micrornas and diabetic complications

MicroRNAs and Diabetic Complications

Rama Natarajan & Sumanth Putta & Mitsuo Kato

Received: 1 March 2012 /Accepted: 12 April 2012 /Published online: 3 May 2012# Springer Science+Business Media, LLC 2012

Abstract Both Type 1 and Type 2 diabetes can lead todebilitating microvascular complications such as retinopa-thy, nephropathy and neuropathy, as well as macrovascularcomplications such as cardiovascular diseases includingatherosclerosis and hypertension. Diabetic complicationshave been attributed to several contributing factors such ashyperglycemia, hyperlipidemia, advanced glycation endproducts, growth factors, and inflammatory cytokines/che-mokines. However, current therapies are not fully effica-cious and hence there is an imperative need for a betterunderstanding of the molecular mechanisms underlying di-abetic complications in order to identify newer therapeutictargets. microRNAs (miRNAs) are short non-coding RNAsthat repress target gene expression via post-transcriptionalmechanisms. Emerging evidence shows that they have di-verse cellular and biological functions and play key roles inseveral diseases. In this review, we explore the role ofmiRNAs in the pathology of diabetic complications and alsodiscuss the potential use of miRNAs as novel diagnostic andtherapeutic targets for diabetic complications.

Keywords microRNAs . Diabetes . Diabeticcomplications . Biomarkers . RNA-based therapeutics

Diabetic Complications

The prevalence of diabetes in the USA is increasing at arapid rate and current estimates from the American Diabetes

Association reveal that approximately 8.3 % of the popula-tion has diabetes. Furthermore, nearly 79 million peoplehave signs of insulin resistance and pre-diabetes whichsuggest an increased risk for future diabetes development,while nearly 1.9 million new cases of diabetes were reportedin people aged 20 years and older in 2010 alone (AmericanDiabetes Association, http://www.diabetes.org/diabetes-basics/diabetes-statistics/).

Diabetes, defined by elevated fasting blood sugar levels,increases the risk for many serious health problems. Type 1and Type 2 diabetes can have devastating effects on thevasculature leading to microvascular complications such asretinopathy (that can lead to blindness), nephropathy (that canresult in end stage renal disease or renal failure), and painfulneuropathy (that can lead to amputations). Diabetes is also aleading cause of macrovascular complications usually associ-ated with cardiovascular diseases such as coronary arterydisease, atherosclerosis, hypertension, and stroke [1, 2]. Fur-thermore, microvascular complications like diabetic nephrop-athy are closely associated with accelerated rates ofcardiovascular disease and atherosclerosis. The increased in-cidence of these complications in the diabetic population hasbeen attributed to several pathological factors such as hyper-glycemia, hyperlipidemia, advanced glycation end products(AGEs), growth factors, and inflammatory cytokines/chemo-kines [3, 4]. Most diabetic complications, if left untreated, canbe life threatening and greatly reduce the quality of life.Furthermore, in some patients, complications seem to prog-ress despite glycemic control, a phenomenon termed metabol-ic memory [5, 6]. Hence there is an imperative need for abetter understanding of the molecular mechanisms underlyingthe accelerated rates of complications under diabetic condi-tions in order to develop more effective therapies. Here wediscuss the emerging role of microRNAs as new players in thedevelopment of various diabetic complications.

R. Natarajan (*) : S. Putta :M. KatoDepartment of Diabetes,Beckman Research Institute of the City of Hope,1500 East Duarte Road,Duarte, CA 91010, USAe-mail: [email protected]

J. of Cardiovasc. Trans. Res. (2012) 5:413–422DOI 10.1007/s12265-012-9368-5

http://www.diabetes.org/diabetes-basics/diabetes-statistics/

http://www.diabetes.org/diabetes-basics/diabetes-statistics/

MicroRNAs

MicroRNAs (miRNAs) are endogenously produced shortnon-coding RNAs of about 20–22 nucleotides that havebeen shown to play an important role in modulating mam-malian gene expression and therefore regulate several keycellular functions [7–12]. miRNAs were discovered in theearly 1990s in a nematode, Caenorhabditis elegans, and thefirst identified miRNA, lin-4 was found to play a critical rolein controlling developmental timing by downregulating itstarget, lin-14, due to the antisense complementarity [13, 14].miRNAs can inhibit the expression of their target genes viapost-transcriptional mechanisms [9, 15, 16]. It is estimatedthat there are over 1,000 human miRNAs and that about 60 %of the human protein-coding genes can be targeted by miR-NAs [9] and thus these small molecules can have profoundeffects on the functions of numerous proteins. Although miR-NAs were discovered only about 20 years ago, they havemade an enormous impact in our understanding of generegulation at the post-transcriptional level, and numerousstudies have now documented their roles in cellular functionssuch as differentiation, growth, proliferation, and apoptosis.

