Role of microRNA 1207-5P and its host gene, the long non-coding RNA Pvt1, as mediators of extracellular matrix accumulation in the kidney: implications for diabetic nephropathy

Abstract

Diabetic nephropathy is the most common cause of chronic kidney failure and end-stage renal disease in the Western World. One of the major characteristics of this disease is the excessive accumulation of extracellular matrix (ECM) in the kidney glomeruli. While both environmental and genetic determinants are recognized for their role in the development of diabetic nephropathy, epigenetic factors, such as DNA methylation, long non-coding RNAs, and microRNAs, have also recently been found to underlie some of the biological mechanisms, including ECM accumulation, leading to the disease. We previously found that a long non-coding RNA, the plasmacytoma variant translocation 1 (PVT1), increases plasminogen activator inhibitor 1 (PAI-1) and transforming growth factor beta 1 (TGF-β1) in mesangial cells, the two main contributors to ECM accumulation in the glomeruli under hyperglycemic conditions, as well as fibronectin 1 (FN1), a major ECM component. Here, we report that miR-1207-5p, a PVT1-derived microRNA, is abundantly expressed in kidney cells, and is upregulated by glucose and TGF-β1. We also found that like PVT1, miR-1207-5p increases expression of TGF-β1, PAI-1, and FN1 but in a manner that is independent of its host gene. In addition, regulation of miR-1207-5p expression by glucose and TGFβ1 is independent of PVT1. These results provide evidence supporting important roles for miR-1207-5p and its host gene in the complex pathogenesis of diabetic nephropathy.

Figure 1. PVT1-derived miRNAs.

A. Location of the validated PVT1-derived miRNAs. The grey bars represent the nine PVT1 exons. Panels B, C and D, relative quantification of PVT1-derived miRNAs in human renal proximal tubule epithelial cells (RPTEC), podocytes, and mesangial cells (MC) by TaqMan qPCR, respectively. RNU6B and RNU44 were used as endogenous controls. E. Relative quantification of miRNAs in MC grown for 48 h in MsBM medium supplemented with 5% FBS, containing either normal glucose (NG: 5.6 mM), NG +3-O-methyl-D-glucose (3-O-MG) to control for osmotic effects (OC: 5.6 mM glucose +24.4 mM 3-O-MG), or high glucose (HG: 30 mM). Results represent average of three independent experiments. Data are means ± S.D. A.U.: arbitrary units. The significance is indicated only for samples that are significant different from all the others. * P<.05; ** P<0.01; *** P<0.001.

Figure 2. Comparison of PVT1 and miR-1207-5p expression.

A. Expression of miR-1207-5p and PVT1 in different normal human tissues using the Human Total RNA Survey Panel (Life Technologies). Relative quantification of miR-1207-5p and PVT1 mRNA was performed by TaqMan qPCR. Results represent average of three quantifications of the same sample and each sample contains tissues from three different donors. Data are means ± S.E.M. B. Relative quantification of miR-1207-5p and PVT1 in whole kidney and different types of kidney cells by TaqMan qPCR. C. Levels of PVT1 mRNA and miR-1207-5p in MC transfected with 30 nM of PVT1 siRNA or negative control (Neg) siRNA. RNU6B, RNU44, UBC and PPIA were used as endogenous controls (see Table S2). In panels B and C, results represent averages from three independent experiments, and data are means ± SD. NT: no treatment. The significance is indicated only for samples that are significant different from all the others. * P<0.05; ** P<0.01; *** P<0.001.

Figure 3. TGF-β1 dose-response and time-course effect on miR-1207-5p.

A. TaqMan qPCR relative quantification of miR-1207-5p in MC treated for 24 h with serum-free medium supplemented with 2.5, 5.0, or 10.0 ng/ml TGF-β1. B. Relative quantification of miR-1207-5p and pri-miR-1207 in MC treated for different times with serum-free medium supplemented with 10 ng/ml TGF-β1. RNU6B, UBC, and 18S RNA were used as endogenous controls. Results represent averages from three independent experiments. Data are means ± SD. A.U.: arbitrary units. The significance is indicated only for samples that are significant different from all the others. * P<0.05; ** P<0.01; *** P<0.001.

Figure 4. Predicted pairing of target region (top) and miR-1207-5p (bottom) according to TargetScan (www.targetscan.org).

A total of 14 miR-1207-5p's target sites are predicted in the four target genes selected: G6PD, PMEPA1, PDPK1, and SMAD7. G6PD, glucose-6-phosphate dehydrogenase; PMEPA1, prostate transmembrane protein, androgen induced 1; PDPK1, 3-phosphoinositide dependent protein kinase-1; SMAD7, SMAD family member 7.

