Inhibition effect of small interfering RNA of connective tissue growth factor on the expression of vascular endothelial growth factor and connective tissue growth factor in cultured human peritoneal mesothelial cells.
BACKGROUND
The peritoneum response to peritoneal dialysis can lead to fibrosis. The transforming growth factor beta1 (TGF-beta1) plays a key role in regulating tissue repair and remodelling after injury. Connective tissue growth factor (CTGF), a downstream mediator of TGF-beta1 inducing fibrosis, has been implicated in peritoneal fibrosis. Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis that can hasten peritoneal fibrosis. In this study, we investigated the effect of small interfering RNA (siRNA) of CTGF by pRETRO-SUPER (PRS) retrovirus vector on the expression of CTGF and VEGF in human peritoneal mesothelial cells.


METHODS
Retrovirus producing CTGF siRNA were constructed from the inverted oligonucleotides and transferred into packaging cell line PT67 with lipofectamine, and the virus supernatant was used to infect human peritoneal mesothelial cell (HPMC). The cells were divided into seven groups: low glucose DMEM, low glucose DMEM + TGF-beta1 5 ng/ml, low glucose DMEM + TGF-beta1 5 ng/ml + PRS-CTGF-siRNA(1-4) and low glucose DMEM + TGF-beta1 5 ng/ml + PRS. The expression of CTGF and VEGF were measured by semiquantitative RT-PCR and Western blot.


RESULTS
Low levels of CTGF and VEGF were detected in confluent HPMCs. Following stimulation with TGF-beta1, the levels of CTGF and VEGF were significantly upregulated (P < 0.01). Introduction of PRS-CTGF-siRNA(1-4) resulted in the significant reduction of CTGF mRNA and protein, and VEGF mRNA (P < 0.01), especially in groups PRS-CTGF-siRNA1 and PRS-CTGF-siRNA4. The introduction of PRS void vector did not have these effects (P > 0.05).


CONCLUSIONS
The expression of CTGF siRNA mediated by PRS retrovirus vector can effectively reduce the level of CTGF and VEGF induced by TGF-beta1 in cultured HPMCs. This study may provide potential therapeutic strategies to prevent the peritoneal fibrosis.
Liu
Fu-you
FY
Division of Nephrology, Second Affiliated Xiangya Hospital, Central South University, Changsha 410002, China.
Xiao
Li
L
Peng
You-ming
YM
Duan
Shao-bin
SB
Liu
Hong
H
Liu
Ying-hong
YH
Ling
Gui-hui
GH
Yuan
Fang
F
Chen
Jun-xiang
JX
Fu
Xiao
X
Zhu
Jian-lian
JL
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins / Q000032:analysis / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010537:Peritoneum / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering / Q000494:pharmacology*; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis / Q000235:genetics*
C526317:CCN2 protein, human; C526318:CCN2 protein, mouse; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A; D055513:Connective Tissue Growth Factor