miRNAs are transcribed by RNA Polymerase II intoprimary transcripts (Pri-miRNAs) in the nucleus. ThesePri-miRNAs are relatively long (up to several kilo bases)and may contain one or more hairpin-like structures. Amicroprocessor complex consisting of the ribonuclease(RNase III) endonuclease, Drosha, and its binding partner,DGCR8, bind to the hairpin structures in Pri-miRNAs andprocess them to precursor miRNAs (Pre-miRNAs), whichare about ∼70 nucleotides and have a stem loop structure[15, 17]. Pre-miRNAs are then exported to the cytoplasm byExportin 5 where they are recognized and cleaved byanother RNase III enzyme, Dicer, in collaboration withTAR RNA binding protein to generate the mature miRNAduplex comprising ∼22 nucleotides [15, 17]. One strand ofthe duplex is selected to be loaded into the RNA-inducedsilencing complex (RISC), while the other gets degraded.RISC is a multiprotein complex that contains Argonaute pro-teins and the mature miRNAwhich guide the RISC to recog-nize the target mRNAs through sequence complementarity.Depending on the complementarity, the target mRNA is eithercleaved (perfect complementarity) or its translation is inhibited(imperfect complementarity to the 3′UTRs) or destined fordegradation in processing bodies (P-bodies) [12, 15, 17].

Since miRNAs can modulate key physiological processesand pathophysiological disease states, there is an imperativeneed to determine the identity of the miRNAs and theirtargets associated with diabetic complications as this wouldprovide a new window of opportunity to identify new bio-markers and therapeutic targets. While several studies haveshown that miRNAs may play a role in diabetes itself and invascular complications unrelated to diabetes [18–22], this

review is focused primarily on miRNAs related to diabeticcomplications (especially diabetic retinopathy, diabetic ne-phropathy and cardiovascular diseases).

MicroRNAs in Diabetes Complications

Diabetic Retinopathy (DR)

DR is a very common microvascular complication of diabe-tes and also one of the leading causes of blindness in theUSA [23, 24]. A few recent studies have demonstrated therole of miRNAs in DR. Kovacs et al. performed the first in-depth miRNA-expression profiling analysis of miRNAs inthe retina and retinal endothelial cells (RECs) from a Strep-tozotocin (STZ) induced diabetic rat model 3 months post-diabetes onset [25]. Among the differentially expressedmiRNAs, interestingly they found that key NF-kB respon-sive miRNAs (such as miR-146, miR-155, miR-132, andmiR-21) were upregulated in the diabetic RECs, and alsothat key vascular endothelial growth factor (VEGF) respon-sive miRNAs and the p53-responsive miR-34 family wereupregulated in both the retinas and RECs of the diabetic rats.Furthermore, due to the negative feedback regulation ofmiR-146 on NF-kB activation in endothelial cells, theysuggest that miR-146 could serve as a potential therapeutictarget for DR through NF-kB inhibition. In a more recentstudy, Feng et al. reported that miR-146a is decreased inendothelial cells treated with HG and also in retinas fromdiabetic animals (STZ injected rats and db/db mice) [26].Furthermore, they demonstrated that under these conditionsthe expression of fibronectin (a miR-146a target that cancontribute to hypertrophy and fibrosis) was increased due todecreases in miR-146a levels, which in turn was mediatedby increases in the co-activator p300. In another study,McArthur et al. reported that miR-200b was downregulatedin endothelial cells treated with high glucose (HG) and inretinas of STZ-induced diabetic rats [27]. They also validat-ed VEGF as a direct target. miRNA-mimic treatments invitro in endothelial cells, or in vivo (intravitreal injection)could ameliorate diabetes induced increases in VEGFmRNA and protein levels. Conversely, miR-200b antago-mirs could increase VEGF production, thus providing fur-ther understanding of the role of this miRNA in thepathogenesis of DR. Silva et al. examined the role of miR-29b and its potential target RAX (an activator of the pro-apoptotic PKR signaling pathway), in the apoptosis of ret-inal neurons related to the pathogenesis of DR [28]. Theyobserved that miR-29b and RAX were localized in theretinal ganglion cells and the cells of the inner nuclear layerof the retinas from normal and STZ-induced diabetic rats.Their results suggest that miR-29b upregulation at the earlystages of diabetes in this model may have protective effects

414 J. of Cardiovasc. Trans. Res. (2012) 5:413–422

against apoptosis of the retinal cells by the PKR pathway.Intravitreal injections of key miRNAs such as miR-29b andmiR-200b could be developed as translational approachesfor the treatment of DR.

