METTL3-mediated EVL M6A Modification Promotes Renal Fibrosis And The Targeted Therapeutic Effect Of Isoforsythiaside

Renal fibrosis is one of the major pathogenic mechanisms and pathologies shared within multiple chronic kidney disease（CKD）processes and a bridge between acute kidney injury（AKI）and CKD.Existing studies have shown that the pathogenesis of renal fibrosis is complex and not yet fully elucidated,which directly leads to the limited effectiveness of specific drugs targeting the pathological process.During long-term mechanistic exploration,RNA epigenetic modifications have been proven to affect renal fibrosis and even the progression of CKD as well as AKI through multiple mechanisms,in which RNA m6A methylation modifications play a nonnegligible role.However,therapeutic strategies that target and regulate RNA m6A methylation modifications to prevent renal fibrosis and related diseases are still under investigation.To elucidate the regulatory mechanisms of RNA epigenetic modifications on renal fibrosis,identify potential therapeutic targets and explore promising drugs:1.This study investigated the alterations and characteristics of RNA m6A modifications in the renal fibrosis process.By exploring human biopsies,mouse fibrosis and cultured HK-2 cell models,we clarified the prominent changes in METTL3-mediated m6A modification in the process of renal fibrosis and confirmed that METTL3 and m6A modification may be important features during renal fibrosis.2.We explored the detailed mechanism of METTL3-mediated m6A modifications in regulating renal fibrosis.Through Me RIP-seq together with RNA-seq and other experiments,we confirmed that METTL3 promotes m6A modification of EVL mRNA in an IGF2BP2-dependent manner.This may be one of the main mechanisms by which m6A modification regulates renal fibrosis.3.We investigated the potential therapeutic effects of traditional Chinese medicine（TCM）monomers on the METTL3-EVL m6A modification axis for renal fibrosis treatment.Through high-throughput virtual screening coupled with high-content imaging technologies,we confirmed the antifibrotic effects and capabilities of isoforsythiaside,which can inhibit the activity of METTL3 and thus regulate the m6A modification of EVL,in both cellular and animal assays.Methods:1.1 Characteristics of RNA m6A modifications/regulators in renal fibrosis1.1.1 To explore the profile of m6A modifications and regulators in human biopsiesAfter approval by the Biomedical Ethics Committee of Anhui Medical University,renal tissues of patients with hydronephrosis/obstructed kidney and paraneoplastic tissues of renal carcinoma were collected based on the demographic and disease diagnostic data of the patients.H&E,Masson and Sirius Red staining were used to observe the histopathological and fibrotic changes in kidney tissues.The m6A methylation quantification assay with immunofluorescence staining was performed to determine the total levels of m6A modification.q RT?PCR was used to detect the expression of m6A methyltransferases and demethylases in kidney tissues.Western blot and immunofluorescence staining were used to measure the fibrosis indicators and METTL3protein expression in kidney tissues.Pearson correlation coefficients were used to assess the correlation between the degree of fibrosis,METTL3 levels and m6A modifications.1.1.2 To investigate the profile of m6A modification/regulators in mouse fibrotic kidneysAfter approval of the Experimental Animal Ethics of Anhui Medical University,UUO-renal fibrosis（UUO-3,7 and 14 days）and AKI-to-CKD-renal fibrosis（I/R-7 and14 days）models were established by unilateral ureteral ligation and ischemia?reperfusion of bilateral renal arteries,respectively.H&E,Masson and Sirius Red staining were used to observe the histopathological and fibrotic changes in kidney tissues.Total m6A levels in kidney tissues were observed by m6A methylation quantification assay and immunofluorescence staining.Western blot and q RT?PCR were used to detect the mRNA and protein levels of m6A-related methyltransferases and demethylases.The distribution and localization of METTL3 were observed by immunofluorescence staining.1.1.3 To explore the profile of METTL3/m6A modification in fibrotic HK-2 cellsThe fibrosis models of renal tubular epithelial（HK-2）cells were established by TGF-β1（5 ng/ml）and hypoxia-reoxygenation[H/R（9/3 h,3 cycle）].The expression and subcellular localization of m6A modification in cultured HK-2 cells were observed by immunofluorescence staining.Western blot and q RT?PCR were used to detect the mRNA and protein changes in METTL3 in kidney tissues.Pearson’s correlation coefficient was used to calculate the correlation between the fibrosis indicators and METTL3 levels.1.2 Role and mechanism of METTL3-mediated EVL m6A modification in renal fibrosis1.2.1 To