lncRNA NR_038323 Suppresses Renal Fibrosis in Diabetic Nephropathy by Targeting the miR-324-3p/DUSP1 Axis

Figures

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  Figure 1. HG Induced the Expression of lncRNA NR_038323 HK-2 cells were treated with NG, mannitol, or HG at indicated time points. (A) The heatmap of lncRNAs. (B) The sequence name and fold change of upregulation of lncRNAs. (C) Intracellular localization of lncRNA NR_038323 in HK-2 cells was carried out using RNA-FISH assay. (D) The expression levels of lncRNA NR_038323 were detected by real-time qPCR. Data are expressed as mean ± SD (n = 6). #p < 0.05, HG at 24–72 h groups versus NG or mannitol group.

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  Figure 2. lncRNA NR_038323 Knockdown Aggravated the HG-Reduced Expression Levels of Collagen I, Collagen IV, and Fibronectin HK-2 cells were transfected with 50 nM lncRNA NR_038323 siRNA or scramble and then treated with or without HG for 72 h. (A) Real-time qPCR analysis of lncRNA NR_038323 expression. (B) Western blot analysis of collagen I, collagen IV, and fibronectin. (C–E) Densitometric measurement of western blot bands for collagen I (C), collagen IV (D), and fibronectin (E). Data are expressed as mean ± SD (n = 6). #p < 0.05, scramble with HG or lncRNA NR_038323 siRNA group versus scramble group; *p < 0.05, lncRNA NR_038323 siRNA with HG group versus scramble with HG group.

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  Figure 3. Overexpression of lncRNA NR_038323 Attenuated the HG-Deduced Expression Levels of Collagen I, Collagen IV, and Fibronectin HK-2 cells were transfected with lncRNA NR_038323 plasmid or control and then treated with or without HG for 72 h. (A) Real-time qPCR analysis of lncRNA NR_038323 expression. (B) Western blot analysis of collagen I, collagen IV, and fibronectin. (C–E) Densitometric measurement of western blot bands for collagen I (C), collagen IV (D), and fibronectin (E). Data are expressed as mean ± SD (n = 6). #p < 0.05, control with HG or lncRNA NR_038323 group versus control group; *p < 0.05, lncRNA NR_038323 with HG group versus control with HG group.

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  Figure 4. lncRNA NR_038323 Directly Bound to miR-324-3p (A) Sequence alignment analysis revealed that lncRNA NR_038323 contained the complementary strand to miR-324-3p. (B) Detection of luciferase activities after co-transfection with lncRNA NR_038323-WT or lncRNA NR_038323-MUT and miR-324-3p or scramble. (C) Intracellular co-localization of lncRNA NR_038323 and miR-324-3p in HK-2 cells and human DN kidney samples. (D and E) Real-time qPCR analysis of lncRNA NR_038323 expression. Data are expressed as mean ± SD (n = 6). #p < 0.05, Scramble with HG group versus Scramble group; * p < 0.05, siRNA NR_038323 or NR_038323 with HG group versus Scramble with HG group or NR_038323 WT/miR-324-3p mimic versus other groups.

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  Figure 5. Overexpression of miR-324-3p Aggravated the HG-Induced Expression Levels of Collagen I, Collagen IV, and Fibronectin HK-2 cells were transfected with 100 nM miR-324-3p mimics or scramble and then treated with or without HG for 72 h. (A) The mRNA expression levels of miR-324-3p were detected by real-time qPCR. (B) The protein expression levels of collagen I, collagen IV, and fibronectin were analyzed by western blotting. (C–E) Densitometric measurement of western blot bands for collagen I (C), collagen IV (D), and fibronectin (E). Data are expressed as mean ± SD (n = 6). #p < 0.05, scramble with HG group versus scramble group; *p < 0.05, miR-324-3p mimics with HG group versus scramble with HG group.

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  Figure 6. DUSP1 Was Identified as a Target Gene of miR-324-3p HK-2 cells were transfected with miR-324-3p analog (100 nM) and then treated with HG for 72 h. (A) Putative miR-301a-5p complementary binding sites in the 3′ UTR of human DUSP1 mRNA. (B) The mRNA expression levels of DUSP1 were detected by RT-qPCR. (C) Western blot analysis of DUSP1 and GAPDH. (D) Densitometric analysis of proteins signals. (E) Measurement of luciferase activities after co-transfection with the 3′ UTR luciferase reporter vector of human WT or MUT-DUSP1 and miR-324-3p or miR-NC. Data are expressed as mean ± SD (n = 6). #p < 0.05, scramble with HG group versus scramble group; *p < 0.05, miR-324-3p mimics with HG group versus scramble with HG group or DUSP1 WT/miR-324-3p mimic versus other groups.

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  Figure 7. DUSP1-Mediated HG-Induced Expression Levels of Collagen I, Collagen IV, and Fibronectin via MAPK Signaling HK-2 cells were transfected with siRNA DUSP1 (100 cnM) or DUSP1 plasmid and then treated with HG for 72 h. (A) Western blot analysis of DUSP1 and GAPDH at indicated time points. (B) Densitometric analysis of proteins signals. (C and G) Western blot analysis of collagen I, collagen IV, fibronectin, and DUSP1. (E and I) Western blot analysis of p-p38MAPK/p38MAPK and p-ERK1/2/ERK1/2. (D, F, H, and J) Densitometric measurement of western blot bands. Data are expressed as mean ± SD (n = 6). #p < 0.05, scramble with HG group or HG at 24–72 h versus scramble group or HG at 0 h; *p < 0.05, siRNA DUSP1 or DUSP1 plasmid with HG group versus scramble with HG group.

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  Figure 8. lncRNA NR_038323 Knockdown Aggravated the HG-Induced Expression Levels of ECM-Related Genes, Which Were Reversed by miR-324-3p Inhibitor HK-2 cells were co-transfected with siRNA lncRNA NR_038323 (100 cnM) and anti-miR-324-3p or scramble and then treated with HG for 72 h. (A) Real-time qPCR analysis of lncRNA NR_038323 expression. (B) Real-time qPCR analysis of miR-324-3p expression. (C) Western blot analysis of collagen I, collagen IV, fibronectin, and DUSP1. (D–G) Densitometric measurement of protein signals in collagen I (D), collagen IV (E), fibronectin (F), and DUSP1 (G). Data are expressed as mean ± SD (n = 6). #p < 0.05, scramble with HG group versus scramble group, lncRNA NR_038323 siRNA with HG group versus scramble with HG group; *p < 0.05, lncRNA NR_038323 plus anti-miR-324-3p with HG group versus lncRNA NR_038323 siRNA with HG group.

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  Figure 9. lncRNA NR_038323/miR-324-3p/DUSP1 Axis in Patients with DN (A) Renal tissues were stained with Masson’s trichrome for fibrosis analysis and immunohistochemical staining for the levels of DUSP1- and ECM-related proteins. (B) Quantitative analysis of tubulointerstitial fibrosis in the kidney cortex. (C–F) Quantification of immunohistochemical staining. (G and H) Real-time qPCR analysis of the expression levels of miR-324-3p and lncRNA NR_038323. Original magnification, ×200. Data are expressed as mean ± SD (n = 6). #p < 0.05, DN group versus MCD group.

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  Figure 10. The Role and Molecular Mechanism of lncRNA NR_038323 in HG-Induced Renal Fibrosis The expression of lncRNA NR_038323 was induced by HG treatment. Mechanistically, lncRNA NR_038323 sponged miR-324-3p to upregulate DUSP1 expression and consequently suppressed the HG-induced activation of p38MAPK and ERK1/2 pathways in renal fibrosis.