A total of 9 groups of key miRNAs that targeted the downregulated DEGs were predicted: miR-506, miR-23a/b, the miR-200 family (miR-200b/c/429), miR-124a, the miR-181 family (miR-181a/b/c/d), the miR-25/32/92/363/367 cluster, the miR-17 family (miR-17-5p, miR-20a/b, miR-106a/b, and miR-519d), miR-27a/b, and the miR-29 family (miR-29a/b/c) (Fig. 3A). Only 2 groups of key miRNAs targeted the upregulated DEGs were found (the miR-17 family and the miR-200 family) (Fig. 3B).
The regulatory relationships between the miRNAs and their target genes were established using Cytoscape, which showed that the single gene was regulated by multiple miRNAs and that the single miRNA could target multiple genes (Fig. 4). Subsequently, miR-29 targeting VEGFA and COL4A5 and miR-200 targeting VEGFA were verified in all 5 databases. A total of 4 databases showed that miR-29 targeted COL4A3, the miR-17 family targeted VEGFA, and miR-181 targeted CBLB. Only 3 databases showed that miR-17 family targeted COL4A3, miR-23 targeted COL4A5, and miR-506 targeted ITGA3, and only 2 databases showed that miR-181 targeted ITGA3 (Table 4). Integrating with the result of KEGG pathway analysis, it was indicated that these key miRNA-target genes were mainly involved in the ECM–receptor interaction, PI3K/Akt signaling pathways, and focal adhesion to influence the pathogenesis and development of DN (Fig. 5).
VEGFA is the most important vascular endothelial growth factor and plays an important role in angiogenesis, tumor growth, and ischemic diseases. The up- and downregulation of the podocyte VEGFA levels during renal development led to glomerular disease. This study identified that VEGFA was a key gene that was downregulated in DN, consistent with previous studies. Sivaskandarajah et al found that selective knockout of VEGFA in glomerular podocytes of diabetic mice resulted in a significant increase in proteinuria with severe glomerular scar formation and increased apoptosis. However, Veron et al found that overexpression of the VEGFA isoform (VEGF164) in the podocytes of adult transgenic mice resulted in proteinuria, glomerular hyperplasia, glomerular basement membrane thickening, and podocyte disappearance, which might be related to different types of diabetes models or the length of studies by different research institutes.
This study showed that VEGFA was regulated by multiple miRNAs, including miR-29 and miR-200. Previous studies have confirmed that VEGFA is a target of miR-29[37,38] and that miR-200 suppresses VEGFA expression.[39,40] The loss of miR-29a/b is thought to be closely related to progressive kidney damage in diabetes. MiR-29a and miR-29b promote resistance to renal fibrosis and prevent podocyte apoptosis.[41,42] MiR-29c can induce apoptosis and increase the accumulation of extracellular matrix proteins, whereas knockdown of miR-29c prevented high glucose-induced apoptosis. Several studies have shown that miR-200 family members can regulate the epithelial-to-mesenchymal transition (EMT) and its reversal process, and that the overexpression of miR-200 family members can prevent the TGF-β-induced EMT.[44,45] The EMT plays an important role in renal fibrosis, and renal interstitial fibrosis is a common feature of the progression of various kidney diseases developing into end-stage renal failure, suggesting that miR-200 can affect the development of DN.
The KEGG pathway analysis showed that VEGFA was mainly enriched in the PI3K/Akt and focal adhesion pathways. Currently, studies in vivo and in vitro have indicated that the PI3K/Akt signaling pathway is involved in DN tubular cell apoptosis, mesangial cell hyperplasia, and podocyte dysfunction. High glucose induces increased Akt expression and accelerates the transdifferentiation of renal tubular epithelial cells, leading to renal fibrosis.[47–49] Previous studies reported that miR-200 ultimately promoted glomerular hypertrophy and renal fibrosis through the PI3K/Akt signaling pathway.[50,51] These studies indicated that the mechanism of miR-200 inducing DN by targeting VEGFA through the PI3K/Akt signaling pathway was feasible. Thus, miR-200 may be a prospective target for the prevention or treatment of renal fibrosis in diabetic patients. This mechanism needs to be further verified in vivo and in vitro experiments as well as large-scale clinical trials in the future.
This study also found that COL4A3 and COL4A5 were downregulated in DN glomeruli. COL4A3 was regulated by miR-29 and miR-17, and COL4A5 was regulated by miR-29 and miR-23. Collagen type IV (COL4) is the main collagen component of the ECM and plays an important role in the renal pathological changes of DN. The accumulation of ECM is considered the key to glomerulosclerosis and renal failure in DN. Zhao et al revealed that miR-23b was downregulated in DN and promoted renal fibrosis and proteinuria via the PI3K/Akt signaling pathway. COL4A3 and COL4A5, which are subtypes of type IV collagen, may be regulated by multiple miRNAs, including miR-29 and miR-23, and participate in the pathogenesis of DN through the ECM–receptor interaction and PI3K/Akt signaling pathways.
