Downregulation of transforming growth factor-β type II receptor prohibit epithelial-to-mesenchymal transition in lens epithelium.
OBJECTIVE
Transforming growth factor-β (TGF-β) is considered to be essential to induce epithelial-to-mesenchymal transition (EMT) which plays central roles in wound healing in ocular fibrotic complication. The present study investigates whether small interference RNAs (siRNAs) targeting the type II receptor of TGF-β (TβRII) could be used to minimize the TGF-β action.


METHODS
TGF-β receptor type II (TβRII) specific siRNAs designed from the Nakamura human gene sequence were used to transfect the cultured lens epithelial cells (LECs). The optimal transfection of scramble siRNA-Cy3 labeled duplexes in cultured LECs were examined by laser scanning confocal microscope and flow cytometry. TβRII protein expression and transcript levels were analyzed by immunofluorescence, western blotting, and real time PCR, respectively. Western blotting was performed to examine protein expression of fibronectin and alpha-smooth muscle actin (α-SMA). Scratch assay was used to determine cell migration. Cell morphology was observed after transfection by inverted microscope.


RESULTS
The optimal transfection rate of scramble siRNA-Cy3 labeled duplexes was efficient in that nearly to 50% in cultured LECs. TβRII specific siRNAs significantly reduced the receptor transcript and protein expression in cultured LECs. The gene knockdown inhibited LECs transdifferentiation, as it abrogated the expression of fibronectin and α-SMA, and retarded cell migration on the scratch assay. In addition, after transfection with TβRII specific siRNA, the cultured LECs did not show fibroblast-like shape which was one of the feature signs of EMT. Wound scratch assays indicated that the number of cultured LECs migrated into the wounded area was significantly lower in TβRII specific siRNA treated group (12.8 ± 3.27/7.85 mm(2)), compared with normal (57.8 ± 3.06/7.85 mm(2)) and scrambled RNA transfected group (50.8 ± 3.64/7.85 mm(2); p<0.0001).


CONCLUSIONS
Our results provided additional evidence to support that TGF-β pathway was involved in the development of EMT of human posterior capsule opacification, while how TβRII was involved should be further investigated.
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Danying
D
Zheng
Tingting
T
Song
Xueying
X
Zhongliu
Mingxing
M
Wu
Jingli
J
Liang
Yizhi
Y
Liu
D000199:Actins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002465:Cell Movement / Q000235:genetics; D054338:Cell Transdifferentiation; D015536:Down-Regulation; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D058750:Epithelial-Mesenchymal Transition; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism*; D018613:Microscopy, Confocal; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000235:genetics; D015398:Signal Transduction; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism
C541116:ACTA2 protein, human; D000199:Actins; D005353:Fibronectins; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II