LncRNA ZFAS1 regulates the proliferation, oxidative stress, fibrosis, and inflammation of high glucose-induced human mesangial cells via the miR-588/ROCK1 axis.

Background: Diabetic nephropathy (DN) is a critical and the most common microvascular complication and its pathogenesis is still faintly understood. Thus, this study was performed to examine the long non-coding RNA ZNFX1 Antisense Gene Protein 1 (lncRNA ZFAS1) biological function and mechanism of regulation in DN.

Method: Human glomerular mesangial cells (HGMC) were induced with high glucose (HG, 25 mM) to establish HG-induced cell viability, pro-inflammation observed in DN. After, target miRNA and mRNA were predicted through Lncbase and Targetscan. Subsequently, the expression of ZFAS1, miR-588, and ROCK1 in DN clinical samples and cell-model was examined through qRT-PCR and western blot analysis. We upheld the targeted interaction between miR-588 and ZFAS1 or ROCK1 through a dual-luciferase reporter assay. The proliferation of the cell was also examined through CCK-8 assay, while the level of HG-induced oxidative stress was established by measuring reactive oxygen species (ROS) level, and also the activities of antioxidant enzymes in the cell. Lastly, the level of accumulated extracellular matrix (ECM) protein-fibronectin and collagen type IV, and inflammatory cytokines produced by the cell was analyzed through western blot analysis and ELISA.

Results: ZFAS1 was significantly upregulated in the DN blood samples and HG-induced HGMC. Prediction result revealed that the ZFAS1 endogenously targets the miR-588 seed sequence while miR-588 plays a role in post-transcriptional regulation of ROCK1 mRNA. Moreover, we found that miR-588 expression was significantly downregulated in DN blood samples and negatively correlates with ZFAS1 expression. Further results show that silencing ZFAS1 had a protective effect on HG-induced proliferation, oxidative stress, fibrosis, and inflammation in HGMC while miR-588 inhibition and ROCK1 overexpression reversed this effect.

Conclusions: Altogether, our data suggest that ZFAS1 regulates the proliferation, oxidative stress, fibrosis, and inflammation of high glucose-induced diabetic nephropathy through the miR-588/ROCK1 axis.