Abstract: TH-PO368

MicroRNA-200c Is Involved in Klotho Reduction by Oxidative Stress in Human Tubular Cells

Session Information

Category: Cell Biology

  • 201 Cell Signaling, Oxidative Stress

Authors

  • Morii, Kenichi, Hiroshima University Hospital, Hiroshima, Japan
  • Yamasaki, Satoshi, Kurume University Medical Center, Kurume, Japan
  • Doi, Shigehiro, Hiroshima University Hospital, Hiroshima, Japan
  • Sasaki, Kensuke, Hiroshima University Hospital, Hiroshima, Japan
  • Ueno, Toshinori, Hiroshima University Hospital, Hiroshima, Japan
  • Nakashima, Ayumu, Hiroshima University Hospital, Hiroshima, Japan
  • Masaki, Takao, Hiroshima University Hospital, Hiroshima, Japan
Background

Klotho deficiency is reportedly associated with the progression of kidney dysfunction, whereas its overexpression exerts renoprotective effects. Previous studies report that oxidative stress suppressed Klotho expression in renal epithelial cells, and that microRNA-200c (miR-200c) is upregulated by oxidative stress in human umbilical vein endothelial cells. In this study, we investigated whether oxidative stress-induced miR-200c is implicated in Klotho reduction in human tubular cells (HK-2).

Methods

HK-2 were stimulated with hydrogen peroxide before Klotho expression was evaluated using western blotting (WB) and quantitative PCR (qRT-PCR). MiR-200c expression was determined by qRT-PCR and the miR-200c binding site at the klotho mRNA 3′-untranslated region (3′-UTR) was characterized using an online prediction tool (microRNA.org). After miR-200c mimic or inhibitor was transfected into HK-2, Klotho expression was examined using WB and qRT-PCR. Luciferase reporter plasmid containing klotho 3′-UTR was transfected into HK-2 to investigate the inhibitory effect of miR-200c on Klotho expression. Histological analysis was performed to examine the correlation between Klotho and oxidative stress markers (8-OHdG, 4-HHE). In situ hybridization was performed to reveal the localization of miR-200c in human kidney biopsy specimens.

Results

Hydrogen peroxide suppressed Klotho expression without any reduction in klotho mRNA levels but upregulated miR-200c expression. Similarly, transfection of miR-200c mimic reduced Klotho expression as well as luciferase activity without any reduction in klotho mRNA levels. In contrast, transfection of miR-200c inhibitor maintained Klotho expression. In human kidney biopsy specimens, Klotho expression inversely correlated with oxidative stress markers (8-OHdG: ρ=−0.44, P=0.010; 4-HHE: ρ=−0.39, P=0.021). MiR-200c was expressed in distal tubular cells whose renal function was lowered.

Conclusion

Oxidative stress-induced miR-200c binds to klotho mRNA 3′-UTR, resulting in a reduction in Klotho expression.