(interleukin-6 receptor),as a key molecule in the activation of the JAK2/STAT1 pathway via binding to the critical inflammatory factor IL-6,can elicit amplification of the inflammatory cascade.However,the upstream regulatory mechanism of IL-6R remains unclear and deserves further study.In recent years,6-methyladenine(m6A)modification has received increasing attention from scholars in the initiation and progression of inflammatory diseases.m6 A,which is a kind of RNA modification in the N6-position of adenosine in eukaryotic cells,can affect gene expression and play an important role in the posttranscriptional regulation.Its regulation is mainly accomplished by three types of molecules,including m6 A binding proteins(m6A readers),methyltransferases(m6A writers)and demethylases(m6A erasers).Among them,YTH Domain Family Member 2(YTHDF2)is a widely studied m6 A reader that can mediate the degradation of m6 A transcripts by specifically recognizing and binding to m6 A transcripts,which is closely related to the pathological and physiological processes of eukaryotic organisms,such as embryonic development,differentiation in pluripotent stem cells,and the initiation and progression of tumors.However,few studies have shown that YTHDF2 is involved in the regulation of inflammatory response in sepsis.Interestingly,our research team found that YTHDF2 can significantly inhibit the release of HMGB1 through pre-experiments,and potential m6 A modification sites recognized by YTHDF2 were found in the 3’-UTR of IL-6R m RNA by using the RMVar database.Based on the above analysis,our study will investigate whether the m6 A reader YTHDF2 can play an anti-inflammatory role in sepsis by recognizing and binding to the m6A-modified site in the IL-6R m RNA,consequently block the activation of the JAK2/STAT1 pathway and finally reduce the release of HMGB1.Part 1 The role of YTHDF2 in regulating inflammatory response by inhibiting the release of HMGB1Methods:1.RAW264.7 cells were treated with 1μg/m L LPS,and were collected at designated times(3,6,12,24,48,72 hours),then the expression of YTHDF2 was detected by q PCR and WB.2.Overexpression or knockdown of YTHDF2 in RAW264.7 cells.RAW264.7 cells were transfected with YTHDF2-overexpressing plasmid or YTHDF2 small interfering RNA(si RNA),then we detected the expression of YTHDF2 by q PCR and WB.3.RAW264.7 cells were transfected with YTHDF2-overexpressing plasmid(OE YTHDF2)or YTHDF2 si RNA(si YTHDF2)for 48 hours and treated with 1μg/m L LPS for24 hours,and then the cell supernatants were collected for detecting the expression of IL-6,TNF-α and IL-1 β by ELISA.4.RAW264.7 cells were transfected with YTHDF2-overexpressing plasmid(OE YTHDF2)or YTHDF2 si RNA(si YTHDF2)for 48 hours and treated with 1μg/m L LPS for24 hours,and then the cell supernatants were collected to detect the expression of HMGB1 by ELISA.RAW264.7 cells were treated with 1μg/m L LPS for 48 hours,cytoplasmic and nuclear proteins were extracted,and the expression of HMGB1 in the cytoplasm and nucleus was detected by WB.RAW264.7 cells were treated with 1μg/m L LPS for 12 hours,and the localization of HMGB1 in cells was visualized by confocal microscopy.5.RAW264.7 cells were transfected with YTHDF2-overexpressing plasmid for 48 hours and then treated with 1μg/m L LPS or 2μg/m L HMGB1 for 16 hours,the levels of TNF-α,IL-1β and IL-6 in the culture supernatants were determined by ELISA.Results:1.YTHDF2 is downregulated in LPS-stimulated RAW264.7 cellsAfter LPS treatment,the m RNA and protein levels of YTHDF2 in RAW264.7 cells were significantly downregulated.2.Overexpression or knockdown of YTHDF2 in RAW264.7 cellsThe results of detecting YTHDF2 by q PCR and WB confirmed that the RAW264.7 cell model with overexpression or knockdown of YTHDF2 was successfully constructed,and si YTHDF2 # 3 had the most significant knockdown effect among three types of YTDHF2 si RNAs(si YTHDF2 # 1,si YTHDF2 # 2,and si YTHDF2 # 3).Therefore,we chose si YTHDF2 # 3 for following experiments.3.YTHDF2 alleviates LPS-induced inflammatory response in RAW264.7 cellsOverexpression of YTHDF2 reduced the levels of TNF-α,IL-1β and IL-6 in cell supernatants,while knockdown of YTHDF2 increased the levels of TNF-α,IL-1β and IL-6in cell supernatants.4.YTHDF2 inhibits the release of HMGB1 in RAW264.7 cellsOverexpression of YTHDF2 decreased the levels of HMGB1 