Post-transcriptional Regulation Of THO Complex Contributes To Vascular Smooth Muscle Cell Phenotype Switch

Backgroud and objective:THO complex including THOC1?THOC2?THOC3?THOC5?THOC6?THOC7 that interacts with other proteins of TREX complex,plays a role in transcriptional elongation,nuclear RNA export and genome stability.THOC5 is required for the recruitment of cleavage and polyadenylation specific factor 100(CPSF100)to the THOC5 target genes and participate in 3 ' end-processing,promoting the expression of IEGs including Idl,Id3 and Wntll,which regulating cell differentiation/proliferation.The depletion of THOC5 in mouse embryonicfibroblasts and bone marrow derived macrophages caused the down regulation of 143(>3-fold)and 99(>2-fold)genes,respectively,suppresses cell growth and proliferation/differentiation.The Ingenuity Pathway Analysis(IPA)of these genes reveals that over 60%of the genes were involved in differentiation/proliferation/motility.In ESC cells,depletion of THOC5 and THOC2 caused the down regulation of 541 genes,suggesting the expression of THOC5 is closely related to the original cell state and cell proliferation/differentiation.As terminally differentiated cells,VSMC is unlike myocardium and skeletal muscle,which can undergo dedifferentiation into a synthetic phenotype when stimulated,synthesize and secrete cytokines,matrix,and promote VSMC proliferation and migration into the intima,leading to pathological changes,and give rise to the occurrence and development of vascular diseases.The current research on the mechanism of phenotypic conversion mainly focuses on the transcriptional regulation and signaling pathways of PI3K/ERKor MAPK et al.Recent studies have begun to reveal that post-transcriptional regulation may play important role in VSMC phenotypic switch.Reveal the role of THO complex in the phenotypic switch in vascular diseases is particularly important,which will provide a identity tool for the target genes.The purpose of this study was to reveal the mechanism of THO complex regulating VSMC marker genes,provide new research pathways and new therapeutic directions for the pathogenesis of vascular diseases.Methods:Immunohistochemical staining was used to detect the expression of THOC5 and THOC2 in CEA versus plaques.The expression of Thoc5 and Thoc2 were further verified by quantitative RT-PCR and Western blot.Quantitative real time-PCR was used to detect the expression of Thoc5 and Thoc2 in dedifferentiated VSMC.Using shRNA knockdown of Thoc5 and Thoc2 expression and detected the expression of VSMC marker genes and the ability of proliferation/migration;RNA-seq analyse VSMC marker genes expression after knockdown Thoc5;using ChIP-seq,Act-D experiments,RIP-seq for mechanism studies;overexpression of THOC5 in the carotid artery of balloon injury model and adventitial fibroblasts,quantitative RT-PCR,IHC,IF detected expression of VSMC marker genes.Results:We performed immunohistochemical staining against THOC5 and THOC2,two subuints of THO complex,and found a dramatic reduction in their expression in human arteries undergoing carotid endarterectomy(CEA)compared to normal internal mammary arteries(IMA),the levels were further verified by quantitative RT-PCR and Western blot.In addition,silencing of Thoc2 or Thoc5 led to dedifferentiation of VSMCs in vitro,characterized by decreased VSMC marker genes expression and increased migration/proliferation.RNA-Seq revealed that Thoc5 silencing closely resembled the gene expression changes induced upon PDGF-BB/PDGF-DD treatments in cultured VSMCs.Mechanistically,both Thoc2 and Thoc5 preferentially interacted with VSMC marker gene mRNAs and mediated their expression.Interestingly,Gene Set Enrichment Analysis(GSEA)showing mRNAs that lost Thoc2 and/or Thoc5 binding during VSMC dedifferentiation were enriched for genes that were highly expressed in PI versus P10 VSMCs or starved VSMC versus FBS-treated VSMC.Lastly,Thoc5 overexpression in injured rat carotid arteries significantly repressed loss of VSMC marker gene expression and neointima formation,Thoc5 overexpression of adventitial fibroblasts can also increase the expression level of smooth muscle marker genes,turning into myofibroblasts.Conclusion:Our data reveal that THO complex specifically regulates VSMC marker genes,introduce dynamic binding of THO to VSMC marker gene mRNAs as a novel mechanism contributing to VSMC fate decisions,revealed a novel posttranscriptional mechanism mediating vascular smooth muscle phenotype switch,and imply Thoc5 as a potential intervention node for vascular diseases,providing new research pathways and new therapeutic directions for vascular diseases.