explore the function of METTL3-mediated m6A modification in fibrotic kidneysMETTL3 conditional knockout（c KO）mice were constructed by Ksp/Cre and METTL3^Flox/Flox and identified by agarose gel electrophoresis and immunofluorescence staining.UUO-7D and I/R-14D models were established,and H&E and Masson staining were used to observe the effects of METTL3 c KO on histopathological and fibrotic alterations in mouse kidneys.Western blot was used to detect the expression of fibrotic indicators and METTL3 in kidney tissues.Immunofluorescence staining was used to observe the effect of conditional knockdown of METTL3 on the fibrotic responses of HK-2 cells induced by TGF-β1（5 ng/ml）and H/R（9/3 h,3 cycles）.1.2.2 Screening and validation of METTL3-mediated m6A modification targetsThe m6A methylation and transcriptome sequencing of mRNA in kidney tissues were performed by Me RIP-seq and RNA-seq.Bioinformatics technology was used to create a correlation profile between mRNA level changes and mRNA m6A modifications.Venn diagrams were plotted with fold change（FC）≥2 and P<0.05,and candidate target genes were filtered.q RT?PCR was used to detect the effect of METTL3 c KO on the mRNAs of candidate target genes for further screening and to validate the expression of candidate target genes in the kidney tissues of patients.To construct candidate target genes,si RNA was used to detect the effect of candidate target genes on TGF-β1-induced fibrotic responses in cultured HK-2 cells to identify the target genes with the most significant changes in expression and antifibrotic effects.Immunofluorescence staining and Western blot were used to detect and verify the expression,localization and changes in candidate target genes in the process of renal fibrosis.1.2.3 Effect and possible mechanism of the downstream target EVL on renal fibrosisWestern blot,q RT?PCR and immunofluorescence staining were performed to detect the effect of EVL silencing on fibrotic indicators in TGF-β1-and H/R-treated HK-2 cells.Western blot and immunofluorescence staining were used to observe the changes in the expression of Smad3 and p-Smad3.Molecular docking was performed to explore the potential interaction between EVL and Smad7.Co-IP assay was carried out to verify the interaction between EVL and Smad7.1.2.4 To investigate the mechanism of METTL3-mediated m6A modification of EVLBioinformatics techniques were used to predict the presence of m6A modification in EVL mRNA.The enrichment of METTL3 in the EVL mRNA was detected by Me RIP-q PCR.The direct relationship between METTL3 and EVL mRNA was verified by rescue experiments.An actinomycin assay was performed to detect the effect of silencing METTL3 on EVL mRNA decay after TGF-β1 and H/R stimulation.IGF2BP1/2/3si RNAs were constructed separately,and the effect of IGF2BP1/2/3 silencing on EVL expression was measured by q RT?PCR after verifying the silencing efficiency,and the IGF2BP subtypes with possible roles were screened.An actinomycin assay was used to detect the effect of silencing IGF2BP2 on EVL mRNA decay after TGF-β1 and H/R stimulation.RIP-q RT?PCR was used to explore the interaction between IGF2BP2 and EVL.Immunofluorescence staining and Western blot were used to detect the effect of silencing METTL3 and IGF2BP2 on EVL and fibrosis indicators,respectively.1.3 Research on the screening and effects of TCM monomers targeting the METTL3-EVL m6A axis for renal fibrosis1.3.1 High-throughput screening of TCM monomersThe METTL3 protein structure data（PDB ID:6TTW）were queried from the PDB database.Shrodinger software（Version 11.5）was used for virtual screening and ranking according to the docking score based on computer-aided design of the traditional Chinese medicine（TCM）monomer database（L6810）.1.3.2 Further screening and validation of candidate TCM monomersThe toxicity of the top 20 TCM monomers in terms of the docking score to HK-2cells was tested by CCK-8 assay,and the appropriate concentrations were selected.The inhibitory effects of the top 20 TCM monomers on the fibrosis indicators of HK-2 cells at different concentrations were detected by high-content imaging.Molecular docking of the candidate TCM monomers was performed,and their effects on METTL3 protein expression were detected by Western blot.Effects of the candidate TCM monomers on the total m6A levels of HK-2 cells,the expression levels of EVL and the fibrosis indicators were detected by cellular immunofluorescence staining.1.3.3 Regulatory mechanisms of isoforsythiaside on the METTL3-EVL m6A axisMolecular docking to demonstrate the binding characteristics of isoforsythiaside and METTL3 protein.A CETSA assay was used to verify the binding of isoforsythiaside to the METTL3 protein.Western blot was conducted to detect the effect of different concentrations of isoforsythiaside on the fibrotic