miRViz: a novel webserver application to visualize and interpret microRNA datasets.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in the regulation of major pathways in eukaryotic cells through their binding to and repression of multiple mRNAs. With high-throughput methodologies, various outcomes can be measured that produce long lists of miRNAs that are often difficult to interpret. A common question is: after differential expression or phenotypic screening of miRNA mimics, which miRNA should be chosen for further investigation? Here, we present miRViz (http://mirviz.prabi.fr/), a webserver application designed to visualize and interpret large miRNA datasets, with no need for programming skills. MiRViz has two main goals: (i) to help biologists to raise data-driven hypotheses and (ii) to share miRNA datasets in a straightforward way through publishable quality data representation, with emphasis on relevant groups of miRNAs. MiRViz can currently handle datasets from 11 eukaryotic species. We present real-case applications of miRViz, and provide both datasets and procedures to reproduce the corresponding figures. MiRViz offers rapid identification of miRNA families, as demonstrated here for the miRNA-320 family, which is significantly exported in exosomes of colon cancer cells. We also visually highlight a group of miRNAs associated with pluripotency that is particularly active in control of a breast cancer stem-cell population in culture.

SARS-CoV-2 receptor ACE2 is upregulated by fatty acids in human MASH.

Background & Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) results in steatosis, inflammation (steatohepatitis), and fibrosis. Patients with MASLD more likely develop liver injury in coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As viral RNA has been identified in liver tissues, we studied expression levels and cellular sources of the viral receptor angiotensin-converting enzyme 2 (ACE2) and coreceptors in MASLD and fibroinflammatory liver diseases. Methods: We built a transcriptomic MASLD meta-dataset (N = 243) to study SARS-CoV-2 receptor expression and verified results in 161 additional cases of fibroinflammatory liver diseases. We assessed the fibroinflammatory microenvironment by deconvoluting immune cell populations. We studied the cellular sources of ACE2 by multiplex immunohistochemistry followed by high-resolution confocal microscopy (N = 9 fatty livers; N = 7 controls), meta-analysis of two single-cell RNA sequencing datasets (N = 5 cirrhotic livers; N = 14 normal livers), and bulk transcriptomics from 745 primary cell samples. In vitro, we tested ACE2 mRNA expression in primary human hepatocytes treated with inflammatory cytokines, bacterial lipopolysaccharides, or long-chain fatty acids. Results: We detected ACE2 at the apical and basal poles of hepatocyte chords, in CLEC4M+ liver sinusoidal endothelial cells, the lumen of ABCC2+ bile canaliculi, HepPar-1+-TMPRSS2+ hepatocytes, cholangiocytes, and CD34+ capillary vessels. ACE2 steeply increased between 30 and 50 years of age; was related to liver fat area, inflammation, high immune reactivity, and fibrogenesis; and was upregulated in steatohepatitis. Although ACE2 mRNA was unmodified in alcoholic or viral hepatitis, it was upregulated in fibroinflammatory livers from overweight patients. In vitro, treatment of primary human hepatocytes with inflammatory cytokines alone downregulated but long chain fatty acids upregulated ACE2 mRNA expression. Conclusions: Lipid overload in fatty liver disease leads to an increased availability of ACE2 receptors. Impact and implications: COVID-19 can be a deadly disease in vulnerable individuals. Patients with fatty liver disease are at a higher risk of experiencing severe COVID-19 and liver injury. Recent studies have indicated that one of the reasons for this vulnerability is the presence of a key cell surface protein called ACE2, which serves as the main SARS-CoV-2 virus receptor. We describe the cellular sources of ACE2 in the liver. In patients with fatty liver disease, ACE2 levels increase with age, liver fat content, fibroinflammatory changes, enhanced positive immune checkpoint levels, and innate immune reactivity. Moreover, we show that long chain fatty acids can induce ACE2 expression in primary human hepatocytes. Understanding the cellular sources of ACE2 in the liver and the factors that influence its availability is crucial. This knowledge will guide further research and help protect potentially vulnerable patients through timely vaccination boosters, dietary adjustments, and improved hygiene practices.

Metabolic Networks: Weighted Gene Correlation Network Analysis.

Weighted Gene Correlation Network Analysis (WGCNA) is used to build weighted gene networks representing direct interconnections among genes. This method is useful to identify gene modules associated with biological functions, revealing core functional differences between samples. Here, we describe a step-by-step methodology to build a WGCNA network associated with a phenotype of interest. The results can be visualized using Cytoscape and other available software or used as a basis for further functional enrichment analyses.

Well-differentiated liver cancers reveal the potential link between ACE2 dysfunction and metabolic breakdown.

Angiotensin-converting enzyme 2 (ACE2) is the receptor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causing Coronavirus disease 2019 (COVID-19). Transmembrane serine protease 2 (TMPRSS2) is a coreceptor. Abnormal hepatic function in COVID-19 suggests specific or bystander liver disease. Because liver cancer cells express the ACE2 viral receptor, they are widely used as models of SARS-CoV-2 infection in vitro. Therefore, the purpose of this study was to analyze ACE2 and TMPRSS2 expression and localization in human liver cancers and in non-tumor livers. We studied ACE2 and TMPRSS2 in transcriptomic datasets totaling 1503 liver cancers, followed by high-resolution confocal multiplex immunohistochemistry and quantitative image analysis of a 41-HCC tissue microarray. In cancers, we detected ACE2 and TMPRSS2 at the biliary pole of tumor hepatocytes. In whole mount sections of five normal liver samples, we identified ACE2 in hepatocyte's bile canaliculi, biliary epithelium, sinusoidal and capillary endothelial cells. Tumors carrying mutated ß-catenin showed ACE2 DNA hypomethylation and higher mRNA and protein expression, consistently with predicted ß-catenin response sites in the ACE2 promoter. Finally, ACE2 and TMPRSS2 co-expression networks highlighted hepatocyte-specific functions, oxidative stress and inflammation, suggesting a link between inflammation, ACE2 dysfunction and metabolic breakdown.