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2022 ◽  
Eleanor Valenzi ◽  
Harinath Bahudhanapati ◽  
Jiangning Tan ◽  
Tracy Tabib ◽  
Daniel I Sullivan ◽  

In idiopathic pulmonary fibrosis (IPF) myofibroblasts are key effectors of fibrosis and architectural distortion by excessive deposition of extracellular matrix and their acquired contractile capacity. Single-cell RNA-sequencing has precisely defined the IPF myofibroblast transcriptome, but identifying critical transcription factor activity by this approach is imprecise. We performed and integrated snATAC-seq and scRNA-seq from human IPF and donor control explants to identify differentially accessible chromatin regions and enriched transcription factor motifs within lung cell populations. TWIST1 and other E-box transcription factor motifs were significantly enriched in IPF myofibroblasts compared to both IPF non-myogenic and control fibroblasts. TWIST1 expression was also selectively upregulated in IPF myofibroblasts. Overexpression of Twist1 in lung COL1A2-expressing fibroblasts in bleomycin-injured mice was associated with increased collagen synthesis. Our studies utilizing human multiomic single-cell analyses combined with in vivo murine disease models confirm a critical regulatory function for TWIST1 in IPF myofibroblast activity in the fibrotic lung. Understanding the global process of opening TWIST1 and other E-box TF motifs that govern myofibroblast differentiation may identify new therapeutic interventions for fibrotic pulmonary diseases.

Cancers ◽  
2021 ◽  
Vol 13 (23) ◽  
pp. 6001
Katharina Koch ◽  
Rudolf Hartmann ◽  
Abigail Kora Suwala ◽  
Dayana Herrera Rios ◽  
Marcel Alexander Kamp ◽  

Cancer stem-like cells mediate tumor initiation, progression, and therapy resistance; however, their identification and selective eradication remain challenging. Herein, we analyze the metabolic dependencies of glioblastoma stem-like cells (GSCs) with high-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy. We stratify our in vitro GSC models into two subtypes primarily based on their relative amount of glutamine in relationship to glutamate (Gln/Glu). Gln/GluHigh GSCs were found to be resistant to glutamine deprivation, whereas Gln/GluLow GSCs respond with significantly decreased in vitro clonogenicity and impaired cell growth. The starvation resistance appeared to be mediated by an increased expression of the glutamate/cystine antiporter SLC7A11/xCT and efficient cellular clearance of reactive oxygen species (ROS). Moreover, we were able to directly correlate xCT-dependent starvation resistance and high Gln/Glu ratios with in vitro clonogenicity, since targeted differentiation of GSCs with bone morphogenic protein 4 (BMP4) impaired xCT expression, decreased the Gln/Glu ratio, and restored the sensitivity to glutamine starvation. Moreover, significantly reduced levels of the oncometabolites lactate (Lac), phosphocholine (PC), total choline (tCho), myo-inositol (Myo-I), and glycine (Gly) were observed in differentiated GSCs. Furthermore, we found a strong association between high Gln/Glu ratios and increased expression of Zinc finger E-box-binding homeobox 1 (ZEB1) and xCT in primary GBM tumor tissues. Our analyses suggest that the inhibition of xCT represents a potential therapeutic target in glioblastoma; thus, we could further extend its importance in GSC biology and stress responses. We also propose that monitoring of the intracellular Gln/Glu ratio can be used to predict nutrient stress resistance.

2021 ◽  
Piwei Huang ◽  
Minghui Wei ◽  
Shufan Ji ◽  
Mitra Fowdur ◽  
maolin he

Abstract BackgroundRibosomal protein L34 (RPL34) is a member of the L34E ribosomal protein family containing zinc finger domains. This protein plays a key role in regulating the apoptosis, cell cycle progression and proliferation of various cancer including osteosarcoma (OS). The purpose of this study is to clarify the expression of RPL34 in osteosarcoma cells and its molecular mechanism of regulating osteosarcoma cells. MethodsThe expression levels of c-Myc and RPL34 were detected by qRT-PCR and Western blot. Luciferase reporter assays and chromatin immunoprecipitation (ChIP) were used to analyse the binding site of c-Myc and RPL34. ResultsThe results showed that c-Myc binds to the E-box region in the RPL34 promoter to regulate RPL34 expression. The results indicated that RPL34 regulates osteosarcoma cells proliferation through c-Myc/RPL34 signaling axis. This research may provide new ideas for targeted therapy of OS. Conclusion RPL34 regulates osteosarcoma cells proliferation through c-Myc/RPL34 signaling axis.

2021 ◽  
Vol 11 ◽  
Yan Jin ◽  
Zhengming Zhang ◽  
Qiao Yu ◽  
Zhu Zeng ◽  
Hong Song ◽  

