mettl3介导的n6-甲基腺苷修饰对胃癌上皮-间充质转化和转移至关重要.ppt

1、METTL3-mediated N6-methyladenosine modification is critical for epithelial-mesenchymal transition and metastasis of gastric cancer,Background: m6A modification: dynamic reversible modification,m6A: The sixth N of adenosine is methylated. plays a role in post-transcriptional regulation： Regulates gen

2、e expression, splicing, RNA editing, and RNA stability Control the lifetime and degradation of mRNA Mediates the translation of cyclic RNAs,Background: m6A modification: dynamic reversible modification,m6A writers：methyltransferase METTL3、METTL14、WTAP、KIAA1429 Knockout of METTL3 decreased m6A peak m

3、RNA in mouse embryonic stem cells, Hela cells, and HepG2 cells m6A erasers：demethyltransferase FTO、ALKBH5 m6A readers：binding protein HNRNPC and HNRNPA2B1 YTHDF2 and YTHDF1 eIF3、HuR,EMT: epithelial-mesenchymal transition the biological process by which epithelial cells are transformed into cells wit

4、h mesenchymal phenotype through specific procedures Plays an important role embryonic development chronic inflammation tissue reconstruction cancer metastasis various fibrotic diseases Main features: Reduced expression of cell adhesion molecules (such as E-cadherin) conversion of cytokeratin cytoske

5、leton to vimentin,Background: EMT: epithelial-mesenchymal transition,As one of the most frequent chemical modifications in eukaryotic mRNAs, N6-methyladenosine(m6A) modification exerts important effects on mRNA stability, splicing, and translation. Recently, the regulatory role of m6A in tumorigenes

6、is has been increasingly recognized. However, dysregulation of m6A and its functions in tumor epithelial-mesenchymal transition (EMT) and metastasis remain obscure.,Introduction:,GC tissues: METTL3 mRNA expression was significantly increased METTL14 expression was decreased The levels of other m6A w

7、riters and erasers in the two data sets were inconsistent,Results:METTL3 overexpression and its prognostic value in GC,writers,erasers,The mRNA levels of METTL3 in GC tissues were significantly overexpressed METTL3 is significantly higher in late (III-IV) GC tissues Increased m6A mRNA levels observe

8、d in GC tissues,Results:METTL3 overexpression and its prognostic value in GC,Immunocytochemistry METTL3 is significantly localized in GC nuclei,Results:METTL3 overexpression and its prognostic value in GC,Patients with high METTL3 expression show poor overall survival (OS) and disease-free survival

9、(DFS),The expression level of METTL3 in GC tissues seems to be negatively correlated with E-cadherin and positively correlated with N-cadherin and vimentin,Results: METTL3 was required for EMT,MKN-28: Epithelial phenotype High E-cadherin Low N-cadherin Low vimentin,To validate whether METTL3 was req

10、uired for the EMT program loss- and gain-of-function studies,Results: METTL3 was required for EMT,Knockdown METTL3: E-cadherin up-regulation N-cadherin down-regulation Vimentin down-regulation Overexpress METTL3: Opposite phenomenon,Results: METTL3 was required for EMT,The global m6A modification le

11、vels in METTL3 knockdown and overexpression GC cells,Wound healing assay Attenuation of METTL3 expression significantly impeded the migratory ability of BGC823 cells Forced expression of METTL3 apparently increased the migration speed of MKN-28 cells,Results: METTL3 promoted GC cell invasion and met

12、astasis in vitro and in vivo,Transwell Matrigel invasion assay,Metastasized efficiency to the lungs of nude: Down-regulation group control group,Orthotopic mouse model assay Larger tumors in the METTL3 overexpression group than in the control group metastatic foci in the liver could be observed in M

13、ETTL3-overexpressing group,Results: METTL3 promoted GC cell invasion and metastasis in vitro and in vivo,Transcriptome-sequencing 798 genes were downregulated m6A-sequencing 850 peaks were diminished,Results: Transcriptome-sequencing and m6A-sequencing identified ZMYM1 as a direct target of METTL3,m

14、apped the m6A methylomes in BGC823 cells m6A consensus sequence GGAC (RRACH) motif was identified to be highly enriched within m6A sites m6A signal was enriched around the stop codon of mRNAs 15 peaks harbored by 13 genes two m6A peaks were detected near the stop codon Both were attenuated after MET

15、TL3 knockdown,Results: Transcriptome-sequencing and m6A-sequencing identified ZMYM1 as a direct target of METTL3,Relationship between ZMYM1 and METTL3 in mRNA and protein level,Results: METTL3 maintained ZMYM11 expression in GC,Immunofluorescence staining depletion of METTL3 induced loss of ZMYM1 ex

16、pression overexpression of METTL3 increased ZMYM1 levels,qRT-PCR ZMYM1 expression was elevated in GC tissues Positively correlated with METTL3 expression,Results: METTL3 maintained ZMYM11 expression in GC,confocal imaging The nuclear colocalization of METTL3 and ZMYM1 in GC tissues,knockdown or over

17、expression of METTL3, dramatically reduced or increased the m6A level of ZMYM1 mRNA,Results: METTL3 enhanced ZMYM1 mRNA expression through the m6A-HuR-dependent pathway,Luc: luciferase mutant ZMYM1: m6A modification was abrogated,Results: METTL3 enhanced ZMYM1 mRNA expression through the m6A-HuR-dep

18、endent pathway,Wild-type: transcription level of ZMYM1 decreased Mutant: essentially unchanged,Wild-type: transcription level of ZMYM1 increased Mutant: essentially unchanged,HuR protein: is one of the few known readers located in the nucleus HuR binding site was located in the m6A modification regi

19、on of ZMYM1,Results: METTL3 enhanced ZMYM1 mRNA expression through the m6A-HuR-dependent pathway,RIP-qPCR using anti-HuR antibody METTL3-silenced cells: reduced affinity of HuR to ZMYM1 mRNA Overexpression of cells: increased affinity of HuR to ZMYM1 mRNA,HuR expression showed no obvious changes in

20、all GC cells with modified METTL3 expression,Results: METTL3 enhanced ZMYM1 mRNA expression through the m6A-HuR-dependent pathway,Flag-tagged ZMYM1 significantly inhibited the reporter activity in a dose-dependent manner in both BGC823 and MKN-28 cells,Results: ZMYM1 was physically associated with t

21、he CtBP/LSD1/CoREST complex in the nucleus,Overexpression of ZMYM1 simply did not influence the activity of Gal4-driven reporter,Results: ZMYM1 was physically associated with the CtBP/LSD1/CoREST complex in the nucleus,co-immunoprecipitation ZMYM1 interacts with all testing proteins,immunoblotting w

22、ith antibodies against ZMYM1 ZMYM1 was efficiently coimmunoprecipitated by all the components of this complex,Re-ChIP assay Identified co-occupancy of ZMYM1, CtBP1, LSD1, and CoREST on the E-cadherin promoter,Results: Transcription repression of E-cadherin by the ZMYM1-associated complex,Validated t

23、he enrichments of all complex components on CRS (consensus recognition sequence),The attenuation of E-cadherin caused by ZMYM1 overexpression was abolished when CtBP1, LSD1, or CoREST was knocked down,Elevation of ZMYM1 expression Recapitulated the levels of Ncadherin and Vimentin Reduce the express

24、ion of E-cadherin in METTL3-knockdown cells Inhibition of ZMYM1: Counteract the acceleration of the EMT process caused by METTL3 overexpression partly,Results: METTL3-mediated epigenetic activation of ZMYM1 was responsible for EMT and metastasis of GC,Transwell invasion assay Knockdown of METTL3 dec

25、reased the invasive ability of GC cells Ectopic expression of ZMYM1 rescued this ability,Results: METTL3-mediated epigenetic activation of ZMYM1 was responsible for EMT and metastasis of GC,overexpression of METTL3 enhanced cell invasion, which was partially attenuated by co-transfection with sh-ZMY

26、M1 plasmid,Results: METTL3-mediated epigenetic activation of ZMYM1 was responsible for EMT and metastasis of GC,The inhibitory effect of METTL3 knockdown on metastasis could be, at partially, offset by introduction of ZMYM1 The increase in the metastatic potential associated with METTL3 overexpressi

27、on could be partially attenuated by depletion of ZMYM1,Results: METTL3-mediated epigenetic activation of ZMYM1 was responsible for EMT and metastasis of GC,Protein level of ZMYM1: Reduced with decreased lung metastasis burden Up-regulated with more lung colonization caused by modified METTL3 expression,Results: METTL3-