Loss of lncRNA MIAT ameliorates proliferation and fibrosis of diabetic nephropathy through reducing E2F3 expression

Figures

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  Figure 1. Expression levels of MIAT in DN. A, The expression levels of MIAT were determined using qRT‐PCR in the kidney tissues of DN patients and healthy controls. N = 30 in each group. B, The expression of MIAT was tested in mesangial cells incubated with HG and NG for various hours. Three independent experiments were conducted. Error bars stand for the mean ± SD of at least triplicate experiments. *P < .05, **P < .01, ***P < .001

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  Figure 2. Effect of MIAT on mesangial cell proliferation and cell cycle. A, Expression of MIAT in nucleus and cytoplasm of mesangial cells. B, Expression of MIAT in mesangial cells indicated by FISH assay. C, The expression of MIAT in mesangial cells transfected with MIAT siRNA. D, Expression of MIAT in mesangial cells transfected with MIAT siRNA after HG incubation. E, CCK8 assay was used to test cell survival. F and G, Flow cytometry assay was carried out to test cell cycle. Three independent experiments were conducted. Error bars stand for the mean ± SD of at least triplicate experiments. *P < .05

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  Figure 3. Effect of MIAT on mesangial cell ECM accumulation. A and B, Collagen IV, FN and TGF‐β1 protein expression in mesangial cells transfected with MIAT siRNA. Three independent experiments were conducted. C, Immunofluorescence indicating the expression of collagen IV, FN and TGF‐β1. Error bars stand for the mean ± SD of at least triplicate experiments. *P < .05

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  Figure 4. miR‐147a was a direct target of MIAT. A, The luciferase reporter constructs containing the WT‐MIAT or MUT‐MIAT sequence. B, Luciferase activity was tested after co‐transfection of construct containing WT or MUT version of MIAT and miR‐147a mimics. C, Anti‐Ago2 RIP was employed to pull down endogenous RNAs associated with Ago2. IgG was used as the control. The levels of MIAT and miR‐147a were measured by qRT‐PCR. Data were exhibited as fold enrichment in Ago2 relative to input. D, The expression levels of miR‐147a determined using qRT‐PCR in the kidney tissues of DN patients and healthy controls. E, The expression of miR‐147a was tested in mesangial cells incubated with HG and LG for various hours. F, The expression levels of miR‐147a in mesangial cells transfected with MIAT siRNA. G, Correlation analysis of MIAT and miR‐147a expressions in 30 cases of DN patient kidney tissues. Three independent experiments were conducted. Error bars stand for the mean ± SD of at least triplicate experiments. *P < .05, **P < .01, ***P < .001

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  Figure 5. E2F3 was a target of miR‐147a. A, The luciferase reporter constructs containing the WT‐E2F3 or MUT‐E2F3 sequence. B, WT‐E2F3 or MUT‐E2F3 was co‐transfected into mesangial cells with miR‐147a mimics or their corresponding negative controls. C and D, E2F3 mRNA expression in DN. E and F, E2F3 protein expression in DN. G, E2F3 mRNA expression in mesangial cells transfected with miR‐147a mimics. H and I, E2F3 protein expression in mesangial cells. Three independent experiments were conducted. Error bars stand for the mean ± SD of at least triplicate experiments. *P < .05, **P < .01, ***P < .001

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  Figure 6. Effect of miR‐147a on mesangial cell proliferation and ECM accumulation. A, The expression of miR‐147a in mesangial cells transfected with MIAT overexpression plasmid and then transfected with miR‐147a mimics. B and C, EdU assay was used to test cell proliferation (D and E) E2F3, collagen IV, FN and TGF‐β1 protein expression in mesangial cells. Three independent experiments were conducted. Error bars stand for the mean ± SD of at least triplicate experiments. *P < .05