A novel aptamer targeting TGF-β receptor II inhibits transdifferentiation of human tenon's fibroblasts into myofibroblast.
OBJECTIVE
To isolate aptamers that were bound to the extracellular segment of TGF-β receptor II (TβRII) and evaluate their effect on the TGF-β-induced transdifferentiation of fibroblasts.


METHODS
TβRII-binding aptamers were screened by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) from a single stranded DNA (ssDNA) library. Human Tenon's fibroblasts (HTFs) were cultured and treated with TGF-β2, TGF-β2 and aptamer S58/68, or aptamer S58/68 alone. Western blot analysis was performed to determine levels of α-smooth muscle actin (α-SMA) and the signaling protein phosphorylated Smad2 (p-Smad2). α-SMA and p-Smad2 subcellular distribution and fibrous actin (F-actin) with rhodamine-phalloidin staining were evaluated by confocal immunofluorescence microscopy. Cell contractility was assessed in collagen gel contraction assays.


RESULTS
Twenty-one sequences were obtained after eight rounds of selection. Two preferential sequences, aptamer S58 and S68, were isolated and used in the following experiments. Aptamer S58 significantly inhibited α-SMA expression and incorporation into actin stress fibers, as induced by TGF-β2. Aptamer S58 also suppressed TGF-β2-induced cell contraction. Furthermore, aptamer S58 inhibited the TGF-β2-induced phosphorylation and nuclear translocation of Smad2. However, we did not find any effect of aptamer S68 on TGF-β2 activity in vitro.


CONCLUSIONS
Our study revealed that a novel aptamer binding TβRII inhibited TGF-β2-induced myofibroblast transdifferentiation in HTFs.
Department of Ophthalmology, College of Basic Medical Sciences, Daping Hospital, Third Military Medical University, Chongqing, People's Republic of China.
Xiaoyan
X
Zhu
Lei
L
Li
Lingyun
L
Zou
Xudong
X
Zhu
Guangjun
G
Xian
Haijun
H
Li
Yan
Y
Tan
Lin
L
Xie
D000199:Actins / Q000378:metabolism; D052157:Aptamers, Nucleotide / Q000494:pharmacology*; D001483:Base Sequence; D015153:Blotting, Western; D054338:Cell Transdifferentiation / Q000187:drug effects*; D002478:Cells, Cultured; D004277:DNA, Single-Stranded / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D058628:Myofibroblasts / Q000166:cytology*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011863:Radioimmunoassay; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000378:metabolism; D052156:SELEX Aptamer Technique; D017422:Sequence Analysis, DNA; D051899:Smad2 Protein / Q000378:metabolism; D058475:Tenon Capsule / Q000166:cytology*; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000378:metabolism
C541116:ACTA2 protein, human; D000199:Actins; D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D018125:Receptors, Transforming Growth Factor beta; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D016212:Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II