Long non-coding RNA CASC2 restrains high glucose-induced proliferation, inflammation and fibrosis in human glomerular mesangial cells through mediating miR-135a-5p/TIMP3 axis and JNK signaling

Qian Xue,Xiang Wu,Dongju Zhu

doi:10.1186/s13098-021-00709-5

Abstract

BackgroundDiabetic nephropathy (DN) is a common complication of diabetes. Long non-coding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) is reported to exert a protective role in DN by a previous study. The working mechanism underlying the protective role of CASC2 in DN progression was further explored in this study.MethodsThe expression of CASC2 and microRNA-135a-5p (miR-135a-5p) was determined by real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation ability was assessed by Cell Counting Kit-8 (CCK8) assay and 5-ethynyl-29-deoxyuridine (EDU) assay. Enzyme-linked immunosorbent assay (ELISA) was conducted to analyze the production of inflammatory cytokines in the supernatant. Western blot assay was performed to analyze protein expression. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to verify the target relationship between miR-135a-5p and CASC2 or tissue inhibitors of metalloproteinase 3 (TIMP3).ResultsHigh glucose (HG) treatment reduced the expression of CASC2 in human glomerular mesangial cells (HMCs) in a time-dependent manner. CASC2 overexpression suppressed HG-induced proliferation, inflammation and fibrosis in HMCs. miR-135a-5p was validated as a target of CASC2, and CASC2 restrained HG-induced influences in HMCs partly by down-regulating miR-135a-5p. miR-135a-5p bound to the 3ʹ untranslated region (3ʹUTR) of TIMP3, and CASC2 positively regulated TIMP3 expression by sponging miR-135a-5p in HMCs. miR-135a-5p silencing inhibited HG-induced effects in HMCs partly by up-regulating its target TIMP3. CASC2 overexpression suppressed HG-induced activation of Jun N-terminal Kinase (JNK) signaling partly through mediating miR-135a-5p/TIMP3 signaling.ConclusionsIn conclusion, CASC2 alleviated proliferation, inflammation and fibrosis in DN cell model by sponging miR-135a-5p to induce TIMP3 expression.

Highlights

Diabetic nephropathy (DN) is a common complication of diabetes
high glucose (HG) treatment promotes the proliferation, inflammation, and fibrosis of human glomerular mesangial cells (HMCs) partly by reducing the level of cancer susceptibility candidate 2 (CASC2) HG (25 mM) treatment decreased the level of CASC2 in HMCs in a time-dependent manner, while normal glucose (NG) (5.5 mM) treatment had no significant effect on CASC2 expression in HMCs (Fig. 1A)
To test whether the down-regulation of CASC2 was important for HG-induced effects, we rescued the expression of CASC2 using its overexpression plasmid in HG-treated HMCs

Summary

Introduction

Long non-coding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) is reported to exert a protective role in DN by a previous study. The working mechanism underlying the protective role of CASC2 in DN progression was further explored in this study. Understanding the molecular mechanism behind the aberrant pathological features of mesangial cells is essential to identify novel targets for DN treatment. Li et al found that KCNQ1OT1 silencing alleviates high glucose (HG)-mediated proliferation, oxidative stress, and deposition of ECM in mesangial cells through mediating miR-18b/HMGA2 signaling [7]. Wang et al demonstrated that lncRNA CTBP1-AS2 attenuates HGmediated oxidative stress, the deposition of ECM, and inflammatory response in mesangial cells by sponging miR-155-5p to induce FOXO1 expression [8]. The working mechanism of CASC2 in DN progression was further explored

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Results

Conclusion