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Inhibitor of growth 4 (ING4) inhibits hypoxia-induced EMT by decreasing HIF-1α and snail in HK2 cells.

Acta histochemica (2019-06-27)
Lingling Lu, Jing Li, Yuan Le, Hong Jiang
ABSTRACT

Renal fibrosis is a common mechanism that leads to all kidney diseases and Epithelial-mesenchymal transition (EMT) is considered as one of the potential mechanisms of renal fibrosis. Inhibitor of growth 4 (ING4) was reported to involve in several diseases; especially it was negatively correlated with lung fibrogenesis parameters. However, the role of ING4 and underlying mechanisms in EMT are still unknown. In this study, we used a UUO rat model to mimic renal fibrosis, which was examined by Masson and HE staining analysis. To explore the effects of ING4 on hypoxia-induced EMT, HK2 cells were treated with hypoxia to induce EMT and ING4 was over-expressed in hypoxia-treated HK2 cells by transfection of pEGFP-N1-ING4. MTT assay was used to describe the cell viability of HK2 cells under the hypoxic condition. The expression levels of ING4, hypoxia-inducible factor-1α (HIF-1α), and EMT markers (E-cadherin, N-cadherin and vimentin) were examined in vivo and in vitro by western blot, qRT-PCR, immunohistochemical staining or Immunofluorescence. Our results showed that, in a UUO rat model, ING4 was decreased and EMT was developed with reduction in E-cadherin and increase in N-cadherin and vimentin, suggesting a significant association between ING4 expression and EMT. Under hypoxia, E-cadherin was down-regulated and N-cadherin and vimentin were up-regulated, indicating that hypoxia induced EMT in HK2 cells. Nonetheless, changes in the expression of EMT biomarkers were inhibited by over-expression of ING4. Moreover, over-expressing ING4 decreased the expression of HIF-1α and snail in HK2 cells. These findings suggest that ING4 may inhibit hypoxia-induced EMT via decreasing HIF-1α and snail in HK2 cells, indicating the potential of ING4 as a therapeutic target for renal fibrosis.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium - high glucose, With 4500 mg/L glucose and L-glutamine, without sodium bicarbonate, powder, suitable for cell culture
Sigma-Aldrich
Dimethyl sulfoxide, ReagentPlus®, ≥99.5%
Sigma-Aldrich
Fetal Bovine Serum, New Zealand origin, sterile-filtered, suitable for cell culture, suitable for hybridoma