Enhancer Reprogramming within Pre-existing Topologically Associated Domains Promotes TGF-β-Induced EMT and Cancer Metastasis

Qiao, Yong; Wang, Zejian; Tan, Fangzhi; Lin, Jianxiang; Yang, Jie; Li, Hui; Wang, Xiongjun; Šali, Andrej; Zhang, Liye; Zhong, Guisheng

doi:10.1016/j.ymthe.2020.05.026

Cited by 27 publications

(24 citation statements)

References 44 publications

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“…Cigarette smoking is a well-known risk factor for the occurrence and progression of malignant diseases [ 82 – 85 ]. Nicotine, the major constituent in cigarette smoke, plays key roles in cancer progression [ 86 – 89 ].…”

Section: Discussionmentioning

confidence: 99%

Identified GNGT1 and NMU as Combined Diagnosis Biomarker of Non-Small-Cell Lung Cancer Utilizing Bioinformatics and Logistic Regression

Zhang

Jiang

et al. 2021

Disease Markers

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Non-small-cell lung cancer (NSCLC) is one of the most devastating diseases worldwide. The study is aimed at identifying reliable prognostic biomarkers and to improve understanding of cancer initiation and progression mechanisms. RNA-Seq data were downloaded from The Cancer Genome Atlas (TCGA) database. Subsequently, comprehensive bioinformatics analysis incorporating gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and the protein-protein interaction (PPI) network was conducted to identify differentially expressed genes (DEGs) closely associated with NSCLC. Eight hub genes were screened out using Molecular Complex Detection (MCODE) and cytoHubba. The prognostic and diagnostic values of the hub genes were further confirmed by survival analysis and receiver operating characteristic (ROC) curve analysis. Hub genes were validated by other datasets, such as the Oncomine, Human Protein Atlas, and cBioPortal databases. Ultimately, logistic regression analysis was conducted to evaluate the diagnostic potential of the two identified biomarkers. Screening removed 1,411 DEGs, including 1,362 upregulated and 49 downregulated genes. Pathway enrichment analysis of the DEGs examined the Ras signaling pathway, alcoholism, and other factors. Ultimately, eight prioritized genes (GNGT1, GNG4, NMU, GCG, TAC1, GAST, GCGR1, and NPSR1) were identified as hub genes. High hub gene expression was significantly associated with worse overall survival in patients with NSCLC. The ROC curves showed that these hub genes had diagnostic value. The mRNA expressions of GNGT1 and NMU were low in the Oncomine database. Their protein expressions and genetic alterations were also revealed. Finally, logistic regression analysis indicated that combining the two biomarkers substantially improved the ability to discriminate NSCLC. GNGT1 and NMU identified in the current study may empower further discovery of the molecular mechanisms underlying NSCLC’s initiation and progression.

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Section: Discussionmentioning

confidence: 99%

Identified GNGT1 and NMU as Combined Diagnosis Biomarker of Non-Small-Cell Lung Cancer Utilizing Bioinformatics and Logistic Regression

Zhang

Jiang

et al. 2021

Disease Markers

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“…TSA has been shown to reduce α SMA expression and collagen type I deposition; inhibit cellular proliferation in fibroblasts; and promote apoptosis in certain experimental skin fibrosis models ( Rombouts et al, 2002 ; Huber et al, 2007 ; Diao et al, 2011 ). HDACs, in general, have been implicated in TGF β 1-induced epithelial-mesenchymal transition (EMT) ( Jones et al, 2019 ; Qiao et al, 2020 ). HDAC suppress epithelial-specific genes and mediate TGF β 1-induced mesenchymal enhancer reprogramming, which results in EMT of the cells ( Qiao et al, 2020 ).…”

Section: Discussionmentioning

confidence: 99%

Adapting the Scar-in-a-Jar to Skin Fibrosis and Screening Traditional and Contemporary Anti-Fibrotic Therapies

Coentro

May

Prince

et al. 2021

Front. Bioeng. Biotechnol.

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Skin fibrosis still constitutes an unmet clinical need. Although pharmacological strategies are at the forefront of scientific and technological research and innovation, their clinical translation is hindered by the poor predictive capacity of the currently available in vitro fibrosis models. Indeed, customarily utilised in vitro scarring models are conducted in a low extracellular matrix milieu, which constitutes an oxymoron for the in-hand pathophysiology. Herein, we coupled macromolecular crowding (enhances and accelerates extracellular matrix deposition) with transforming growth factor β1 (TGFβ1; induces trans-differentiation of fibroblasts to myofibroblasts) in human dermal fibroblast cultures to develop a skin fibrosis in vitro model and to screen a range of anti-fibrotic families (corticosteroids, inhibitors of histone deacetylases, inhibitors of collagen crosslinking, inhibitors of TGFβ1 and pleiotropic inhibitors of fibrotic activation). Data obtained demonstrated that macromolecular crowding combined with TGFβ1 significantly enhanced collagen deposition and myofibroblast transformation. Among the anti-fibrotic compounds assessed, trichostatin A (inhibitors of histone deacetylases); serelaxin and pirfenidone (pleiotropic inhibitors of fibrotic activation); and soluble TGFβ receptor trap (inhibitor of TGFβ signalling) resulted in the highest decrease of collagen type I deposition (even higher than triamcinolone acetonide, the gold standard in clinical practice). This study further advocates the potential of macromolecular crowding in the development of in vitro pathophysiology models.

