LncRNA CASC15 inhibition relieves renal fibrosis in diabetic nephropathy through down-regulating SP-A by sponging to miR-424

Abstract

Study has demonstrated the abnormal expression and role of lncRNA CASC15 in diabetes patients with chronic renal failure. However, its role in diabetes nephropathy (DN) is still unclear. This study aimed to investigate the potential mechanism and role of lncRNA CASC15 in DN. The relationship between miR-424 and CASC15/SP-A was predicted by Starbase software and verified by luciferase reporter assay. HK-2 cells were treated with 25 mM glucose (HG) for 24 h to establish DN cell model. MTT and flow cytometry analysis were carried out to test cell proliferation and apoptosis. Epithelial-to-mesenchymal transition (EMT) markers were analyzed by RT-qPCR and western blot assay. We proved that CASC15 could interact with miR-424, and SP-A was a target of miR-424. HG-treatment significantly enhanced lncRNA CASC15 level and decreased miR-424 level in HK-2 cells. LncRNA CASC15-siRNA significantly improved cell viability, repressed apoptosis, promoted E-cadherin expression, and inhibited N-cadherin expression in HG-treated HK-2 cells, and these effects were reversed by miR-424 inhibitor. SP-A was highly expressed in HG-treated HK-2 cells. The biological effects of miR-424 mimic on HG-treated HK-2 cells were reversed by SP-A-plasmid. In conclusion, lncRNA CASC15 inhibition relieved HG-induced HK-2 cell injury and EMT through miR-424/SP-A axis.

Keywords: DN; HK-2 cells; LncRNA CASC15; MiRNA-424; SP-A.

Figure 1

LncRNA CASC15 directly targets miR-424. (a) Binding region was shown between lncRNA CASC15 and miR-424. (b) Dual luciferase reporter assay was used to verify the binding sites between CASC15 and miR-424. **p < 0.01 vs mimic control.

Figure 2

HG-treatment increased lncRNA CASC15 expression and reduced miR-424 expression in HK-2 cells. (a) RT-qPCR analysis of CASC15 expression in HG-treated HK-2 cells and normal group. (b) RT-qPCR analysis of miR-424 expression. **p < 0.01 vs normal group.

Figure 3

LncRNA CASC15 negatively regulated miR-424 expression in HK-2 cells. (a) RT-qPCR assay was used to measure lncRNA CASC15 expression when HK-2 cells were transfected with lncRNA CASC15-siRNA. (b) RT-qPCR analysis of miR-424 expression when HK-2 cells were transfected with miR-424 inhibitor or inhibitor control. (c) RT-qPCR analysis of miR-424 expression when HK-2 cells were transfected with inhibitor control, miR-424 inhibitor, lncRNA CASC15-siRNA + inhibitor control, or lncRNA CASC15-siRNA + miR-424 inhibitor. **p < 0.01 vs control-siRNA; ^## p < 0.01 vs inhibitor control; ^&& p < 0.01 vs lncRNA CASC15-siRNA + inhibitor control.

Figure 4

LncRNA CASC15-siRNA relieved HG-induced HK-2 cell injury. (a) MTT proliferation assay in HK-2 cells. (b and c) FCM was used to detect the cell apoptosis and quantitative results. (d) Western blot analysis of E-cadherin and N-cadherin expression in HK-2 cells. (e) RT-qPCR analysis was used to detect E-cadherin expression. (f) RT-qPCR analysis of N-cadherin expression at mRNA level. **p < 0.01 vs normal; ^## p < 0.01 vs HG + control-siRNA; ^&& p < 0.01 vs HG + lncRNA CASC15-siRNA + inhibitor control.

Figure 5

SP-A was a target of miR-424. (a) Binding site was shown between miR-424 and SP-A. (b) The luciferase activity of SP-A-WT or SP-A-MUT. **p < 0.01 vs mimic control.

Figure 6

HG-treatment elevated SP-A expression in HK-2 cells. (a) Western blot analysis of SP-A expression. (b) RT-qPCR analysis of SP-A expression. **p < 0.01 vs Normal group.

Figure 7

MiR-424 mimic reduced SP-A expression in HK-2 cells. (a) RT-qPCR analysis of miR-424 expression in HK-2 cells 24 h post transfection with mimic control or miR-424 mimic alone. (b) RT-qPCR analysis of SP-A expression in HK-2 cells 24 h post transfection with control plasmid or SP-A-plasmid alone. (c and d) Western blot analysis and RT-qPCR analysis of SP-A expression in HK-2 cells 24 h post transfection with control plasmid or SP-A-plasmid alone, or miR-424 mimic and control-plasmid together, or miR-424 mimic and SP-A-plasmid together. **p < 0.01 vs mimic control; ^## p < 0.01 vs control-plasmid; ^&& p < 0.01 vs miR-424 mimic + control-plasmid.

Figure 8

MiR-424 overexpression relieved HG-induced HK-2 cell injury by targeting SP-A. (a) MTT assay was used to measure HK-2 cell viability. (b and c) FCM analysis of cell apoptosis and quantitative results. (d) Western Blot assay was used to examine E-cadherin and N-cadherin expression. (e) RT-qPCR analysis of E-cadherin expression. (f) RT-qPCR analysis of N-cadherin expression. **p < 0.01 vs Normal; ^## p < 0.01 vs HG + mimic control; ^&& p < 0.01 vs HG + miR-424 mimic + control-plasmid.