MicroRNA-29b Overexpression Decreases Extracellular Matrix mRNA and Protein Production in Human Corneal Endothelial Cells

Tetsuya Toyono, Tomohiko Usui, Guadalupe Villarreal, Laura Kallay, Mario Matthaei, Lucas M M Vianna, Angela Y. Zhu, Masahiko Kuroda, Shiro Amano, Albert Jun

Research output: Contribution to journalArticle

Abstract

PURPOSE:: MicroRNAs are small noncoding RNAs that regulate gene expression at the posttranscriptional level. We reported that levels of microRNA (miR)-29 family are decreased in corneas of patients with Fuchs endothelial corneal dystrophy (FECD). The miR-29 family regulates the production of extracellular matrix (ECM) proteins. Accumulation of ECM proteins in Descemet membrane is an important pathologic change in FECD. In this study, we transfected miR-29b into human corneal endothelial cells and tissues and evaluated ECM protein expression levels. METHODS:: An immortalized Fuchs human corneal endothelial cell line (iFECD) was established by infection of corneal endothelial cells from patients with FECD with hTERT lentivirus. MiR-29b was transfected into iFECD, and the expression levels of ECMs collagen type 1 alpha 1 (COL1A1), collagen type 4 alpha 1 (COL4A1), and laminin gamma 1 (LAMC1) were evaluated with quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) and Western blot. Expression level of LAMC1 protein in miR-29b−transfected donor corneal endothelium was also evaluated by Western blot. RESULTS:: Compared with control, miR-29b expression level after transfection of iFECD was increased to 335.6% (±91.0%), and ECM expression levels were significantly decreased. Compared with control, qRT-PCR demonstrated reduction of ECM to the following levels: COL1A1: 1.9% (±0.4%); COL4A1: 7.1% (±1.7%); and LAMC1: 21.5% (±2.7%). Western blot showed reduced protein expression: COL1A1: 4.8% (±3.2%); COL4A1: 42.5% (±25.0%); and LAMC1: 44.8% (±3.1%). In miR-29b−transfected corneal tissue, LAMC1 protein expression level was decreased to 14.4% (±20.5%). CONCLUSIONS:: Overexpression of miR-29b decreased ECM protein production in human corneal endothelial cells. Thus, miR-29 replacement therapy might be a new treatment strategy for FECD aimed at reducing pathologic production of ECM proteins in Descemet membrane.

Original languageEnglish (US)
JournalCornea
DOIs
StateAccepted/In press - Aug 3 2016

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Extracellular Matrix Proteins
MicroRNAs
Endothelial Cells
Fuchs' Endothelial Dystrophy
Messenger RNA
Collagen Type IV
Collagen Type I
Descemet Membrane
Corneal Endothelium
Western Blotting
Extracellular Matrix
Small Untranslated RNA
Lentivirus
Cornea
Transfection
Tissue Donors
Gene Expression
Cell Line
Therapeutics

ASJC Scopus subject areas

  • Medicine(all)
  • Ophthalmology

Cite this

MicroRNA-29b Overexpression Decreases Extracellular Matrix mRNA and Protein Production in Human Corneal Endothelial Cells. / Toyono, Tetsuya; Usui, Tomohiko; Villarreal, Guadalupe; Kallay, Laura; Matthaei, Mario; Vianna, Lucas M M; Zhu, Angela Y.; Kuroda, Masahiko; Amano, Shiro; Jun, Albert.

In: Cornea, 03.08.2016.

Research output: Contribution to journalArticle

Toyono, Tetsuya ; Usui, Tomohiko ; Villarreal, Guadalupe ; Kallay, Laura ; Matthaei, Mario ; Vianna, Lucas M M ; Zhu, Angela Y. ; Kuroda, Masahiko ; Amano, Shiro ; Jun, Albert. / MicroRNA-29b Overexpression Decreases Extracellular Matrix mRNA and Protein Production in Human Corneal Endothelial Cells. In: Cornea. 2016.
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abstract = "PURPOSE:: MicroRNAs are small noncoding RNAs that regulate gene expression at the posttranscriptional level. We reported that levels of microRNA (miR)-29 family are decreased in corneas of patients with Fuchs endothelial corneal dystrophy (FECD). The miR-29 family regulates the production of extracellular matrix (ECM) proteins. Accumulation of ECM proteins in Descemet membrane is an important pathologic change in FECD. In this study, we transfected miR-29b into human corneal endothelial cells and tissues and evaluated ECM protein expression levels. METHODS:: An immortalized Fuchs human corneal endothelial cell line (iFECD) was established by infection of corneal endothelial cells from patients with FECD with hTERT lentivirus. MiR-29b was transfected into iFECD, and the expression levels of ECMs collagen type 1 alpha 1 (COL1A1), collagen type 4 alpha 1 (COL4A1), and laminin gamma 1 (LAMC1) were evaluated with quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) and Western blot. Expression level of LAMC1 protein in miR-29b−transfected donor corneal endothelium was also evaluated by Western blot. RESULTS:: Compared with control, miR-29b expression level after transfection of iFECD was increased to 335.6{\%} (±91.0{\%}), and ECM expression levels were significantly decreased. Compared with control, qRT-PCR demonstrated reduction of ECM to the following levels: COL1A1: 1.9{\%} (±0.4{\%}); COL4A1: 7.1{\%} (±1.7{\%}); and LAMC1: 21.5{\%} (±2.7{\%}). Western blot showed reduced protein expression: COL1A1: 4.8{\%} (±3.2{\%}); COL4A1: 42.5{\%} (±25.0{\%}); and LAMC1: 44.8{\%} (±3.1{\%}). In miR-29b−transfected corneal tissue, LAMC1 protein expression level was decreased to 14.4{\%} (±20.5{\%}). CONCLUSIONS:: Overexpression of miR-29b decreased ECM protein production in human corneal endothelial cells. Thus, miR-29 replacement therapy might be a new treatment strategy for FECD aimed at reducing pathologic production of ECM proteins in Descemet membrane.",
author = "Tetsuya Toyono and Tomohiko Usui and Guadalupe Villarreal and Laura Kallay and Mario Matthaei and Vianna, {Lucas M M} and Zhu, {Angela Y.} and Masahiko Kuroda and Shiro Amano and Albert Jun",
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AU - Toyono, Tetsuya

