Inhibition of the lncRNA MIAT prevents podocyte injury and mitotic catastrophe in diabetic nephropathy

Figure 1. MIAT is expressed at high levels in individuals with DN and participates in DN progression (A) RNA-FISH revealed the location and expression of Miat under LG or HG conditions by confocal laser scanning microscope (n = 4). Scale bar, 25 μm. (B) Real-time PCR showed the mRNA levels of Miat in podocytes after LG or HG stimulation for 48 h (n = 7). (C) RNA-FISH illustrated the distribution of Miat and its relative position to podocin in paraffin-embedded mouse sections by confocal laser scanning microscope (n = 3). The podocytes were labeled with arrows. Scale bar, 25 μm. (D) The expression of Miat in primary podocytes from WT-CTL and WT-STZ mice at 12 weeks after the onset of diabetes (n = 3). (E) Expression of Miat in the kidney tissues from WT-CTL mice at 4 (n = 7), 6 (n = 7), 8 (n = 7), and 12 weeks (n = 8) and WT-STZ mice at 4 (n = 8), 6 (n = 8), 8 (n = 9), and 12 weeks (n = 8) after the onset of diabetes. (F) Levels of MIAT in the plasma from clinical patients with DN (n = 61) and their healthy counterparts (n = 22). (G) MIAT expression in the plasma from control subjects (n = 22) and patients with DN presenting normal urinary albumin levels (n = 20), microalbuminuria (n = 21),…

Figure 2. Effects of Miat on podocyte injury (A) Real-time PCR detected the levels of podocalyxin (left panel) and synaptopodin (right panel) in the urinary sediment from patients with DN (n = 25) and healthy individuals (n = 17). (B–E) Immunofluorescence staining showed the intensities of Desmin (B), synaptopodin (C), podocin (D), and ZO-1 (E) (n = 4). Scale bar, 50 μm. (F) Western blotting demonstrated the expression of Desmin, synaptopodin, podocin, and ZO-1 under different conditions (n = 3). (G) The motility of podocytes was determined using Transwell assays (n = 5). Scale bars, 100 μm. (H) FITC-phalloidin staining showed the organization of F-actin in podocytes (n = 3). Scale bar, 50 μm. (I) Podocyte injury-associated proteins were detected using western blotting (n = 3). (J) The motility of podocytes under different conditions was detected using Transwell assays (n = 5). Scale bars, 100 μm. (K) The organization of F-actin was determined using FITC-phalloidin staining (n = 3). Scale bar, 50 μm. Error bars represent ±SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 3. Miat depletion ameliorates podocyte injury in vivo (A) Morphological changes in the FPs and GBM were observed under TEM. The GBM thickness and podocyte effacement were qualified (n = 3). Scale bar, 1 μm. (B) PAS staining of tissues from WT and KO mice. The relative mesangial area and glomerular volume were qualified (n = 4). Scale bar, 50 μm. (C) Masson’s trichrome staining of tissues from WT and KO mice (n = 4). Scale bar, 50 μm. (D) PAS staining of tissues from clinical patients with different levels of progression. Scale bar, 100 μm. (E) TEM image of tissues from clinical patients with different levels of progression. Scale bar, 2 μm. (F–I) Immunofluorescence staining for Desmin (F), podocin (G), synaptopodin (H), and ZO-1 (I) in WT and KO mice at 12 weeks (n = 3). Scale bar, 50 μm. (J) The expression of WT-1 in glomeruli of WT and KO mice at 12 weeks (n = 3). Scale bar, 50 μm. (K) Western blotting exemplified the expression of Desmin, podocin, synaptopodin, and ZO-1 in primary podocytes of WT and KO mice at 12 weeks (n = 3). Error bars represent ±SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 4. Effects of Miat on podocyte mitotic catastrophe are mediated by Sox4 in vitro (A) Flow cytometry showed the apoptosis rate of podocytes (n = 4). (B) Flow cytometry illustrated the cell-cycle progression of podocytes (n = 3). (C) Immunostaining of mitosis with antibodies against α-tubulin (green; to indicate mitotic spindles) and histone-3 phosphorylated at serine 10 (H3-Ser10) (red; to indicate metaphase) (n = 3). Scale bar, 50 μm. (D) The percentages of podocytes with normal or abnormal mitotic processes were monitored and analyzed (n = 3). Scale bar, 10 μm. (E) Western blotting depicted the expression of Sox4, p53, p21cip1/waf1, CyclinB, and cdc2 (n = 3). (F–J) Levels of the Sox4 (n = 3), p53 (n = 5), p21cip1/waf1 (n = 4), CyclinB (n = 4), and cdc2 (n = 3) mRNAs. Error bars represent ±SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 5. Podocyte injury and mitotic catastrophe require Sox4 in vitro (A) Western-blot analysis showed the expression of Sox4, p53, p21cip1/waf1, CyclinB, and cdc2 (n = 3). (B) Flow cytometry analysis illustrated the cell-cycle progression of podocytes (n = 3). (C) Immunostaining of mitosis with antibodies against α-tubulin (green) and H3-Ser10 (red) (n = 3). Scale bar, 10 μm. (D)The percentages of podocytes with normal or abnormal mitotic processes were monitored and analyzed (n = 3). (E) Morphological changes in the podocyte cytoskeleton (n = 3). Scale bar, 50 μm. (F) The expression of Desmin, podocin, synaptopodin, and ZO-1 was measured using western blotting (n = 3). (G–I) Immunofluorescence staining was performed to determine the intensities of podocin (G), Desmin (H), and p-cadherin (I) under HG conditions (n = 4). Scale bar, 50 μm. (J) Cell migration was detected using Transwell assays (n = 5). Scale bar, 100 μm. (K) Flow cytometry revealed the apoptosis rate of podocytes (n = 5). (L) Flow cytometry clarified the proportion of podocytes in G2/M phase (n = 4). (M) Immunostaining illustrated morphological changes associated with normal or abnormal mitosis using antibodies against…

