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

Abstract:

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 P<0.0001), the expressions of α-SMA, SQSTM1/p62 increased with time(F=53.37, 302.1; all P<0.0001), and cell migration also increased compared with the cells in NC group(all P<0.001), indicating that high glucose stimulated EMT and suppressed autophagy. After treatment with rapamycin, the expressions of LC3 Ⅱ/Ⅰ and E-cadherin increased, the expressions of α-SMA, p-Smad2/Smad2, p-Smad3/Smad3 and Snail decreased(all P<0.05), and the expressions of TGF-β2 did not change(all P>0.05)in RAPA group compared with HG group and DMSO group, cell migration was also suppressed(all P<0.001), indicating that Rapamycin down regulated the expressions of molecules in TGF-βsignaling pathway after activation of autophagy, which resulted in inhibiting EMT. Immunofluorescence staining showed co-localization of SQSTM1/p62 and Smad2/3 in cytoplasm. Co-immunoprecipitation confirmed the combination between SQSTM1/p62 and Smad2/3.

CONCLUSION: 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.

Key words:autophagy; epithelial-mesenchymal transition; lens epithelial cells; rapamycin; TGF-β signaling pathway