Other key miRNAs identified in this study were miR-25, miR-27, and miR-124a. Previous studies have reported that miR-25 downregulation plays an important part in diabetic kidney injury, such as high glucose-induced podocyte apoptosis, persistent proteinuria, and secondary hypertension.[63–65] In addition, high glucose stimulation has been shown to increase miR-27a expression, leading to the worsening of renal function and increasing tubulointerstitial fibrosis in DN patients.[66–68]
All the data contributors are sincerely appreciated for data submitted in the GEO and other databases.
fengying yang orcid: 0000-0002-1428-0395.
. Umanath K, Lewis JB. Update on diabetic nephropathy
: core Curriculum 2018. Am J Kidney Dis 2018;71:884–95.
. Tuttle KR, Bakris GL, Bilous RW, et al. Diabetic kidney disease: a report from an ADA consensus conference. Am J Kidney Dis 2014;64:510–33.
. Doshi SM, Friedman AN. Diagnosis and management of type 2 diabetic kidney disease. Clin J Am Soc Nephrol 2017;12:1366–73.
. Alvarez ML, DiStefano JK. Towards microRNA-based therapeutics for diabetic nephropathy
. Diabetologia 2013;56:444–56.
. Simpson K, Wonnacott A, Fraser DJ, et al. MicroRNAs
in diabetic nephropathy
: from biomarkers to therapy. Curr Diab Rep 2016;16:35.
. Kato M, Natarajan R. MicroRNAs
in diabetic nephropathy
: functions, biomarkers, and therapeutic targets. Ann N Y Acad Sci 2015;1353:72–88.
. Wu H, Kong L, Zhou S, et al. The role of microRNAs
in diabetic nephropathy
. J Diabetes Res 2014;2014:920134.
. Wang WN, Zhang WL, Zhou GY, et al. Prediction of the molecular mechanisms and potential therapeutic targets for diabetic nephropathy
methods. Int J Mol Med 2016;37:1181–8.
. Qu W, Han C, Li M, et al. Revealing the underlying mechanism of diabetic nephropathy
viewed by microarray analysis. Exp Clin Endocrinol Diabetes 2015;123:353–9.
. Cui C, Cui Y, Fu Y, et al. Microarray analysis reveals gene and microRNA signatures in diabetic kidney disease. Mol Med Rep 2018;17:2161–8.
. Baelde HJ, Eikmans M, Doran PP, et al. Gene expression profiling in glomeruli from human kidneys with diabetic nephropathy
. Am J Kidney Dis 2004;43:636–50.
. Woroniecka KI, Park AS, Mohtat D, et al. Transcriptome analysis of human diabetic kidney disease. Diabetes 2011;60:2354–69.
. Davis S, Meltzer PS. GEOquery: a bridge between the Gene Expression Omnibus (GEO) and BioConductor. Bioinformatics
. Szklarczyk D, Franceschini A, Kuhn M, et al. The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res 2011;39(Database issue):D561–568.
. Chin CH, Chen SH, Wu HH, et al. cytoHubba: identifying hub objects and sub-networks from complex interactome. BMC Syst Biol 2014;8(Suppl 4):S11.
. Kohl M, Wiese S, Warscheid B. Cytoscape: software for visualization and analysis of biological networks. Methods in molecular biology (Clifton, NJ) 2011;696:291–303.
. Huang da W, Sherman BT, Lempicki RA. Bioinformatics
enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 2009;37:1–3.
. Wang J, Duncan D, Shi Z, et al. WEB-based GEne SeT AnaLysis Toolkit (WebGestalt): update 2013. Nucleic Acids Res 2013;41(Web Server issue):W77–83.
. Tang D, Chen Y, He H, et al. Integrated analysis of mRNA, microRNA and protein in systemic lupus erythematosus-specific induced pluripotent stem cells from urine. BMC Genomics 2016;17:488.
. Carlsen AL, Schetter AJ, Nielsen CT, et al. Circulating microRNA expression profiles associated with systemic lupus erythematosus. Arthritis Rheum 2013;65:1324–34.
. Shen N, Liang D, Tang Y, et al. MicroRNAs
—novel regulators of systemic lupus erythematosus pathogenesis. Nature Rev Rheumatol 2012;8:701–9.
. Wang W, Mou S, Wang L, et al. Up-regulation of Serum MiR-130b-3p Level is Associated with Renal Damage in Early Lupus Nephritis. Sci Rep 2015;5:12644.
