The Role And Mechanism Of METTL3 And E2F1 In Mouse Depression Model

Objective:RNA methylation is an important form of post-transcriptional modification regulation and plays a key role in regulation of gene expression.N6-methyladenosine(m6A),a universal reversible modification of mRNA in mammalian cells,has been shown to regulate mRNA processing,translation,and decay.Recent studies have found that m6A in the adult mammalian brain represents a new window in the regulation of gene expression after stress,and dysregulation of m6A expression may contribute to the pathophysiological regulation and recovery of stress-related mental diseases.However,the role and underlying mechanism of m6A methylation in depression is largely unknown.Based on the above research background,this study mainly focused on the role m6A methyltransferase METTL3 in chronic unpredicted stress(CUS)and lipopolysaccharide(LPS)induced depression-like behaviors in mice.Methods:(1)CUS and LPS that are animal models of depression were used to induce depression-like behaviors in mice,respectively.Real-time quantitative PCR(qPCR)and Western blot(WB)were used to determine the effects of CUS and LPS on the levels of METTL3 and E2F1 in the mouse medial prefrontal cortex(mPFC).Pearson correlation analysis was used to determine correlation of METTL3 and E2F1 mRNA levels.(2)Dot blot hybridization and m6A methylation quantification kit were used to detect the methylation level of m6A in the mPFC.(3)To determine the effect of METTL3 overexpression on the levels of synaptic proteins and inflammatory factors in the mPFC in the mouse model of depression induced by LPS,WB and qPCR were used.(4)The effect of E2F1 knockdown on the expression of synaptic proteins and inflammatory factors in the mPFC was evaluated by WB and qPCR in the LPS-induced mouse depression model.(5)Biosignal analysis and RNA methylation immunoprecipitation(RIP)were used to determine whether E2F1 contains the m6A methylation modification sites.(6)In vitro transfection of siRNA or shRNA was used to study the effect of reducing endogenous METTL3 on E2F1 expression.(7)Determined whether E2F1 is a METTL3 substrate by dot blot hybridization,m6A-RIP and luciferase reporter gene.(8)In vitro transfection and immunofluorescence staining were used to explore the mechanism through which METTL3 and YTHDF2 regulated E2F1 expression.(9)ChIP-qPCR was used to determine the mechanism through which METTL3 and E2F1 regulated the expression of inflammatory factors.(10)By infecting cells with lentivirus in vitro to overexpress METTL3 or knock down endogenous E2F1 expression level,studied the effect of METTL3/E2F1 pathway on LPS-induced inflammatory factor expression levels in vitro.Results:(1)CUS and LPS induced depression-like behaviors in mice,respectively;CUS and LPS respectively decreased the level of METTL3 and increased the level of E2F1 in the mouse mPFC,the levels of METTL3 and E2F1 were negatively correlated.(2)CUS and LPS increaseed m6A methylation levels in mPFC of mice,respectively.(3)METTL3 overexpression effectively reversed the increase in the levels of NMDA receptors and inflammatory cytokiness induced by LPS.(4)Reducing endogenous E2F1 inhibited the LPS-induced increase in NMDA receptor and inflammatory factor expression levels.(5)There is a potential m6A methylation site in E2F1.(6)Knockdown of METTL3 in vitro up-regulated the level of E2F1.(7)E2F1 is a substrate of m6A methyltransferase METTL3.(8)METTL3 reduced.the expression of E2F1 in YTHDF2-dependent manner.(9)E2F1 as a transcription factor promoted the expression of inflammatory cytokines.(10)The METTL3/E2F1 pathway inhibited the expression of inflammatory factors induced by LPS in vitro.Conclusions:(1)CUS and LPS induced depression-like behavior in mice.(2)CUS and LPS reduced the level of METTL3 and increased the level of E2F1 in the mouse mPFC,respectively,the levels of METTL3 and E2F1 were negatively correlated.(3)CUS and LPS caused a decrease in m6A methylation level in the mPFC.(4)E2F1 is the downstream signal molecule of METTL3.(5)The METTL3/E2F1 pathway regulated the expression of NMDA receptors by inhibiting inflammatory factors.(6)METTL3 decreased the expression of E2F1 via YTHDF2.(7)METTL3/E2F1 inflammatory cytokinesmay play an important regulatory role in LPS-induced depression-like behavior in mice.