CYR61, regulated by miR-22-3p and MALAT1, promotes autophagy in HK-2 cell inflammatory model.

Abstract

BackgroundRenal tubular epithelial cells play an important role in renal function and are a major site of injury from inflammation. Emerging evidence suggests that CYR61 is involved in the regulation of autophagy. However, there are few studies on CYR61 in nephropathy and associated inflammation. This study aimed to clarify how CYR61 regulates autophagy in human renal epithelial cells while in an inflammatory state and regulates the upstream pathway of CYR61 levels.MethodsThe human renal tubular epithelial cells (HK-2) cell line treated by lipopolysaccharide (LPS) was used as an inflammatory model of human epithelial cells. Short hairpin RNA (shRNA) was used to down-regulate CYR61, and the changes in the transcription and expression levels of related molecules, as well as the morphological changes of HK-2 cells, were detected by quantitative real time-PCR (qRT-PCR), western blot (WB), and transmission electron microscopy. Either CYR61 or MALAT1 were up-regulated by overexpression vectors, or MALAT1 was down-regulated by miR-22-3p mimics. Subsequently, the levels of CYR61, MALAT1, related inflammatory factors, and autophagy factors were measured by qPCR, WB, and enzyme-linked immunosorbent assay (ELISA). Cell apoptosis was detected by flow cytometry and acridine-orange assay.ResultsWe observed that down-regulation of CYR61 could down-regulate 1B-light chain 3 (LC3) level and inhibit autophagy in the LPS-induced inflammation model of HK-2 cells. The expression levels of CYR61, Beclin1, Atg5, LC3, interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) were significantly increased by upregulating CYR61 or MALAT1 by overexpression vector, while the expression level of p62 was significantly decreased, intracellular reactive oxygen species (ROS) content was increased, and the proportion of autophagy and apoptosis was increased. The use of miR-22-3p mimics significantly reversed the changes induced by up-regulation of CYR61 or MALAT1 at the molecular and cellular levels.ConclusionsOur data indicated that CYR61 positively regulates autophagy of HK-2 cells under an inflammatory state, and was negatively regulated by miR-22-3p, while miR-22-3p and MALAT1 were negatively regulated by each other.