. Li Y, Fang X, Li QZ. Biomarker profiling for lupus nephritis. Genom Proteomics Bioinformatics
. Zeng Y, Liu JX, Yan ZP, et al. Potential microRNA biomarkers for acute ischemic stroke. Int J Mol Med 2015;36:1639–47.
. Li P, Teng F, Gao F, et al. Identification of circulating microRNAs
as potential biomarkers for detecting acute ischemic stroke. Cell Mol Neurobiol 2015;35:433–47.
. Peng G, Yuan Y, Wu S, et al. MicroRNA let-7e is a potential circulating biomarker of acute stage ischemic stroke. Transl Stroke Res 2015;6:437–45.
. Tiedt S, Prestel M, Malik R, et al. RNA-Seq identifies circulating miR-125a-5p, miR-125b-5p, and miR-143-3p as potential biomarkers for acute ischemic stroke. Circul Res 2017;121:970–80.
. Zhang Y, Mo WJ, Wang X, et al. Microarraybased bioinformatics
analysis of the prospective target gene network of key miRNAs influenced by long noncoding RNA PVT1 in HCC. Oncol Rep 2018;40:226–40.
. Li Q, Li J, Dai W, et al. Differential regulation analysis reveals dysfunctional regulatory mechanism involving transcription factors and microRNAs
in gastric carcinogenesis. Artif Intell Med 2017;77:12–22.
. Zhang GM, Goyal H, Song LL. Bioinformatics
analysis of differentially expressed miRNA-related mRNAs and their prognostic value in breast carcinoma. Oncol Rep 2018;39:2865–72.
. Li D, Hao X, Song Y. Identification of the key MicroRNAs
and the miRNA-mRNA regulatory pathways in prostate cancer by bioinformatics
methods. Biomed Res Int 2018;2018:6204128.
. Zhang B, Kirov S, Snoddy J. WebGestalt: an integrated system for exploring gene sets in various biological contexts. Nucleic Acids Res 2005;33(Web Server issue):W741–748.
. Saito R, Smoot ME, Ono K, et al. A travel guide to Cytoscape plugins. Nat Methods 2012;9:1069–76.
. Carranza K, Veron D, Cercado A, et al. Cellular and molecular aspects of diabetic nephropathy
; the role of VEGF-A. Nefrologia 2015;35:131–8.
. Sivaskandarajah GA, Jeansson M, Maezawa Y, et al. Vegfa protects the glomerular microvasculature in diabetes. Diabetes 2012;61:2958–66.
. Veron D, Reidy KJ, Bertuccio C, et al. Overexpression of VEGF-A in podocytes of adult mice causes glomerular disease. Kidney Int 2010;77:989–99.
. Liu L, Bi N, Wu L, et al. MicroRNA-29c functions as a tumor suppressor by targeting VEGFA in lung adenocarcinoma. Mol Cancer 2017;16:50.
. Liu Q, Liao F, Wu H, et al. Different expression of miR-29b and VEGFA in glioma. Artif Cells Nanomed Biotechnol 2016;44:1927–32.
. Mitra RN, Nichols CA, Guo J, et al. Nanoparticle-mediated miR200-b delivery for the treatment of diabetic retinopathy. J Control Release 2016;236:31–7.
. Liu GT, Chen HT, Tsou HK, et al. CCL5 promotes VEGF-dependent angiogenesis by down-regulating miR-200b through PI3K/Akt signaling pathway in human chondrosarcoma cells. Oncotarget 2014;5:10718–31.
. Lin CL, Lee PH, Hsu YC, et al. MicroRNA-29a promotion of nephrin acetylation ameliorates hyperglycemia-induced podocyte dysfunction. J Am Soc Nephrol 2014;25:1698–709.
. Wang B, Komers R, Carew R, et al. Suppression of microRNA-29 expression by TGF-beta1 promotes collagen expression and renal fibrosis. J Am Soc Nephrol 2012;23:252–65.
. Long J, Wang Y, Wang W, et al. MicroRNA-29c is a signature microRNA under high glucose conditions that targets Sprouty homolog 1, and its in vivo knockdown prevents progression of diabetic nephropathy
. J Biol Chem 2011;286:11837–48.
. Kato M, Putta S, Wang M, et al. TGF-beta activates Akt kinase through a microRNA-dependent amplifying circuit targeting PTEN. Nat Cell Biol 2009;11:881–9.
. Gregory PA, Bert AG, Paterson EL, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 2008;10:593–601.
. Zhao L, Wang X, Sun L, et al. Critical role of serum response factor in podocyte epithelial-mesenchymal transition of diabetic nephropathy
. Diab Vasc Dis Res 2016;13:81–92.