Figure 5. Validation of putative miR-1207-5p target genes.

miR-1207-5p target genes were identified by TargetScan software and four genes were selected using Pathway Studio software based on potential biological effects relevant to ECM accumulation. The four genes selected, G6PD, PMEPA1, PDPK1, and SMAD7, were validated as putative miR-1207-5p target genes in MC using 25 nM miR-1207-5p or NC mimic (A), and 50 nM anti-miR-1207-5p or negative control (B). Results in A and B represent averages from three independent experiments. Data are means ± SD. About 20,000 human embryonic kidney 293 (HEK293) cells were seeded per well into a white 96-well plate and co-transfected with 100 ng of the indicated 3′UTR luciferase reporter vectors and 30 nM miR-1207-5p or negative control mimic (Dharmacon) using 0.2 µl per well of Lipofectamine 2000 (C). HEK293 cells were also co-transfected with reporter vectors and 50 nM miR-1207-5p inhibitor or negative control (Dharmacon) (D). Luciferase activity was measured 48 h after transfection using the Dual-Glo Luciferase Assay System (Promega). Firefly luciferase activity was normalized to the corresponding renilla luciferase activity and plotted as a percentage of the control (HEK293 cells co-transfected with plasmid and control mimic or inhibitor). Approximately 70,000 MC/well were seeded in a 12-well plate and transfected with 3 µl Lipofectamine RNAiMAX (Life Technologies) mixed with 30 nM miR-1207-5p or negative control mimic, and 50 nM anti-miR-1207-5p or negative control. About 24 h after transfection, cells were stimulated with high glucose [30 mM] and incubated for another 24 h. Afterwards, cell culture media was removed, and cells were lysed and phosphorylathed Smad3 was quantified using phospho-SMAD3 (ser423/425) Instant One ELISA (eBiosciences), as per manufacturer's instructions (E). Experiments showed in C, D, and E were performed in quadruplicate. The significance is indicated only for samples that are significant different from all the others. * P<0.05; ** P<0.01; *** P<0.001. A.U.: arbitrary units. G6PD, glucose-6-phosphate dehydrogenase; PMEPA1, prostate transmembrane protein, androgen induced 1; PDPK1, 3-phosphoinositide dependent protein kinase-1; SMAD7, SMAD family member 7; TGF-β, transforming growth factor beta; PAI-1, plasminogen-activator inhibitor 1; MC, mesangial cells; NC, negative control; p-Smad3, phosphorylated Smad3.

Figure 6. Effect of miR-1207-5p on TGF-β1, PAI-1 and FN1.

Relative quantification of TGF-β1 (A) and PAI-1 (B) mRNA and secreted protein in MC over-expressing miR-1207-5p compared to control. (C) Relative TGF-β1 and PAI-1 protein secreted by MC transfected with miR-1207-5p inhibitor or NC inhibitor compared to control. (D) Relative quantification of secreted FN1 from MC with miR-1207-5p over-expression or knockdown compared to control. Cells were transfected with 3 µl Lipofectamine RNAiMAX mixed with 30 nM miR-1207-5p or negative control (NC) mimic, or 50 nM of miR-1207-5p inhibitor or NC inhibitor. Specific mRNAs were quantified by TaqMan qPCR using PPIA and UBC as endogenous controls. Secreted TGF-β1, PAI-1, and FN1 were determined by ELISA. Results represent averages from three independent experiments. Data are means ± SD. A.U.: arbitrary units. The significance is indicated only for samples that are significant different from all the others. * P<0.05; ** P<0.01; *** P<0.001.

Figure 7. Effect of PVT1 on TGF-β1, PAI-1 and FN1.

Relative quantification of TGF-β1, PAI-1, and FN1 mRNA (A) or secreted protein (B) in MC with PVT1 over-expression. Cells were transfected with plasmid pCMV-PVT1 or empty vector pCMV (NC, negative control) by electroporation using the Neon System. (C) Changes in expression of FN1 mRNA in MC treated with 30 nM PVT1 or NC siRNA, in the presence or absence of the TGF-β signaling inhibitor SB431542. NC siRNA comprised of sequence not found in the human genome. MC were transfected using 5 µl Lipofectamine RNAiMax (Life Technologies) per ml of MsBM media. Results represent averages from three independent experiments. Data are means ± SD. A.U.: arbitrary units. The significance is indicated only for samples that are significantly different from all the others. * P<0.05; ** P<0.01; *** P<0.001.

Figure 8. Potential mechanisms for miR-1207-5p and PVT1 contribution to ECM accumulation in the kidney under hyperglycemic conditions.

(A) Hyperglycemia increases miR-1207-5p level, which downregulates G6PD, PMEPA1, PDPK1 and SMAD7. G6PD (glucose-6-phosphate dehydrogenase) is the first and rate-limiting enzyme of the pentose pathway, which results in the production of NADPH and ribose-5-phosphate. PMEPA1 (prostate transmembrane protein, androgen induced 1) antagonizes TGF-β signaling by sequestering phosphorylated Smad2 and Smad3 to prevent their nuclear translocation . PMEPA1 negatively impact on the duration and intensity of TGF- β/Smad signaling. PDPK1 (3-phosphoinositide-dependent protein kinase-1) negatively regulates TGF-β signaling by preventing translocation of Smad3 and Smad4 from the cytoplasm to the nucleus, and Smad7 from the nucleus to the cytoplasm . Smad7 antagonizes TGF-β family by forming a stable complex with TGF-β type I receptor and, therefore, leading to inhibition of Smad2/3 phosphorylation . Direct downregulation of G6PD by miR-1207-5p may induce increased production of reactive oxygen species (ROS). Increased ROS up-regulates proteins involved in ECM accumulation in the kidney, including TGF-β and PAI-1 (B) Proposed mechanism for a joint and independent effect of miR-1207-5p and PVT1 on ECM accumulation under hyperglycemic conditions. PAI-1, plasminogen-activator inhibitor 1; TGF-β, transforming growth factor beta; FN1, fibronectin 1; ECM, extracellular matrix.