response of HK-2 cells.HE,Masson and Sirius Red staining were performed to observe the effects of different doses of isoforsythiaside on renal fibrosis in mice.Immunohistochemistry was performed to detect the changes in EVL expression in fibrotic kidney tissues,and immunofluorescence staining was performed to observe the effects of isoforsythiaside on total m6A levels.Results:2.1 Profile of regulators and m6A modifications in renal fibrosis2.1.1 Profile of m6A modifications and regulators in human biopsiesCompared with the control group（renal cell carcinoma adjacent tissue）,the fibrotic kidney tissue of patients with hydronephrosis/obstruction（obstructed nephropathy,ON）showed obvious fibrotic features such as inflammatory cell infiltration,reddish homogeneous tubular pattern with transparent tubular shape,and loss of tubular structure.Masson staining showed a significant increase in collagen fibers in the ON renal tissues.Sirius Red staining also confirmed these changes.Renal fibrosis indicators included type I collagen alpha 1（COL1A1）,alpha-smooth muscle actin（α-SMA）and fibronectin（FN）,methylation transferases METTL3,METTL14,WTAP,and VIRMA,and demethylases.VIRMA and the demethylase FTO were significantly increased,while the mRNA levels of the demethylase ALKBH5 were significantly decreased.Meanwhile,the total m6A levels in renal tissues of ON patients significantly increased and showed a positive correlation with fibrosis.Consistently,the expression of METTL3 in renal tissues of ON patients was significantly elevated mainly in the nucleus of tubular epithelial cells and showed a positive correlation with total m6A levels and fibrosis indicators to a moderate degree.These results suggest that multiple regulators are involved in enhancing m6A modification and ultimately promoting renal fibrosis.2.1.2 Profile of regulators and m6A modification in mouse fibrotic renal tissuesCompared with the sham-operated group（Sham）,renal tissues of UUO（3D-7D-14D）and I/R（7D-14D）mice showed renal fibrosis features,which progressively increased with the disease duration.The increase in collagen fibers was also observed with Masson and Sirius red staining,and it progressively increased with disease duration.The levels of total m6A modification and METTL3 in the kidney tissues of UUO and I/R mice increased gradually with disease progression and were mainly concentrated in the tubular region.With the prolongation of the disease,the protein levels of renal fibrosis indicators,including COL1A1,α-SMA and FN,and METTL3,increased significantly,while WTAP decreased significantly,and the trends of METTL14,WTAP,VIRMA,FTO and ALKBH5were not significant.Renal fibrosis indicators and METTL3 protein levels were significantly increased in I/R（7D-14D）mice.These results suggest that m6A modification was significantly enhanced in both UUO and AKI-to-CKD renal fibrosis,closely related to the progression of renal fibrosis and mainly regulated by METTL3.2.2 METTL3 regulates EVL m6A modification to promote renal fibrosis2.2.1 Function of METTL3 and m6A modification in fibrotic kidneysFluorescent dual-staining results showed that METTL3 was mainly expressed in the tubular region of fibrotic kidney tissues,especially near the nucleus.Conditional knockout of METTL3 in the kidney significantly attenuated histopathology as well as fibrosis,which was consistent with the Western blot assay results.Meanwhile,METTL3silencing significantly reduced TGF-β1（5 ng/ml）-and H/R（9/3 h,3 cycle）-stimulated overactivation of m6A modification and fibrotic responses.These results suggest that METTL3 and m6A modification are overexpressed and activated in the tubular region,especially in the cytosolic region of tubular epithelial cells,in response to multiple factors and stimuli-induced renal fibrosis and HK-2 fibrosis.Silencing and knocking out METTL3 significantly reduced m6A modification in HK-2 cells and the cellular fibrosis response in HK-2 cells,ultimately alleviating renal fibrosis.2.2.2 METTL3 mediates the m6A modification of downstream target genes of EVLIn normal and fibrotic kidney tissues,3909 mRNAs showed elevated m6A modification and self-expression,and 2978 mRNAs showed decreased m6A modification and self-expression.A total of 3094 genes were significantly upregulated and correlated with m6A modification levels,while 409 genes were significantly downregulated and correlated with m6A modification levels in METTL3 knockout kidneys.Venn analysis screened 21 significantly changed candidate target genes,of which Pola2 and Wfdc15B were excluded as illogical.q RT?PCR results showed that ACTA1,EVL,LSP1,NPY6R,PGLYRP2,TCEAL5 and VGF were consistent with sequencing results in mouse and human biopsies.EVL silencing more significantly reduced the fibrotic response than ACTA1,LSP1,NPY6R,PGLYRP2,TCEAL5 and VGF