Inhibition of TGF-beta1 expression in human peritoneal mesothelial cells by pcDU6 vector-mediated TGF-beta1 shRNA.
OBJECTIVE
Transforming growth factor-beta(1) (TGF-beta(1)) is an important mediator of the fibrosis process. High expression of TGF-beta(1) is closely related to peritoneal fibrosis. RNA interference using short hairpin RNA (shRNA) can mediate sequence-specific inhibition of gene expression in mammalian cells. The aim of this study was to assess the effect of shRNA targeting TGF-beta(1) on the expression of TGF-beta(1) in human peritoneal mesothelial cells (HPMC).


METHODS
TGF-beta(1) specific shRNA expression vectors were constructed and introduced to HPMC stimulated with 4.25% D-glucose (Gs) and 10 microg/mL of lipopolysaccharide (LPS). Expression of TGF-beta(1) mRNA was assessed by semiquantification reverse transcription polymerase chain reaction (RT-PCR). The TGF-beta(1) protein level in the culture supernatant was determined by sandwich enzyme-linked immunosorbent assay.


RESULTS
The expression of TGF-beta(1) was upregulated significantly in HPMC stimulated with 4.25% D-Gs and 10 microg/mL LPS P < 0.01. TGF-beta(1) expression in pcDU6 plasmid vector-mediated TGF-beta(1) shRNA groups were obviously downregulated when compared to the 4.25% D-Gs and 10 microg/mL LPS group (P < 0.01) and the pcDU6 void vector group (P < 0.05), with no significant difference among pcDU6 plasmid vector -mediated TGF-beta(1) shRNA groups (P > 0.05). No significant difference was found between teh pcDNA3.1(-) vector plasmid-mediated TGF-beta(1) antisense RNA group and pcDU6 void vector group (P > 0.05). The expression of TGF-beta(1) in pcDU6 plasmid vector-mediated TGF-beta(1) shRNA groups were obviously downregulated when compared to the pcDNA3.1(-) plasmid vector-mediated TGF-beta(1) antisense RNA group (P < 0.05).


CONCLUSIONS
TGF-beta(1)-specific shRNA can significantly inhibit the expression of TGF-beta(1) in HPMC stimulated with 4.25% D-Gs and 10 microg/mL LPS in HPMC. These results suggest the possible application of TGF-beta(1)-specific shRNA in preventing peritoneal fibrosis in patients receiving peritoneal dialysis.
Liu
Fuyou
F
Nephrology Department, 2nd Xiangya Hospital, Central South University, Changsha City, Hunan Province, China.
Liu
Hong
H
Peng
Youming
Y
Liu
Yinghong
Y
Li
Jun
J
Chen
Xing
X
D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D010537:Peritoneum / Q000166:cytology*; D010957:Plasmids; D012313:RNA; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics*; D053773:Transforming Growth Factor beta1
C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D012313:RNA