BackgroundMany studies have reported the roles of the extracellular hypoxia microenvironment in the tumorigenesis and metastasis of multiple cancers. However, long noncoding RNAs (lncRNAs) that induce cancer oncogenicity and metastasis of pancreatic cancer (PC) under hypoxia conditions remain unclear.MethodsIn PC cells, the expression levels of lncRNAs in different conditions (normoxia or hypoxia) were compared through RNA sequencing (RNA-seq). The effects of the zinc finger E-box-binding homeobox 1 (ZEB1-AS1) antisense lncRNA on PC cells cultured in normoxia/hypoxia medium were measured through gain and loss-of-function experiments. Fluorescence in situ hybridization and luciferase reporter assays in addition to in vivo studies were utilized to explore the adaptive mechanisms of ZEB1-AS1 in the hypoxia-promoted proliferation, migration, and invasion ability of PC cells. Moreover, the level of ZEB1-AS1 and its associated targets or pathways were investigated in both PC and pancreatic normal tissues.ResultsRNA-seq revealed that ZEB1-AS1 was significantly upregulated in PC cells under hypoxia conditions. The ZEB1-AS1 expression level was closely associated with poor prognosis of PC patients. Knockdown of ZEB1-AS1 suppressed the proliferation, migration, and invasion of PC cells in vitro as well as PC xenograft tumor growth in vivo. In PC cells, RNAi-mediated reduction of ZEB1-AS1 inhibited zinc finger E-box-binding homeobox 1 (ZEB1), while ZEB1-AS1 overexpression rescued ZEB1 expression, indicating that ZEB1-AS1 promotes ZEB1 expression. Moreover, hypoxia-inducible factor-1α (HIF-1α)induced the expression of ZEB1-AS1 by binding to the ZEB1-AS1 promoter, which contains a putative hypoxia response element (HRE). Mechanistically, ZEB1-AS1 scaffolded the interaction among HIF-1α, ZEB1, and histone deacetylase 1 (HDAC1), leading to deacetylation-mediated stabilization of HIF-1α. We further revealed that ZEB1 induced the deacetylase capacity of HDAC1 to suppress the acetylation or degradation of HIF-1α, improving HIF-1α assembly. Thus, hypoxia-induced ZEB1-AS1 facilitated ZEB1 transcription and the stability of HIF-1α, which promoted the metastasis of PC cells. Clinically, dysregulated ZEB1 and HIF-1α expression was significantly correlated with histological grade, lymphatic metastasis, and distant metastasis in PC patients.ConclusionsOur results emphasized that the positive reciprocal loop of HIF-1α/ZEB1-AS1/ZEB1/HDAC1 contributes to hypoxia-promoted oncogenicity and PC metastasis, indicating that it might be a novel therapeutic target for PC.

Tinghui Shao ◽  
Yujia Xue ◽  
Mingming Fang

Cardiac fibrosis is a key pathophysiological process that contributes to heart failure. Cardiac resident fibroblasts, exposed to various stimuli, are able to trans-differentiate into myofibroblasts and mediate the pro-fibrogenic response in the heart. The present study aims to investigate the mechanism whereby transcription of chloride channel accessory 2 (Clca2) is regulated in cardiac fibroblast and its potential implication in fibroblast-myofibroblast transition (FMyT). We report that Clca2 expression was down-regulated in activated cardiac fibroblasts (myofibroblasts) compared to quiescent cardiac fibroblasts in two different animal models of cardiac fibrosis. Clca2 expression was also down-regulated by TGF-β, a potent inducer of FMyT. TGF-β repressed Clca2 expression at the transcriptional level likely via the E-box element between −516 and −224 of the Clca2 promoter. Further analysis revealed that Twist1 bound directly to the E-box element whereas Twist1 depletion abrogated TGF-β induced Clca2 trans-repression. Twist1-mediated Clca2 repression was accompanied by erasure of histone H3/H4 acetylation from the Clca2 promoter. Mechanistically Twist1 interacted with HDAC1 and recruited HDAC1 to the Clca2 promoter to repress Clca2 transcription. Finally, it was observed that Clca2 over-expression attenuated whereas Clca2 knockdown enhanced FMyT. In conclusion, our data demonstrate that a Twist1-HDAC1 complex represses Clca2 transcription in cardiac fibroblasts, which may contribute to FMyT and cardiac fibrosis.

2021 ◽  
Paivi Pihlajamaa ◽  
Otto Kauko ◽  
Biswajyoti Sahu ◽  
Teemu Kivioja ◽  
Jussi Taipale

The two major limitations of applying CRISPR/Cas9-technology for analysis of the effect of genotype on phenotype are the difficulty of cutting DNA exactly at the intended site, and the decreased cell proliferation and other phenotypic effects caused by the DNA cuts themselves. Here we report a novel competitive genome editing assay that allows analysis of the functional consequence of precise mutations. The method is based on precision genome editing, where a target sequence close to a feature of interest is cut, and the DNA is then repaired using a template that either reconstitutes the original feature, or introduces an altered sequence. Introducing sequence labels to both types of repair templates generates a large number of replicate cultures, increasing statistical power. In addition, the labels identify edited cells, allowing direct comparison between cells that carry wild-type and mutant features. Here, we apply the assay for multiplexed analysis of the role of E-box sequences on MYC binding and cellular fitness.

2021 ◽  
Vol 11 (2) ◽  
pp. 194-213
Paulo Henrique Couto Simões ◽  
José Fabiano da Serra Costa ◽  
Marcello Montillo Provenza ◽  
Vinicius Layter Xavier ◽  
Jorge Luiz de Jesus Goulart

Os mais de sete mil quilômetros do litoral brasileiro constituem uma zona de riqueza ímpar do nosso país onde se concentra grande parte da população brasileira. É na região costeira onde se realiza grande parte da pesca, da navegação, da extração de petróleo, do lazer e do turismo do Brasil. Nesse cenário, um conhecimento das condições oceânicas existentes ao longo do nosso litoral torna-se cada vez mais necessário. Diversos programas incentivam e executam atividades relacionadas ao estudo dos oceanos. Nesse trabalho, será analisada a base de dados do GOOS-BRASIL que é um sistema nacional de observação dos oceanos. O objetivo do presente estudo é modelar a série temporal entre 2005 e 2014 das temperaturas e salinidades médias a um metro de profundidade, coletadas da boia ATLAS (posição geográfica 19°00S34°00W) da rede PIRATA. Inicialmente foram feitas as análises de estacionariedade e sazonalidade, para posteriormente elaborar as previsões. Foram utilizados os modelos de alisamento exponencial e Box-Jenkins, os quais foram avaliados pelas métricas do Erro Quadrático Médio e da Média Absoluta Percentual de Erro.

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