AU - Usui, Tomohiko

AU - Villarreal, Guadalupe

AU - Kallay, Laura

AU - Matthaei, Mario

AU - Vianna, Lucas M M

AU - Zhu, Angela Y.

AU - Kuroda, Masahiko

AU - Amano, Shiro

AU - Jun, Albert

PY - 2016/8/3

Y1 - 2016/8/3

N2 - PURPOSE:: MicroRNAs are small noncoding RNAs that regulate gene expression at the posttranscriptional level. We reported that levels of microRNA (miR)-29 family are decreased in corneas of patients with Fuchs endothelial corneal dystrophy (FECD). The miR-29 family regulates the production of extracellular matrix (ECM) proteins. Accumulation of ECM proteins in Descemet membrane is an important pathologic change in FECD. In this study, we transfected miR-29b into human corneal endothelial cells and tissues and evaluated ECM protein expression levels. METHODS:: An immortalized Fuchs human corneal endothelial cell line (iFECD) was established by infection of corneal endothelial cells from patients with FECD with hTERT lentivirus. MiR-29b was transfected into iFECD, and the expression levels of ECMs collagen type 1 alpha 1 (COL1A1), collagen type 4 alpha 1 (COL4A1), and laminin gamma 1 (LAMC1) were evaluated with quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) and Western blot. Expression level of LAMC1 protein in miR-29b−transfected donor corneal endothelium was also evaluated by Western blot. RESULTS:: Compared with control, miR-29b expression level after transfection of iFECD was increased to 335.6% (±91.0%), and ECM expression levels were significantly decreased. Compared with control, qRT-PCR demonstrated reduction of ECM to the following levels: COL1A1: 1.9% (±0.4%); COL4A1: 7.1% (±1.7%); and LAMC1: 21.5% (±2.7%). Western blot showed reduced protein expression: COL1A1: 4.8% (±3.2%); COL4A1: 42.5% (±25.0%); and LAMC1: 44.8% (±3.1%). In miR-29b−transfected corneal tissue, LAMC1 protein expression level was decreased to 14.4% (±20.5%). CONCLUSIONS:: Overexpression of miR-29b decreased ECM protein production in human corneal endothelial cells. Thus, miR-29 replacement therapy might be a new treatment strategy for FECD aimed at reducing pathologic production of ECM proteins in Descemet membrane.

AB - PURPOSE:: MicroRNAs are small noncoding RNAs that regulate gene expression at the posttranscriptional level. We reported that levels of microRNA (miR)-29 family are decreased in corneas of patients with Fuchs endothelial corneal dystrophy (FECD). The miR-29 family regulates the production of extracellular matrix (ECM) proteins. Accumulation of ECM proteins in Descemet membrane is an important pathologic change in FECD. In this study, we transfected miR-29b into human corneal endothelial cells and tissues and evaluated ECM protein expression levels. METHODS:: An immortalized Fuchs human corneal endothelial cell line (iFECD) was established by infection of corneal endothelial cells from patients with FECD with hTERT lentivirus. MiR-29b was transfected into iFECD, and the expression levels of ECMs collagen type 1 alpha 1 (COL1A1), collagen type 4 alpha 1 (COL4A1), and laminin gamma 1 (LAMC1) were evaluated with quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) and Western blot. Expression level of LAMC1 protein in miR-29b−transfected donor corneal endothelium was also evaluated by Western blot. RESULTS:: Compared with control, miR-29b expression level after transfection of iFECD was increased to 335.6% (±91.0%), and ECM expression levels were significantly decreased. Compared with control, qRT-PCR demonstrated reduction of ECM to the following levels: COL1A1: 1.9% (±0.4%); COL4A1: 7.1% (±1.7%); and LAMC1: 21.5% (±2.7%). Western blot showed reduced protein expression: COL1A1: 4.8% (±3.2%); COL4A1: 42.5% (±25.0%); and LAMC1: 44.8% (±3.1%). In miR-29b−transfected corneal tissue, LAMC1 protein expression level was decreased to 14.4% (±20.5%). CONCLUSIONS:: Overexpression of miR-29b decreased ECM protein production in human corneal endothelial cells. Thus, miR-29 replacement therapy might be a new treatment strategy for FECD aimed at reducing pathologic production of ECM proteins in Descemet membrane.

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