in cell supernatants,while knockdown of YTHDF2 increased the levels of HMGB1 in cell supernatants.Western blot analysis revealed that overexpression of YTHDF2 reduced the cytoplasmic HMGB1 expression and conversely up-regulated nuclear HMGB1 expression under LPS induction.However,when we inhibited the expression of YTHDF2,the cytoplasmic and nuclear HMGB1 expressions were increased and decreased respectively.Immunofluorescent staining confirmed that overexpression of YTHDF2 inhibited the translocation of HMGB1 from nucleus to cytoplasm under LPS treatment while knockdown of YTHDF2 promoted the HMGB1 translocation.5.HMGB1 can reverse the inhibitory effect of YTHDF2 on inflammatory responseAfter treatment with LPS,the levels of TNF-α,IL-1β and IL-6 were up-regulated,while after overexpression of YTHDF2,the levels of TNF-α,IL-1β and IL-6 were down-regulated.However,administration of HMGB1 remarkably restrained the inhibitory effect of YTHDF2 on the secretion of TNF-α,IL-1β and IL-6.The data illustrated that treating cells with HMGB1 could reverse the inhibitory effect of YTHDF2 on LPS-induced inflammatory response.Part 2 Study on the molecular mechanism of YTHDF2 inhibiting the release of HMGB1Methods:1.RAW264.7 cells were transfected with YTHDF2-overexpressing plasmid or YTHDF2 si RNA for 48 hours and then treated with 1μg/m L LPS for 48 hours,the phosphorylation levels of STAT1 and JAK2 were determined via Western blotting.RAW264.7 cells were transfected with YTHDF2-overexpressing plasmid or YTHDF2 si RNA for 48 hours and treated with 1μg/m L LPS for 24 hours,then incubated with actinomycin D for 3,6 and 9 hours,the JAK2 and STAT1 m RNA levels were analyzed by q RT-PCR.2.RAW264.7 cells were transfected with YTHDF2-overexpressing plasmid or JAK2-overexpression plasmid for 48 hours and then treated with 1μg/m L LPS for 12 hours,the levels of HMGB1,TNF-α,IL-1β and IL-6 in the culture supernatants were determined by ELISA.3.RAW264.7 cells were transfected with YTHDF2-overexpressing plasmid or YTHDF2 si RNA for 48 hours and then treated with 1μg/m L LPS for 24 hours,the m RNA levels of IL-6R were analyzed by q RT-PCR.RAW264.7 cells were transfected with YTHDF2-overexpressing plasmid or YTHDF2 si RNA for 48 hours and then treated with1μg/m L LPS for 48 hours,the protein levels of IL-6R were analyzed by WB.RAW264.7cells were transfected with YTHDF2-overexpressing plasmid or YTHDF2 si RNA for 48 hours and treated with 1μg/m L LPS for 24 hours,then incubated with actinomycin D for 3,6 and 9 hours,the IL-6R m RNA levels were analyzed by q RT-PCR.4.We predicted the potential m6 A modification sites in IL-6R m RNA using the RMVar database.According to the predicted sequences,the dual-luciferase reporter containing fragments of m6 A modification sites(wild-type)or its mutant reporter was constructed,and then the luciferase reporter assays were performed.The cells were co-transfected with reporter vector(IL6R-3’-UTR WT or IL6R-3’-UTR)and plasmid [overexpressed YTHDF2(OE Ythdf2)or Control] and relative luciferase activity was measured.RIP-q PCR analysis was performed to identify the targeted effect of YTHDF2 on IL-6R m RNA.Results:1.YTHDF2 inhibits the phosphorylation of JAK2 and STAT1,but has no effect on the stability of JAK2 and STAT1WB results suggested that overexpressing YTHDF2 suppressed the phosphorylation of JAK2 and STAT1,while knockdown of YTHDF2 enhanced the phosphorylation of JAK2 and STAT1.Overexpression or knockdown of YTHDF2 had no effect on the stability of STAT1 and JAK2.These results showed that YTHDF2 inhibited the phosphorylation of JAK2 and STAT1,but as a m6 A "reader",YTHDF2 did not directly bind with JAK2 and STAT1 m RNA to regulate the JAK2/STAT1 signaling pathway.2.Overexpression of JAK2 can reverse the inhibitory effect of YTHDF2 on inflammatory responseAfter LPS treatment,the levels of HMGB1,IL-6,TNF-α and IL-1β in cell supernatants were up-regulated,overexpression of YTHDF2 reduced the levels of HMGB1,IL-6,TNF-αand IL-1β.However,overexpression of JAK2 remarkably attenuated the YTHDF2-mediated inhibition of HMGB1 and other inflammatory cytokines including TNF-α,IL-6 and IL-1β.These results suggested that overexpression of JAK2 can reverse the inhibitory effect of YTHDF2 on LPS-induced inflammatory response.3.YTHDF2 reduces the expression and stability of IL-6ROverexpression of YTHDF2 