Silencing of long noncoding RNA PVT1 inhibits podocyte damage and apoptosis in diabetic nephropathy by upregulating FOXA1

Fig. 1. LncRNA PVT1 has high expression in diabetic nephropathy. a The expression of lncRNA PVT1 in DN patients, MPC5 cells, and primary podocytes in HG medium as determined by RT-qPCR. b, c The subcellular location of lncRNA PVT1 was primarily located in the nucleus, predicted by the lncATLAS website and detected by FISH (the original magnification is ×200); *p < 0.05 vs. the normal control and the NG group, respectively. The measurement data are expressed as the mean ± standard deviation, and the comparisons between the two groups were analyzed by Student’s t test. The experiment was repeated three times. DN, diabetic nephropathy; MPC5, mouse podocyte clone 5; NG, normal glucose; HG, high glucose; DAPI, 4′,6-diamidino-2-phenylindole; PVT1, plasmacytoma variant translocation 1; lncRNA PVT1, long noncoding RNA plasmacytoma variant translocation 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; FISH, fluorescence in situ hybridization

Fig. 2. The poor expression of lncRNA PVT1 inhibits the damage to and apoptosis of podocytes. a, b The cellular morphology of MPC5 and primary podocytes in NG and HG media (the original magnification is ×400). c, d The apoptosis of MPC5 and primary podocytes in NG and HG media. e The apoptosis of MPC5 and primary podocytes in NG and HG media; *p < 0.05 vs. the OE-NC group; #p < 0.05 vs. the sh-NC group. The measurement data are expressed as the mean ± standard deviation, and the comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA). The experiment was repeated three times. MPC5, mouse podocyte clone 5; NG, normal glucose; HG, high glucose; OE, overexpressed; NC, negative control; lncRNA, long noncoding RNA; PVT1, plasmacytoma variant translocation 1

Fig. 3. LncRNA PVT1 inhibits the expression of FOXA1. a The target binding site between PVT1 and FOXA1 predicted by the biological prediction website microRNA.org. b The measurement of luciferase activity to verify the relationship of PVT1 and FOXA1; *p < 0.05 vs. the NC group; c the enrichment of IgG and EZH2 by PVT1; *p < 0.05 vs. IgG. d The protein levels of EZH2 and FOXA1; *p < 0.05 vs. the si-NC group. e The CpG island sites in the FOXA1 promoter region. f The methylation levels in cells cultured in HG medium and NG medium as detected by MSP assay. g The methylation level of EZH2 and H3K27me3 as detected by ChIP; *p < 0.05 vs. the sh-NC group. The measurement data are expressed as the mean ± standard deviation, and the comparisons between the two groups were analyzed by a t test. The experiment was repeated three times. LncRNA PVT1, long noncoding RNA plasmacytoma variant translocation 1; FOXA1, forkhead box A1; CpG, C-phosphate-G; NG, normal glucose; HG, high glucose; EZH2, enhancer of zeste homolog 2; IgG, immunoglobulin G; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; DN, diabetic nephropathy; MPC5, mouse podocyte clone 5

Fig. 4. Silencing of PVT1 or overexpression of FOXA1 inhibited the apoptosis and damage of podocytes in DN. a Immunofluorescence staining revealing the expression of synaptopodin and podocin in MPC5 cells with normal culture medium (the original magnification is ×400). b Flow cytometry map demonstrating MPC5 cell apoptosis conditions with normal culture medium. c Apoptosis rate analysis for cells with normal culture medium in response to the treatment of OE-PVT1, OE-PVT1 + GSK126, sh-NC, sh-FOXA1, OE-PVT1 + sh-FOXA1; *p < 0.05 vs. the OE-NC group; #p < 0.05 vs. the sh-NC group; &p < 0.05 vs. the OE-PVT1 group and the sh-FOXA1 group. d Immunofluorescence staining revealing the expression of synaptopodin and podocin in MPC5 cells with HG culture medium (×400). e Flow cytometry map demonstrating MPC5 cell apoptosis conditions with HG culture medium. f Apoptosis rate analysis for cells with HG culture medium in response to the treatment of OE-PVT1, OE-PVT1 + GSK126, sh-NC, sh-FOXA1, and OE-PVT1 + sh-FOXA1. g The protein levels of apoptosis-related factors (Bcl-2, Bax, and cleaved caspase-3) in response to the treatment of OE-PVT1, OE-PVT1 + GSK126, sh-NC, sh-FOXA1, and OE-PVT1 + sh-FOXA1…

Fig. 5. Silencing of PVT1 or the overexpression of FOXA1 inhibited the apoptosis and damage of podocytes in DN in vivo. a The expression of PVT1 and FOXA1 in mice with DN in vivo. b FOXA1 promoter recruitment in vivo detected by ChIP assay, n = 10. c The degree of glomerulosclerosis and renal tubule atrophy in response to the treatment of sh-PVT1 and/or GSK126 assessed by periodic acid-Schiff (PAS) staining (the original magnification is ×400). d Podocyte apoptosis in response to the treatment of sh-PVT1 and/or GSK126 in vivo. e Immunofluorescence staining displaying the expression of podocyte marker Nephrin and FOXA1 in vivo (the original magnification is ×400). f The protein level of FOXA1 in vivo detected by western blot analysis; *p < 0.05 vs. the sh-NC group. The measurement data are expressed as the mean ± standard deviation, and the comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA). The experiment was repeated three times. DN, diabetic nephropathy; PVT1, plasmacytoma variant translocation 1; IgG, immunoglobulin G; FOXA1, forkhead box A1; NC, negative control; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;…

Fig. 6. The mechanism map illustrating the regulatory role of lncRNA PVT1 in the damage of podocytes by inhibiting FOXA1. PVT1 can promote the recruitment of H3K27me3 in the FOXA1 promoter region by recruiting EZH2 to inhibit the expression of FOXA1, thereby promoting the damage and apoptosis of podocytes in diabetic nephropathy. LncRNA PVT1, long noncoding RNA; PVT1, plasmacytoma variant translocation 1; FOXA1, forkhead box A1; EZH2, enhancer of zeste homolog 2; H3K27me3, histone 3 lysine 27 trimethylation