. Liu L, Hu X, Cai GY, et al. High glucose-induced hypertrophy of mesangial cells is reversed by connexin43 overexpression via PTEN/Akt/mTOR signaling. Nephrol Dial Transplant 2012;27:90–100.
. Rane MJ, Song Y, Jin S, et al. Interplay between Akt and p38 MAPK pathways in the regulation of renal tubular cell apoptosis associated with diabetic nephropathy
. Am J Physiol Renal Physiol 2010;298:F49–61.
. Ha TS. High-glucose and advanced glycosylation end products increased podocyte permeability via PI3-K/Akt signaling. J Mol Med (Berl) 2010;88:391–400.
. Park JT, Kato M, Yuan H, et al. FOG2 protein down-regulation by transforming growth factor-beta1-induced microRNA-200b/c leads to Akt kinase activation and glomerular mesangial hypertrophy related to diabetic nephropathy
. J Biol Chem 2013;288:22469–80.
. Hyun S, Lee JH, Jin H, et al. Conserved MicroRNA miR-8/miR-200 and its target USH/FOG2 control growth by regulating PI3K. Cell 2009;139:1096–108.
. Watanabe H, Sanada H, Shigetomi S, et al. Urinary excretion of type IV collagen as a specific indicator of the progression of diabetic nephropathy
. Nephron 2000;86:27–35.
. Kolset SO, Reinholt FP, Jenssen T. Diabetic nephropathy
and extracellular matrix. J Histochem Cytochem 2012;60:976–86.
. Zhao B, Li H, Liu J, et al. MicroRNA-23b targets Ras GTPase-activating protein SH3 domain-binding protein 2 to alleviate fibrosis and albuminuria in diabetic nephropathy
. J Am Soc Nephrol 2016;27:2597–608.
. Liu H, Wang X, Liu S, et al. Effects and mechanism of miR-23b on glucose-mediated epithelial-to-mesenchymal transition in diabetic nephropathy
. Int J Biochem Cell Biol 2016;70:149–60.
. Cosio FG. Cell-matrix adhesion receptors: relevance to glomerular pathology. Am J Kidney Dis 1992;20:294–305.
. Pozzi A, Jarad G, Moeckel GW, et al. Beta1 integrin expression by podocytes is required to maintain glomerular structural integrity. Dev Biol 2008;316:288–301.
. Wolf G, Chen S, Ziyadeh FN. From the periphery of the glomerular capillary wall toward the center of disease: podocyte injury comes of age in diabetic nephropathy
. Diabetes 2005;54:1626–34.
. Sawada K, Toyoda M, Kaneyama N, et al. Upregulation of alpha3 beta1-integrin in podocytes in early-stage diabetic nephropathy
. J Diabetes Res 2016;2016:9265074.
. Elewa U, Sanchez-Nino MD, Mahillo-Fernandez I, et al. Circulating CXCL16 in diabetic kidney disease. Kidney Blood Press Res 2016;41:663–71.
. Lin Z, Gong Q, Zhou Z, et al. Increased plasma CXCL16 levels in patients with chronic kidney diseases. Eur J Clin Invest 2011;41:836–45.
. Ruster C, Wolf G. The role of chemokines and chemokine receptors in diabetic nephropathy
. Front Biosci 2008;13:944–55.
. Liu Y, Li H, Liu J, et al. Variations in microRNA-25 expression influence the severity of diabetic kidney disease. J Am Soc Nephrol 2017;28:3627–38.
. Li H, Zhu X, Zhang J, et al. MicroRNA-25 inhibits high glucose-induced apoptosis in renal tubular epithelial cells via PTEN/AKT pathway. Biomed Pharmacother 2017;96:471–9.
. Oh HJ, Kato M, Deshpande S, et al. Inhibition of the processing of miR-25 by HIPK2-Phosphorylated-MeCP2 induces NOX4 in early diabetic nephropathy
. Sci Rep 2016;6:38789.
. Zhou Z, Wan J, Hou X, et al. MicroRNA-27a promotes podocyte injury via PPARgamma-mediated beta-catenin activation in diabetic nephropathy
. Cell Death Dis 2017;8:e2658.
. Hou X, Tian J, Geng J, et al. MicroRNA-27a promotes renal tubulointerstitial fibrosis via suppressing PPARgamma pathway in diabetic nephropathy
. Oncotarget 2016;7:47760–76.
. Wu L, Wang Q, Guo F, et al. MicroRNA-27a induces mesangial cell injury by targeting of PPARgamma, and its in vivo knockdown prevents progression of diabetic nephropathy
. Sci Rep 2016;6:26072.