obviously downregulated IL-6R at both m RNA and protein levels in RAW264.7 cells,on the contrary,knockdown of YTHDF2 increased IL-6R levels.Overexpression of YTHDF2 decreased the stability of IL-6R m RNA,while knockdown of YTHDF2 increased the stability of IL-6R m RNA.4.YTHDF2 recognizes and binds to the m6 A binding site in IL-6R m RNAIn our study,the luciferase activity of wild-type reporter was significantly reduced compared to the vector control,while the mutant reporter abolished the reduction of luciferase activity in YTHDF2-over-expressing cells.RIP assays confirmed that IL-6R m RNA was enriched in the YTHDF2 group compared with Ig G group.The above results illustrated that YTHDF2 inhibited JAK2/STAT1 signaling by binding to the m6 A modification site of IL-6R 3’-UTR,leading to IL-6R m RNA degradation.Part 3 The role of YTHDF2/IL6R-JAK2/STAT1/HMGB1 pathway in inflammatory response in sepsisMethods:1.The levels of YTHDF2 in peripheral blood mononuclear cells of sepsis patients and sepsis mouse models were determined via q RT-PCR and WB.2.C57BL/6 mice were injected with AVV-YTHDF2(adeno-associated virus with YTHDF2 overexpression)via the tail vein to construct sepsis mouse models with YTHDF2 overexpression.Immunohistochemistry and HE staining were performed in mice lung tissues.Mice serums were collected to detect the levels of TNF-α and IL-6 by ELISA.Survival analysis of mice was performed using data processing software.3.The phosphorylation levels of JAK2 and STAT1 and the expression of IL-6R in mice lung tissues were detected by WB.Immunofluorescence staining was used to observe the cellular localization of HMGB1 in mice lung tissues and ELISA experiment was conducted to detect the expression of HMGB1 in mice serums.Results:1.YTHDF2 is downregulated in peripheral blood mononuclear cells of sepsisYTHDF2 expression was notably downregulated in peripheral blood mononuclear cells of sepsis patients and LPS-induced sepsis mouse models.2.YTHDF2 alleviates LPS-induced inflammatory response in septic mice and increases the survival rate of septic miceThe results of immunohistochemistry in mice lung tissues suggested that compared with the PBS group(control),YTHDF2 expression in the lung tissues of mice injected intraperitoneally with LPS induced sepsis was down-regulated,while the tail vein injection of AAV-YTHDF2(adeno-associated virus overexpressing YTHDF2)significantly increased the expression level of YTHDF2 in lung tissues.The results of HE staining and lung injury score suggested that overexpression of YTHDF2 alleviated lung injury in septic mice.ELISA assays showed that overexpression of YTHDF2 decreased the levels of HMGB1,TNF-α and IL-6 in serum.The results of survival analysis showed that overexpression of YTHDF2 increased the survival rate of septic mice.3.YTHDF2 inhibits IL-6R-JAK2/STAT1-HMGB1 pathway in mouse modelsOverexpression of YTHDF2 inhibited the activation of JAK2/STAT1 signaling pathway in alveolar cells and decreased the expression of IL-6R in mice.The results of immunofluorescence staining of lung tissues showed that compared with the control group mice injected intraperitoneally with PBS,mice injected intraperitoneally with LPS induced sepsis showed more translocation of HMGB1 from nucleus to cytoplasm,while overexpression of YTHDF2 in mice inhibited the translocation of HMGB1.Overexpression of YTHDF2 can reduce the levels of HMGB1 in mouse serum.Conclusion(1)YTHDF2 is involved in regulating the inflammatory response in sepsis,and alleviates the inflammatory response by inhibiting the release of HMGB1.(2)Mechanistically,YTHDF2 reduces the stability of IL-6R m RNA and inhibits the activation of JAK2/STAT1 signaling pathway to inhibit the release of HMGB1 by directly recognizing the m6A-modified site in IL-6R m RNA,and finally alleviates inflammatory response.(3)In vivo experiments have shown that YTHDF2 alleviates inflammatory responses in septic mice by inhibiting the IL-6R-JAK2/STAT1-HMGB1 signaling pathway,and increases the survival rate of septic mice.This study reveals the molecular mechanisms of YTHDF2 in regulating the inflammatory response in sepsis,which provides an experimental basis for the involvement of RNA methylation in sepsis,and provides useful clues for the prevention and treatment of sepsis by targeting the “YTHDF2/IL-6R m RNA m6 A modification /HMGB1” pathway.