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Elucidating the molecular basis of PPCD: Effects of decreased ZEB1 expression on corneal endothelial cell function.

ABSTRACT: To investigate the functional role that the zinc e-box binding homeobox 1 (ZEB1) gene, which underlies the genetic basis of posterior polymorphous corneal dystrophy 3 (PPCD3), plays in corneal endothelial cell proliferation, apoptosis, migration, and barrier function.A human corneal endothelial cell line (HCEnC-21T) was transfected with siRNA targeting ZEB1 mRNA. Cell proliferation, apoptosis, migration, and barrier assays were performed: Cell proliferation was assessed with cell counting using a hemocytometer; cell apoptosis, induced by either ultraviolet C (UVC) radiation or doxorubicin treatment, was quantified by measuring cleaved caspase 3 (cCASP3) protein levels; and cell migration and barrier function were monitored with electric cell-substrate impedance sensing (ECIS).ZEB1 knockdown in HCEnC-21T cells transfected with siRNA targeting ZEB1 did not result in a significant difference in cell proliferation when compared with control. Although knockdown of ZEB1 in HCEnC-21T cells sensitized the cells to UV-induced apoptosis, ZEB1 knockdown did not alter the cells' susceptibility to doxorubicin-induced apoptosis, as measured with cCASP3 protein levels, compared with controls. Similarly, no difference was observed in cell migration following ZEB1 knockdown. However, cell barrier function increased significantly following ZEB1 knockdown.The corneal endothelium in PPCD3 is characterized by morphologic, anatomic, and molecular features that are more consistent with an epithelial-like rather than an endothelial-like phenotype. Although these characteristics have been well documented, we demonstrate for the first time that susceptibility to UV-induced apoptosis and cell barrier function are significantly altered in the setting of reduced ZEB1. The significance of an altered cellular response to apoptotic stimuli and increased cell barrier function in the pathobiology of PPCD remains to be fully elucidated.

SUBMITTER: Zakharevich M

PROVIDER: S-EPMC5644665 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Elucidating the molecular basis of PPCD: Effects of decreased ZEB1 expression on corneal endothelial cell function.

Zakharevich Marina M Kattan Jaffer M JM Chen Judy L JL Lin Benjamin R BR Cervantes Aleck E AE Chung Doug D DD Frausto Ricardo F RF Aldave Anthony J AJ

Molecular vision 20171014

<h4>Purpose</h4>To investigate the functional role that the <i>zinc e-box binding homeobox 1</i> (<i>ZEB1</i>) gene, which underlies the genetic basis of posterior polymorphous corneal dystrophy 3 (PPCD3), plays in corneal endothelial cell proliferation, apoptosis, migration, and barrier function.<h4>Methods</h4>A human corneal endothelial cell line (HCEnC-21T) was transfected with siRNA targeting <i>ZEB1</i> mRNA. Cell proliferation, apoptosis, migration, and barrier assays were performed: Cell ...[more]

PMID: 29046608

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Project description:Licorice (Glycyrrhiza spp.) is used widely in traditional Chinese medicine (TCM) due to its numerous pharmacologic effects. However, the mechanisms of action of the chemical constituents of licorice and their structure-function relationships are not fully understood. To address these points, we analyzed the chemical compounds in licorice listed in the TCM Systems Pharmacology database and TCM Integrated database. Target proteins of the compounds were predicted using Integrative Pharmacology-based Research Platform of TCM v2.0. Information on the pharmacologic effects of licorice was obtained from the 2020 Chinese Pharmacopoeia, and disease-related genes that have been linked to these effects were identified from the Encyclopedia of TCM database. Pathway analyses using the Kyoto Encyclopedia of Genes and Genomes database were carried out for target proteins, and pharmacologic networks were constructed based on drug target-disease-related gene and protein-protein interactions. A total of 451 compounds were analyzed, of which 211 were from the medicinal parts of the licorice plant. The 241 putative targets of 106 bioactive compounds in licorice comprised 52 flavonoids, 47 triterpenoids, and seven coumarins. Four distinct pharmacologic effects of licorice were defined: 61 major hubs were the putative targets of 23 compounds in heat-clearing and detoxifying effects; 68 were targets of six compounds in spleen-invigorating and qi-replenishing effects; 28 were targets of six compounds in phlegm-expulsion and cough-suppressant effects; 25 compounds were targets of six compounds in spasm-relieving and analgesic effects. The major bioactive compounds of licorice were identified by ultra-high-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry. The anti-inflammatory properties of liquiritin apioside, liquiritigenin, glycyrrhizic acid and isoliquiritin apioside were demonstrated by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Liquiritin apioside, liquiritigenin, isoliquiritin, isoliquiritin apioside, kaempferol, and kumatakenin were the main active flavonoids, and 18α- and 18β-glycyrrhetinic acid were the main active triterpenoids of licorice. The former were associated with heat-clearing and detoxifying effects, whereas the latter were implicated in the other three pharmacologic effects. Thus, the compounds in licorice have distinct pharmacologic effects according to their chemical structure. These results provide a reference for investigating the potential of licorice in treatment of various diseases.

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Elucidating the molecular basis of PPCD: Effects of decreased ZEB1 expression on corneal endothelial cell function.

Publications

Elucidating the molecular basis of PPCD: Effects of decreased ZEB1 expression on corneal endothelial cell function.

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