Long noncoding RNA protein-disulfide isomerase-associated 3 regulated high glucose-induced podocyte apoptosis in diabetic nephropathy through targeting miR-139-3p

Figure 1 RNA-sequencing analysis indicated a total of 51 long noncoding RNAs that were differentially expressed between the normal glucose and high glucose groups, which included 20 upregulated and 31 downregulated long noncoding RNAs. lncRNA: Long noncoding RNA; HG: High glucose; NG: Normal glucose; Pdia3: Protein-disulfide isomerase-associated 3.

Figure 2 The upregulation of long noncoding RNA Pdia3 attenuated podocyte apoptosis and endoplasmic reticulum stress in high glucose-cultured podocytes. The downregulation of long noncoding RNA Pdia3 aggravated podocyte apoptosis and endoplasmic reticulum stress in normal glucose-cultured podocytes. A: The transfection efficiency of pcDNA3.1-long noncoding RNA (lncRNA) Pdia3 was detected by quantitative real-time polymerase chain reaction (qRT-PCR); B: The transfection efficiency of small interfering RNA-lncRNA Pdia3 was detected by qRT-PCR; C: The expressions of lncRNA Pdia3 were measured by qRT-PCR. GAPDH served as a loading control; D: Quantitative analysis of the relative cell viability; E: Podocyte apoptosis was detected by flow cytometry; F: Quantitative analysis of the cell apoptotic rate; G and H: The expressions of nephrin and podocin in podocytes were analyzed by immunofluorescence. Scale bar = 50 μm; I: The protein levels of endoplasmic reticulum stress-related factors (glucose-regulated protein 78, C/EBP homologous protein, and caspase-12) were analyzed by western blotting. β-Tubulin was used as an internal control; J: Quantitative analysis of glucose-regulated protein 78, C/EBP homologous protein and caspase-12. The data were presented as mean ± SD. ^aP < 0.05. lncRNA: Long noncoding RNA; HG: High glucose; NG: Normal glucose; siRNA: Small interfering RNA; Pdia3: Protein-disulfide isomerase-associated 3; GRP78: Glucose-regulated protein 78; CHOP: C/EBP homologous protein.

Figure 3 The direct interaction between long noncoding RNA Pdia3 and miR-139-3p was revealed. A: The subcellular location of long noncoding RNA (lncRNA) Pdia3 was examined by fluorescence in situ hybridization. LncRNA Pdia3 was mainly distributed in the cytoplasm; B: The target binding site between lncRNA Pdia3 and miR-139-3p was revealed; C: Dual-luciferase reporter assay was performed to measure the luciferase activity of co-transfecting with miR-139-3p mimics or NC mimics and lncRNA Pdia3 wt or mut luciferase reporters. The data were presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. ^aP < 0.05. ns: No significance. lncRNA: Long noncoding RNA; HG: High glucose; NG: Normal glucose; Pdia3: Protein-disulfide isomerase-associated 3; DAPI: 6-diamidino-2-phenylindole.

Figure 4 Inhibition of miR-139-3p attenuated podocyte apoptosis and endoplasmic reticulum stress in high glucose-cultured podocytes. The overexpression of miR-139-3p aggravated podocyte apoptosis and endoplasmic reticulum stress in normal glucose-cultured podocytes. A: The transfection efficiency of miR-139-3p mimics was detected by quantitative real-time polymerase chain reaction (qRT-PCR); B: The transfection efficiency of miR-139-3p inhibitors was detected by qRT-PCR; C: The expression of miR-139-3p was measured by qRT-PCR. U6 served as the loading control; D: Quantitative analysis of the relative cell viability; E: Podocyte apoptosis was detected by flow cytometry; F: Quantitative analysis of cell apoptotic rate; G and H: The expressions of nephrin and podocin in podocytes were analyzed by immunofluorescence. Scale bar = 50 μm; I: The protein expression of endoplasmic reticulum stress-related factors (glucose-regulated protein 78, C/EBP homologous protein, and caspase-12) was analyzed by western blotting. β-Tubulin served as an internal control; J: Quantitative analysis of glucose-regulated protein 78, C/EBP homologous protein and caspase-12. The data were presented as mean ± SD. ^aP < 0.05. lncRNA: Long noncoding RNA; HG: High glucose; NG: Normal glucose; Pdia3: Protein-disulfide isomerase-associated 3; GRP78: Glucose-regulated protein 78; CHOP: C/EBP homologous protein.

Figure 5 Mechanistic depiction of the role of long noncoding RNA Pdia3 in endoplasmic reticulum stress and podocyte apoptosis is illustrated. Our finding revealed that long noncoding RNA Pdia3 downregulation induced endoplasmic reticulum stress and podocyte injury by acting as a competing endogenous RNA of miR-139-3p, which led to diabetic nephropathy progression. lncRNA: Long noncoding RNA; Pdia3: Protein-disulfide isomerase-associated 3.