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“…Total RNA extracted from HCT116 cells and zebrafish tissues was subjected to RNA‐seq using Illumina Nova‐seq platform at Novogene, China. RNA‐seq data analysis was performed as we previously described 22,23 . The paired‐end RNA‐Seq reads were mapped to the GRCh38/hg38 reference genome using STAR (v2.5.3a), and gene expression was quantified to FPKM for each gene using RSEM (v1.3.0).…”

Section: Methodsmentioning

confidence: 99%

“…RNA‐seq data analysis was performed as we previously described. 22 , 23 The paired‐end RNA‐Seq reads were mapped to the GRCh38/hg38 reference genome using STAR (v2.5.3a), and gene expression was quantified to FPKM for each gene using RSEM (v1.3.0). Raw read counts were estimated by featureCounts (v1.5.2), and DESeq2 (v1.22.1) was used for differential expression analysis.…”

Section: Methodsmentioning

confidence: 99%

Base editing‐mediated perturbation of endogenous PKM1/2 splicing facilitates isoform‐specific functional analysis in vitro and in vivo

Lin

Shen

et al. 2021

Cell Proliferation

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Objectives PKM1 and PKM2, which are generated from the alternative splicing of PKM gene, play important roles in tumourigenesis and embryonic development as rate‐limiting enzymes in glycolytic pathway. However, because of the lack of appropriate techniques, the specific functions of the 2 PKM splicing isoforms have not been clarified endogenously yet. Materials and methods In this study, we used CRISPR‐based base editors to perturbate the endogenous alternative splicing of PKM by introducing mutations into the splicing junction sites in HCT116 cells and zebrafish embryos. Sanger sequencing, agarose gel electrophoresis and targeted deep sequencing assays were utilized for identifying mutation efficiencies and detecting PKM1/2 splicing isoforms. Cell proliferation assays and RNA‐seq analysis were performed to describe the effects of perturbation of PKM1/2 splicing in tumour cell growth and zebrafish embryo development. Results The splicing sites of PKM, a 5’ donor site of GT and a 3’ acceptor site of AG, were efficiently mutated by cytosine base editor (CBE; BE4max) and adenine base editor (ABE; ABEmax‐NG) with guide RNAs (gRNAs) targeting the splicing sites flanking exons 9 and 10 in HCT116 cells and/or zebrafish embryos. The mutations of the 5’ donor sites of GT flanking exons 9 or 10 into GC resulted in specific loss of PKM1 or PKM2 expression as well as the increase in PKM2 or PKM1 respectively. Specific loss of PKM1 promoted cell proliferation of HCT116 cells and upregulated the expression of cell cycle regulators related to DNA replication and cell cycle phase transition. In contrast, specific loss of PKM2 suppressed cell growth of HCT116 cells and resulted in growth retardation of zebrafish. Meanwhile, we found that mutation of PKM1/2 splicing sites also perturbated the expression of non‐canonical PKM isoforms and produced some novel splicing isoforms. Conclusions This work proved that CRISPR‐based base editing strategy can be used to disrupt the endogenous alternative splicing of genes of interest to study the function of specific splicing isoforms in vitro and in vivo. It also reminded us to notice some novel or undesirable splicing isoforms by targeting the splicing junction sites using base editors. In sum, we establish a platform to perturbate endogenous RNA splicing for functional investigation or genetic correction of abnormal splicing events in human diseases.

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Enhancer Reprogramming within Pre-existing Topologically Associated Domains Promotes TGF-β-Induced EMT and Cancer Metastasis

Cited by 27 publications

References 44 publications

Identified GNGT1 and NMU as Combined Diagnosis Biomarker of Non-Small-Cell Lung Cancer Utilizing Bioinformatics and Logistic Regression

Identified GNGT1 and NMU as Combined Diagnosis Biomarker of Non-Small-Cell Lung Cancer Utilizing Bioinformatics and Logistic Regression

Adapting the Scar-in-a-Jar to Skin Fibrosis and Screening Traditional and Contemporary Anti-Fibrotic Therapies

Base editing‐mediated perturbation of endogenous PKM1/2 splicing facilitates isoform‐specific functional analysis in vitro and in vivo

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