Regulatory effect of miR-29b on OPN/TGF-β pathway and the change of this pathway in a high glucose environment in a renal cell co-culture system
10.3969/j.issn.1672-8467.2024.06.007
- VernacularTitle:肾细胞共培养体系中miR-29b对OPN/TGF-β通路的调控作用及高糖环境下该通路的变化
- Author:
Juan LIU
1
;
Ming-Zheng YANG
;
Xiao-Ying LI
;
Ting-Ting JI
;
Xiao-Yan XIONG
;
Ying-Chun ZHU
;
Shou-Jun BAI
Author Information
1. 复旦大学附属中山医院青浦分院肾内科 上海 201700
- Keywords:
diabetic nephropathy(DN);
renal cell;
miR-29b-3p;
osteopontin(OPN);
transforming growth factor β(TGF-β);
high glucose;
co-culture
- From:
Fudan University Journal of Medical Sciences
2024;51(6):921-930
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a renal cell co-culture system to simulate the renal barrier system,and to test its responsiveness to different glucose concentrations,and to investigate the regulatory effect of miR-29b-3p on osteopontin(OPN)/transforming growth factor β(TGF-β)pathway and the changes of this pathway under high glucose condition.Methods The three-cell co-culture system consisting of human renal podocytes,human glomerular mesangial cells and human renal tubular epithelial cells was established to test the cell viability and glucose consumption value at glucose concentrations of 5,8,12 and 16 mmol/L.The content of TGF-β and OPN in cell supernatant was measured.The recombinant plasmid and siRNA of OPN were transfected,and the expressions of TGF-β and OPN were detected by Q-PCR and Western blot.Results The mRNA expressions of OPN,TGF-β and miR-29b were significantly increased at 12 mmol/L glucose conditions.Western blot results showed that the protein expression of OPN increased in high glucose conditions,while the protein expression of TGF-β did not change significantly.After adding miR-29b-3p activator,the mRNA levels of OPN and TGF-β in the cell supernatant were significantly increased.After adding miR-29b-3p inhibitor,the mRNA levels of OPN and TGF-β in the cell supernatant were significantly decreased.Western blot results showed that compared with 5 mmol/L glucose,the protein expressions of OPN and TGF-β were increased by miR-29b-3p activator,and the protein expressions of OPN and TGF-β were decreased by miR-29b-3p inhibitor.After transfection with OPN recombinant plasmid,the content of TGF-β in the cell supernatant was significantly increased,and the mRNA expressions of OPN and TGF-β in the cells were significantly increased.After transfection with OPN siRNA,the content of TGF-β in the cell supernatant was decreased,and the expression of OPN mRNA in the cells was significantly decreased,but the expression of TGF-β mRNA was not significantly increased.Conclusion The renal cell co-culture system can mimic the complex renal environment in vivo.When induced by high glucose,cell proliferation is inhibited,glucose consumption is increased,and the content of TGF-β in the cell supernatant is increased,and miR-29b-3p has a regulatory effect on OPN/TGF-β signaling pathway in the co-culture system.
