title: Inhibition effect of short hairpin RNA of transforming growth factor beta1 on cytokines expression induced by albumin in HK2 cells.
abstract: 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.
authors.affiliation: Department of Nephrology, Second Xiangya Hospital, Central South University, Changsha, China.
authors.forename: Shaobin
authors.initials: S
authors.lastname: Duan
authors.forename: Fuyou
authors.initials: F
authors.lastname: Liu
authors.forename: Yinyin
authors.initials: Y
authors.lastname: Chen
authors.forename: Fang
authors.initials: F
authors.lastname: Liu
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.forename: Guanghui
authors.initials: G
authors.lastname: Ling
authors.forename: Li
authors.initials: L
authors.lastname: Xiao
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.forename: Youming
authors.initials: Y
authors.lastname: Peng
mesh_terms: 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*
chemical_list: 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