SMAD3 Dependent Phase Separation Regulates Super Enhancer Related Genes To Promote The Development Of Hepatocellular Carcinoma Cells(HCC)

Objective The altered transcriptome of HCC and its impact on the hallmarks of cancer have been extensively reviewed.However,the mechanisms that mediate the aberrant transcription programs in cancer cells are not fully understood.Recently,emerging evidence has indicated that the continuous and robust transcription of oncogenes in cancers is often driven by a remarkably large cluster of enhancer regions,namely,the super enhancers(SEs)The TGF-β signaling pathway coordinates many cellular processes,including growth,invasion,immunosuppression,and extracellular matrix remodeling,its key signaling factor SMAD3 can enter the phase formed by transcriptional co-activator MED1 on super enhancer with other transcription factors in mouse embryonic stem cells(mESCs).However,SMAD3 was not further investigated in the text The aim of this paper is to analyze and find mutations that disrupt SMAD3 phase separation,to demonstrate the functional role of phase separation in tumor growth using cell phenotyping experiments,and to explore the molecular mechanisms by which SMAD3 phase separation regulates aberrant transcription of HCC-specific super enhancers related genes through histological experiments.Method1.The amino acid sequence of SMAD3 was predicted to analyze whether it has the Intrinsically disordered region(IDR)necessary for phase formation;the amino acid composition in IDR was counted,and the Python package localCIDER(version 0.1.10)was used to analyze the amino acid charge and construct mutants that can affect the phase separation ability.2.Prokaryotic expression vectors with EGFP tags were constructed by molecular cloning,and the in vitro purification of the proteins simulated a crowded intracellular environment with the addition of 1,6-hexanediol,which can disrupt phase separation,to verify the phase formation ability of SMAD3 in vitro.3.Stable knockdown HepG2 were constructed using shRNA,and the knockdown effect was verified by Western blot;overexpression stable transfectants were constructed using the piggy Bac transposon system,and the overexpression effect was verified after screening for brighter fluorescent polyclones.4.Overexpression of exogenous EGFP-SMAD3 in low-expressing cell lines was demonstrated by live cell imaging as well as fluorescence recovery after photobleaching(FRAP)experiments to form droplets with mobility in vivo.5.Cell phenotype verification of SMAD3 phase separation and the effect of disrupted phase separation on tumor cell proliferation and migration.6.Co-localization of SMAD3-EGFP with the active enhancer signal H3K27 ac was observed by immunofluorescence.The target genes regulated by phase separation were screened by RNA-seq;H3K27ac was used to define super enhancers,and find HCCspecific super enhancer-associated genes,then take the intersection to find super enhancer-associated genes regulated by phase separation.7.Live cell imaging to observe the co-localization of SMAD3-EGFP with nucleolar granular component nucleophosmin and Dense Fibrillar Component Fibrillarin.RTPCR was performed to detect ribosomal RNA expression.Result1.SMAD3 can undergo phase separation in vitro,and the phase formation ability becomes stronger with increasing concentration at a certain concentration,1,6-hexanediol will destroy the phase.2.Mutant SMAD3 will destroy its phase forming ability,and the phase forming ability of the mutant can be regained by FUS IDR remediation.3.Successfully constructed SMAD3 knockdown cell line with about 50% knockdown effect;successfully constructed overexpression cell line.4.SMAD3 droplets in vivo are mobile and can recover quickly after photobleaching.5.Cell phenotyping experiments demonstrate that SMAD3 promotes tumour cell proliferation and migration through phase separation.6.Immunofluorescence showed co-localization of wild-type SMAD3 condensates with H3K27 ac and significantly reduced co-localization of mutant SMAD3;SMAD3regulates high expression of the HCC-specific super-enhancer-associated gene SLC39A7 by phase separation.7.Most SMAD3 proteins localize in the nucleoplasm,and a few SMAD3 can colocalize with fibronectin FBL,and mutations disrupt this co-localization.SMAD3 also inhibits rRNA splicing.Conclusion The intrinsically disordered region(IDR)is required for SMAD3 to form phase separation,and mutating glutamate and aspartate in the IDR to alanine or deleting them all would disrupt the independent phase formation ability of SMAD3.SMAD3 promotes tumor cell growth and migration by phase-separated regulation of hepatocellular carcinoma-specific SE-associated poor prognosis SLC39A7 overexpression.SMAD3 co-localizes with Fibrillarin in Dense Fibrillar Component and inhibits rRNA splicing.