Inhibition of TGF-beta1 expression in human peritoneal mesothelial cells by pcDU6 vector-mediated TGF-beta1 shRNA.

Abstract

BACKGROUND AND AIM:

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).

CONCLUSION:

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.

• Authors: Fuyou Liu, Hong Liu, Youming Peng, Yinghong Liu, Jun Li, Xing Chen
• Journal: Nephrology (Carlton, Vic.) (Nephrology (Carlton)) Vol. 11 Issue 1 Pg. 23-8 (Feb 2006) ISSN: 1320-5358 [Print] Australia
• PMID: 16509928 (Publication Type: Journal Article)

Chemical References

• TGFB1 protein, human
• Transforming Growth Factor beta
• Transforming Growth Factor beta1
• RNA

Topics

• Epithelial Cells (metabolism)
• Humans
• Peritoneum (cytology)
• Plasmids
• RNA
• Transforming Growth Factor beta (antagonists & inhibitors, biosynthesis, genetics)
• Transforming Growth Factor beta1