silencing.Based on this,EVL is a potential target gene of METTL3-mediated m6A modification.Immunofluorescence staining showed that EVL was mainly enriched in the tubular region of fibrotic kidneys in patients and mice,which was consistent with the METTL3-mediated m6A modification profile,suggesting that METTL3 may regulate the m6A modification of EVL mRNA involved in renal fibrosis.2.2.3 Effect and possible mechanism of EVL on the renal fibrosis processWestern blot and q RT?PCR showed that silencing EVL not only significantly reduced the mRNA levels of fibrotic indicators in HK-2 cells but also obviously reduced their protein levels.Meanwhile,silencing EVL significantly reduced the expression and nuclear translocation of p-Smad3 but had no significant effect on Smad3.Molecular docking showed an interaction between EVL and Smad7 and between Smad7 and TGFβR1,which was also verified by Co-IP assays.2.2.4 METTL3 regulates the m6A modification of EVL mRNA in an IGF2BP2-dependent mannerBioinformatic predictions revealed multiple m6A modification sites on EVL mRNA.Me RIP-q PCR identified that METTL3 was enriched in the mRNA of the downstream target gene EVL.Rescue experiments showed that silencing EVL significantly reduced the fibrosis caused by METTL3 overexpression,which verified that METTL3 affects the fibrotic process by regulating the m6A modification of EVL mRNA.Actinomycin assays showed that METTL3 silencing shortened the half-life of EVL mRNA.Only IGF2BP2silencing significantly reduced EVL mRNA levels when IGF2BP1,2 and 3 were silenced,and RIP assays further confirmed the interaction between IGF2BP2 and EVL mRNA in TGF-β1（5 ng/ml）and H/R（9/3 h,3 cycle）stimulated HK-2 cells.Meanwhile,silencing IGF2BP2 significantly inhibited the protein expression of EVL.Silencing IGF2BP2significantly decreased the stability of EVL mRNA in actinomycin D-treated HK-2 cells.2.3 Effects of the isoforsythiaside-regulated METTL3-EVL m6A axis on renal fibrosis2.3.1 High-throughput screening of TCM monomers that target METTL3The METTL3 small molecule inhibitor STM2457 was used as a positive control,and the METTL3 protein structure was molecularly docked with TCM monomers for virtual screening.After sorting according to docking score,several TCM monomers were found to have binding scores close to the STM2457 binding score,and the top 20 TCM monomers were selected for follow-up study2.3.2 Screening and validation of the candidate TCM monomersAn MTT assay was performed to select the appropriate cellular drug concentrations（0.39,0.78 and 1.56μM）.High-content imaging results showed that T5796,T3S1088 and T2993 could inhibit the protein expression of COL1A1 andα-SMA to different degrees.Western blot analysis showed that T5796,T3S1088 and T2993 did not affect the protein expression of METTL3,suggesting that all three monomers may affect the fibrotic response by affecting the enzyme activity of METTL3.Immunofluorescence staining showed that T3S1088 could most significantly reduce the level of total m6A modification in TGF-β1（5 ng/ml）and H/R（9/3 h,3 cycle）-stimulated HK-2 cells,suggesting that T3S1088（isoforsythiaside）may be the most promising TCM monomer for targeted regulation of the METTL3-EVL m6A axis.2.3.3 Isoforsythiaside inhibits the METTL3-EVL m6A axis to alleviate renal fibrosisMolecular docking supported the potential interaction between isoforsythiaside and METTL3 protein,which was further confirmed by the CETSA assay.There were significant differences in the denaturation temperature（from room temperature to 62°C）of METTL3 protein with and without isoforsythiaside treatment,i.e.,isoforsythiaside caused significant thermal transfer of METTL3 protein in HK-2 cells.Isoforsythiaside（0.5,1.0 and 2.5μM）differentially inhibited the elevated EVL expression as well as the fibrotic response in HK-2 cells induced by TGF-β1（5 ng/ml）and H/R（9/3 h 3 cycle）.The antifibrotic effect of isoforsythiaside was confirmed by HE,Masson,and Sirius Red staining.In vitro studies showed that isoforsythiaside（10,25 and 50 mg/kg）attenuated renal pathological changes,fibrosis degree,total m6A modification and EVL expression levels to varying degrees.These results suggest that isoforsythiaside could be a potential anti-renal fibrosis agent by inhibiting the METTL3-EVL m6A modification axis.Conclusions:1.Overexpression of METTL3 and m6A modification are strongly associated with renal fibrosis.2.METTL3 regulates the m6A modification of EVL mRNA to enhance its stability and expression in an IGF2BP2-dependent manner.3.Overactivated METTL3-EVL m6A axis promotes the fibrotic response of HK-2 cells and renal fibrosis.4.Inhibition of METTL3 and the METTL3-EVL m6A axis by isoforsythiaside attenuates renal fibrosis.5.Targeted modulation of the METTL3-EVL m6A axis is a potential therapeutic strategy against renal fibrosis.