Figure 6. Sox4 is critical for p53 stabilization and function (A–D) Western blotting (A) and real-time PCR illustrated the expression of Sox4 (B), p53 (C), and p21cip1/waf1 (D) under HG conditions (n = 3). (E) Lysates from scramble shRNA or Sox4 shRNA-transfected podocytes treated with 20 mg/mL cycloheximide (CHX) at the indicated time points (0, 20, 40, and 60 min; left panel), and quantification of the relative p53 levels were quantified (right panel; n = 3). (F) Luciferase reporter assay revealed the interaction between Sox4 and p53 transcriptional activity in podocytes (n = 3). (G) Sox4 and p53 were endogenously expressed in podocytes, as shown by immunoprecipitation (IP) with an anti-Sox4 antibody (n = 3). (H) Podocytes were transfected with His-ubiquitin with or without Sox4 shRNA for 24 h and were incubated in the presence or absence of HG for another 48 h, followed by treatment with 20 μmmol/L MG-132 for 8 h. Cell lysates were immunoprecipitated and analyzed using immunoblotting with an anti-p53 antibody (n = 3). IB, immunoblotting. (I) Podocytes were transfected with FLAG-p53, GFP, HA-Mdm2, and Myc-Sox4 vectors (1, 2, and 4 μg) for 24 h, and the expression of p53 in podocyte…

Figure 7. Miat promotes Sox4 expression by targeting miR-130b-3p in vitro (A) The levels of miR-130b-3p in the plasma from healthy controls (n = 22, −0.8066 ± 0.4477) and clinical patients with DN (n = 61, −1.124 ± 0.4351). (B) A luciferase reporter assay was performed to evaluate the interaction between miR-130b-3p and Miat in HEK293 cells (n = 3). (C) RIP revealed the interaction of Miat and miR-130b-3p in podocytes (n = 3). (D) Real-time PCR revealed the expression of miR-130b-3p in podocytes in the presence of miR-130b-3p mimic and Miat-WT plasmid or Miat-MUT plasmid (n = 3). (E) Dual-luciferase reporter assay showed the interaction between miR-130b-3p and the Sox4 3′ UTR in HEK293 cells (n = 3). (F) Western blotting manifested the expression of Sox4 in podocytes after pretreatment with the miR-130b-3p mimic (n = 3). (G) Real-time PCR revealed Sox4 mRNA expression in podocytes (n = 5). (H) Western blotting was used to assess Sox4 expression in different groups of podocytes (n = 3). EV, empty vector. (I) The expression of Sox4 in podocytes was revealed by western blotting (n = 3). (J) The expression of Sox4 was measured in podocytes using western blotting (n = 3). Error bars represent…

Figure 8. Miat enhances podocyte injury and G2/M-phase arrest by binding to miR-130b-3p in vivo (A) Morphological changes in the FPs and GBM of the mice at 12 weeks were observed under TEM. The GBM thickness and podocyte effacement were qualified (n = 3). Scale bar, 1 μm. (B) PAS staining of mouse tissues at 12 weeks. The relative mesangial area and glomerular volume were qualified (n = 4). Scale bar, 50 μm. (C) Masson’s trichrome staining of mouse tissues at 12 weeks (n = 4). Scale bar, 50 μm. (D–G) Immunofluorescence staining for the podocyte-specific markers Desmin (D), podocin (E), synaptopodin (F), and ZO-1 (G) in mice at 12 weeks (n = 3). Scale bar, 50 μm. (H) Immunofluorescence staining showed the expression of WT-1 in the mouse glomeruli at 12 weeks (n = 3). Scale bar, 50 μm. (I) Levels of injury markers were confirmed in primary mouse podocytes at 12 weeks using western blotting (n = 3). (J) Western-blot analysis showed the expression of Sox4, p53, p21cip1/waf1, CyclinB, and cdc2 in primary mouse podocytes at 12 weeks (n = 3). (K) Cell-cycle progression of primary mouse podocytes at 12 weeks was assessed using flow cytometry (n = 4). Error bars represent ±SD. ∗p < 0.05, ∗∗p <…