Guoji Yanke Zazhi (May 2020)

Autophagy regulates high glucose-induced epithelial-mesenchymal transition in human lens epithelial cells

  • Ji-Yuan Ma,
  • Wei Ye,
  • Ji Li,
  • Rui Pei,
  • Meng-Mei He,
  • Jing-Bo Su,
  • Dong-Jie Sun,
  • Qi-Wu Zhou,
  • Jian Zhou

DOI
https://doi.org/10.3980/j.issn.1672-5123.2020.5.04
Journal volume & issue
Vol. 20, no. 5
pp. 759 – 767

Abstract

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AIM: To investigate the regulation of autophagy on high glucose-induced epithelial-mesenchymal transition(EMT)in human lens epithelial cells.METHODS: In order to investigate the changes of EMT and autophagy induced by high glucose, HLE-B3 cells were divided into two groups. In NC group, cells were cultured in DMEM with 5.5mmol/L glucose, and in HG group, cells were treated with DMEM in addition with 30mmol/L glucose for 12h, 24h, and 48h. Western blot was used to detect the expression of EMT-marker proteins(E-cadherin and α-SMA)and autophagy-marker proteins(LC3, Beclin 1 and SQSTM1/p62). Wound healing assay was conducted to observe the migration ability. To investigate the regulation of autophagy on EMT, we employed rapamycin, an agonist of autophagy. HLE-B3 cells were divided into 4 groups. Two of them were mentioned as above, and the other two groups were treated with high glucose combined with DMSO(DMSO)and high glucose combined with 200nmol/L rapamycin(RAPA), respectively. Migration ability of cells was evaluated by Transwell assay. Expressions of proteins, such as EMT marker proteins, molecules in TGF-β signaling pathway(TGF-β2, Smad2/3, p-Smad2/3, Snail), and autophagy markers were detected by Western blot. The intracellular co-localization of SQSTM1/p62 and Smad2/3 was observed by immunofluorescence staining, and their interaction was confirmed by co-immunoprecipitation assay. RESULTS: The expression of E-cadherin, LC3 Ⅱ/Ⅰ, and Beclin 1 in HLE-B3 cells of HG group gradually decreased(F=67.52, 163, 206; all PF=53.37, 302.1; all PPPP>0.05)in RAPA group compared with HG group and DMSO group, cell migration was also suppressed(all PCONCLUSION: High glucose stimulates the process of EMT and suppresses the autophagy in HLE-B3 cells. Autophagy regulates EMT by interacting with Smad2/3 via SQSTM1/p62, altering the amount of Smad2/3 which works in the TGF-β signaling pathway.

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