Inhibition effect of short hairpin RNA of transforming growth factor beta1 on cytokines expression induced by albumin in HK2 cells.
OBJECTIVE
To determine the effect of 2 transforming growth factor beta1 (TGF-beta1) short hairpin RNA (shRNA) expression plasmids (pcDU6-A1-A2 and pcDU6-B1-B2) on proliferation, TGF-beta1, connective tissue growth factor (CTGF), and fibronectin (FN) expression induced by human serum albumin (HSA) in HK2 cells.


METHODS
A vector plasmid containing the TGF-beta1 shRNA was generated. An HK2 cell line was used in the study. The 2 TGF-beta1 shRNA expression plasmids were transfected into cultured HK2 cells by lipofectamine 2000. Cellular proliferation was assessed by tetrazolium salt colorimetry. The semi-quantitative reverse transcriptive PCR was performed to detect the expression of TGF-beta1, CTGF, and FN mRNA. Levels of TGF-beta1 and FN protein were measured with a sandwich enzyme-linked immunosorbent assay.


RESULTS
After treating with 5 g/L HSA for 24 hours in HK2 cells, cellular proliferating capacity increased significantly (P<0.05). The expression levels of TGF-beta1, CTGF, and FN mRNA were upregulated in HK2 cells stimulated by 5 g/L HSA, and levels of TGF-beta1 and FN protein in the culture supernatant increased (P<0.05). The introduction of pcDU6-A1-A2 and pcDU6-B1-B2 resulted in significant reduction of cellular proliferation activity, and the expression levels of TGF-beta1, CTGF, and FN mRNA were downregulated (P<0.05). Levels of TGF-beta1 and FN protein in the culture supernatant decreased (P<0.05) after 12 or 24 hours of TGF-beta1 shRNA transfection into HK2 cells There was no significant difference in the expression levels of TGF-beta1, CTGF, and FN mRNA between the 2 pcDU6 vector plasmid mediated TGF-beta1 shRNA groups (P>0.05).


CONCLUSIONS
pcDU6 vector plasmid mediated TGF-beta1 shRNAs could obviously inhibit the expression levels of TGF-beta1, CTGF, FN and cellular proliferation stimulated by HSA in HK2 cells, which may be related to the mediation of TGF-beta1.
Duan
Shaobin
S
Department of Nephrology, Second Xiangya Hospital, Central South University, Changsha, China.
Liu
Fuyou
F
Chen
Yinyin
Y
Liu
Fang
F
Li
Ying
Y
Ling
Guanghui
G
Xiao
Li
L
Liu
Hong
H
Peng
Youming
Y
D002460:Cell Line; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D012709:Serum Albumin / Q000494:pharmacology*; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000235:genetics*
C526317:CCN2 protein, human; D016207:Cytokines; D005353:Fibronectins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012709:Serum Albumin; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor