lncRNA MALAT1 Promotes Renal Fibrosis in Diabetic Nephropathy by Targeting the miR-2355-3p/IL6ST Axis

Figures

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  FIGURE 1. lncRNA MALAT1 was upregulated in STZ-induced diabetic rats and HG-treated HK-2 or 293T cells. (A) Expression of the MALAT1 level was detected by RT-qPCR in STZ-induced diabetic rats (n = 10) and control rats (n = 10). HK-2 cells were treated with NG, mannitol, and HG for 24, 48, and 72 h, respectively. (B and C) Effects of different treatments on cell proliferation and invasion of HK-2 cells at 0, 24, 48, and 72 h were detected by CCK-8 and Transwell assays. *p < 0.05 vs. NG or mannitol group, **p < 0.01 vs. NG or mannitol group. (D and E) Effects of different treatments on the expression of col I, col IV, FN, and LN in HK-2 cells were detected by densitometric analysis. *p < 0.05 or **p < 0.01 vs. NG group or mannitol group. (F and G) lncRNA MALAT1 expression was detected by RT-qPCR in HK-2 or 293T cells. *p < 0.05 or **p < 0.01 vs. NG or mannitol group.

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  FIGURE 2. Knockdown of lncRNA MALAT1 downregulated col I, col IV, FN, and LN protein levels and inhibited cell proliferation and invasion in HG-treated HK-2 cells. MALAT1 siRNA and NC siRNA were transfected into cells and then treated with NG and HG, respectively: (A) RT-qPCR was used to determine the expression of MALAT1 in cells. **p < 0.01 vs. NG + NC siRNA group or HG + NC siRNA group. CCK-8 (B) and Transwell assays (C) were used to detect the effects of these two vectors on cell proliferation and invasion. *p < 0.05 vs. NG + NC siRNA group or HG + NC siRNA group, **p < 0.01 vs. NG + NC siRNA group or HG + NC siRNA group. (D and E) ELISA was used to determine the contents of IL-6 and TNF-α in cell lysate when these two vectors were transfected into NG- or HG-treated cells. *p < 0.05 vs. NG + NC siRNA group or HG + NC siRNA group, **p < 0.01 vs. NG + NC siRNA group. (F) Western blotting was used to detect the expression of col Ⅰ, col Ⅳ, FN, and LN in NG- or HG-treated cells after transfection with these two vectors. The histogram in (G–J) shows the densitometric analysis of the blots (col I, col IV, FN, and LN) normalized to GAPDH. *p < 0.05 vs. NG + NC siRNA group or HG + NC siRNA…

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  FIGURE 3. Overexpression of lncRNA MALAT1 upregulated col I, col IV, FN, and LN protein levels in HG-treated HK-2 cells. Cells were transfected with pcDNA-MALAT1 (MALAT1) and pcDNA3.1 (vector) and then treated with NG or HG, respectively: (A) RT-qPCR was used to determine the expression of MALAT1 in cells transfected with these two vectors. **p < 0.01 vs. NG + vector group or HG + vector group. (B) Expression of col I, col IV, FN, and LN in cells transfected with these two vectors was detected by Western blotting. The histogram in (C–F) shows the densitometric analysis of the bands (col I, col IV, FN, and LN) normalized to GAPDH. **p < 0.01 vs. NG + vector group or HG + vector group.

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  FIGURE 4. lncRNA MALAT1 directly bound to miR-2355-3p, and overexpression of miR-2355-3p downregulated col I, col IV, FN, and LN protein levels in HG-treated HK-2 cells. (A) StarBase (http://starbase.sysu.edu.cn/) was used to predict the binding between MALAT1 and miR-2355-3p. (B) Luciferase reporter gene assay was used to verify the binding between MALAT1 and miR-2355-3p. **p < 0.01 vs. NC mimic group. (C) Pull-down assay was used to detect the binding ability of miR-2355-3p and lncRNA MALAT1. **p < 0.01 vs. control or biotin-labeled miR-2355-3p group. (D) miR-2355-3p level in STZ-induced diabetic rats (n = 10) and control rats (n = 10) was detected by RT-qPCR. **p < 0.01 vs. control rats. (E and F) miR-2355-3p level was measured with RT-qPCR in NG- or HG-treated cells transfected with MALAT1 siRNA, NC siRNA, pcDNA-MALAT1, or pcDNA3.1. *p < 0.05 vs. NG + vector group, **p < 0.01 vs. NG + NC siRNA group, HG + NC siRNA group, NG + vector group, or HG + vector group. (G) miR-2355-3p level was measured with Western blot assay in NG- or HG-treated cells transfected with miR-2355-3p mimic or NC mimic. *p < 0.05 vs. NG + NC mimic, **p < 0.01 vs. NG + NC mimic group or HG + NC mimic group. (H)…

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  FIGURE 5. IL6ST was a target of miR-2355-3p. (A) StarBase (http://starbase.sysu.edu.cn/) was used to predict the binding between IL6ST and miR-2355-3p. (B) Luciferase reporter gene assay in HEK293T cells was used to verify the binding between IL6ST and miR-2355-3p. **p < 0.01 vs. NC mimic group. (C and D) The expression of IL6ST was detected by RT-qPCR and Western blotting in NG- or HG-treated cells transfected with miR-2355-3p mimic or NC mimic. **p < 0.01 vs. NG + NC mimic group or HG + NC mimic group. The histogram in (E) presents the densitometric analysis of the blots (IL6ST) normalized to GAPDH. **p < 0.01 vs. NG + NC mimic group or HG + NC mimic group.

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  FIGURE 6. IL6ST knockdown downregulated col I, col IV, FN, and LN protein levels and blocked the STAT3/NF-kB signaling pathway in HG-treated HK-2 cells. (A) Western blotting was used to detect the IL6ST level in cells treated by HG for 0, 24, 48, and 72 h. The histogram in (B) shows the densitometric analysis of the blots (IL6ST) normalized to GAPDH. **p < 0.01 vs. 0 h group. (C) col I, col IV, FN, LN, and IL6ST protein levels in cells transfected with IL6ST siRNA or NC siRNA were detected by Western blotting. (D) Densitometric analysis of the blots (col I, col IV, FN, LN, and IL6ST) normalized to GAPDH. **p < 0.01 vs. NG + NC siRNA or HG + NC siRNA group. (E) Western blotting was used to detect the STAT3 phosphorylation level, total STAT3, and NF-kB p65 protein levels in NG- or HG-treated cells. The histogram in (F) presents the densitometric analysis of the blots (p-STAT3/STAT3 and NF-kB p65) normalized to GAPDH. **p < 0.01 vs. NG + NC siRNA group or HG + NC siRNA group.

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  FIGURE 7. miR-2355-3p knockdown reversed the inhibitory effect of lncRNA MALAT1 knockdown on col I, col IV, FN, LN, and IL6ST protein levels in HG-treated HK-2 cells. Cells were transfected with pcDNA-IL6ST (IL6ST) or pcDNA3.1 (vector) and then treated by NG or HG, respectively: (A) Western blotting was used to detect the STAT3 phosphorylation level, total STAT3, and NF-kB p65 protein levels in cells transfected with these two vectors. (B) Densitometric analysis of the blots (p-STAT3/STAT3 and NF-kB p65) normalized to GAPDH. **p < 0.01 vs. NG + vector group or HG + vector group, *p < 0.05 vs. HG + vector group. (C) col I, col IV, FN, LN, and IL6ST protein levels were measured by Western blotting in cells transfected with these two vectors. The histogram in (D) shows the densitometric analysis of the blots (col I, col IV, FN, LN, and IL6ST) normalized to GAPDH. **p < 0.01 vs. NG + vector group or HG + vector group. Cells were transfected with MALAT1 siRNA together with the NC inhibitor or the miR-2355-3p inhibitor and NC siRNA together with the NC inhibitor and then treated by NG or HG, respectively: (E and F) RT-qPCR was used to examine the expression of MALAT1 and miR-2355-3p in cells.…

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  FIGURE 8. STZ-induced diabetic rats aggravated renal injury and promoted renal fibrosis. Contents of (A) blood glucose, (B) serum creatinine, and (C) urinary ACR were detected by ELISA in STZ-induced diabetic rats (n = 10) and control rats (n = 10). **p < 0.01 vs. control rats. (D and E) Glomerular and tubular damage scores were evaluated by the experimenter. **p < 0.01 vs. control rats. (F) Western blot assay was used to detect the col I, col IV, FN, and LN protein levels in STZ-induced diabetic rats administrated with MALAT1 siRNA or miR-2355-3p inhibitor. The histogram in (G) shows the densitometric analysis of the blots (col I, col IV, FN, and LN) normalized to GAPDH. **p < 0.01 vs. scramble group. (H) Renal tissues of rats were collected for H&E (×200, scale bar = 50 µm), Masson’s trichrome (×200, scale bar = 50 µm), PAS (×400, scale bar = 25 µm), and PAM staining (×200, scale bar = 50 µm). miR-2355-3p levels were measured in heart, liver, bladder, kidney and peritoneal tissues of STZ-induced diabetic rats administrated with LNA-anti-miR-2355-3p and scramble (Supplementary Figure S1). **p < 0.01 vs. scramble group. (I) Western blot assay was used to measure the IL6ST, p-STAT3, total…

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  FIGURE 9. Schematic diagram for the functions and mechanism of lncRNA MALAT1 in regulating renal fibrosis in diabetic rats. MALAT1 is induced by high glucose in renal tubular epithelial cells in vitro and in renal tubular epithelium in STZ-diabetic rats in vivo, which sponges miR-2355-3p and positively regulates expression of IL6ST gene. IL6ST is an identified regulator to phosphorylate STAT3 and activates its downstream master pro-inflammatory transcription factor NF-kB. Then, NF-kB transcriptionally upregulates the fibrogenic marker genes, including LN, FN, coi I, and col IV, as well as secretion of pro-inflammatory cytokines IL-6 and TNF-α, thus promoting renal fibrosis in diabetic rats.