Diabetic Nephropathy

Several studies to date have demonstrated a role for miR-NAs in diabetic nephropathy (DN), a severe microvascularcomplication that can lead to end-stage renal disease andpainful dialysis. Increased expansion (hypertrophy) and ac-cumulation of extracellular matrix (ECM) proteins such ascollagen (fibrosis) in the glomerular mesangium along withglomerular podocyte dysfunction are major features of DN[29, 30]. DN is associated with increased expression oftransforming growth factor-β1 (TGF-β) which is a potentinducer of these fibrotic events and renal dysfunction[30–33]. We observed that a group of miRNAs (miR-192,miR-200b/c, miR-216a, and miR-217) were increased inmouse renal mesangial cells (MMC) treated with TGF-β,and in glomeruli of mouse models of diabetes [type 1(STZ-induced) and type2 (db/db)] [31, 34–36]. Our studiesshowed that miR-192 regulates Collagen type I alpha2(Col1a2) and Col4a1 genes as well as downstream miRNAs,miR-216a/217 and miR-200b/c, by targeting the E-boxrepressor, Zeb1/2. In addition, we found that the miR-216a/miR-217 cluster activates Akt kinase by targeting Ptenin MMC treated with TGF-β and induces hypertrophy inMMC [36]. miR-216a also regulates Col1a2 gene throughmechanisms involving inhibition of the RNA binding pro-tein Ybx1 [34]. Recently, we found that miR-200b/c are alsoinvolved in collagen expression and TGF-β auto-regulationby targeting Zeb1 in MMC [35]. Together these cascades ofmiRNAs are initiated from miR-192 which is upregulatedby TGF-β and under diabetic conditions in the mesangium,and contribute to major features of DN such as glomerularhypertrophy and ECM accumulation via key signaling andgene regulation mechanisms [35–37].

Other miRNAs have also been implicated in renal fibro-sis associated with DN. miR-377 was found to inducefibronectin (ECM protein) expression in MCs via down-regulation of manganese superoxide dismutase and p21-activated kinase [38]. In this study, the authors also foundthat HG treatment of the MCs increased the expression ofmiR-192. Wang et al. reported that miR-192 levels were alsoincreased in kidneys of STZ-injected type 1 diabetic micefed with a high fat diet and this was accelerated in FXRknockout mice [39]. miR-93 levels were reported by Long etal. to be lower in glomeruli of diabetic db/db mice comparedto control mice, and also in HG treated podocytes and renalmicrovascular endothelial cells [40]. The parallel increase inthe expression of VEGF-A suggested that it was a directtarget of miR-93. More recent work by these authors

showed that miR-29c as well as miR-192 and miR-200bwere upregulated in glomeruli from db/db mice, and inendothelial cells and podocytes treated with HG [41]. Usingextensive in vitro and in vivo approaches, they demonstrat-ed that the decrease in miR-29c activates Rho kinase bytargeting Spry1 and contributes to DN (enhanced ECMaccumulation and podocyte apoptosis). On the other hand,recently Wang et al. reported that members of the miR-29family were downregulated, along with increases in colla-gens I, III, IV (validated miR-29 family targets), in diabeticApoE−/− mice kidneys and in proximal tubule epithelialcells, podocytes and MC treated with TGF-β [42]. Thedifferences in these two studies with miR-29 were attributedto the different animal models of diabetes used and types ofcell stimulations. In addition, a report suggested that de-creased miR-192 levels was associated with severity of DNand fibrosis in diabetic patients, although normal levels ofmiR-192 in healthy kidneys were not provided[43]. miR-192 also regulates E-cadherin gene expression in tubularepithelial cells through Zeb1/2 and Wang et al. reporteddecreased levels of miR-192 in diabetic ApoE−/− mice aswell as in TGF-β treated tubular and other renal cells [44]. Ittherefore appears that the nature of the animal models usedas well renal cell-specific responses could play a key role inthe regulation of miRNAs in the kidney. In another study,Wang et al. showed that miR-200a targets TGF-β2 in cul-tured proximal-tubular epithelial cells, creating another cir-cuit in the TGF-β response since TGF-β increased TGF-β2expression via decreases in miR-200a in these cells [45].miR-21 has attracted a lot of attention and Dey et al.reported that miR-21 is increased in the renal cortex of theOVE26 type 1 diabetic mice and activates mTOR signalingrelated to DN pathogenesis by targeting Pten [46]. On theother hand, another study found that miR-21 levels weredecreased in db/db mice and that its overexpression couldinhibit the proliferation of MC and decrease the 24 hour urinealbumin excretion rate in the diabetic mice [47]. miR-25 wasalso reported to be decreased in diabetic rat kidneys and inMC treated with HG [48]. These authors found that miR-25targets Nox4 which has been shown to promote oxidant stressassociated with the pathogenesis of DN. Thus, decrease ofmiR-25 can induce Nox4 and contribute to the developmentof DN in rats. Taken together, several miRNAs have nowbeen identified that may promote or inhibit the progression ofDN. As discussed below, these miRNAs could be evaluatedas potential therapeutic targets for DN in the future.

Limb Ischemia

Limb ischemia due to poor circulation and endothelial dys-function is another complication of diabetes. Caporali et al.found that miR-503 expression levels were upregulated inendothelial cells (ECs) under HG culture conditions and

J. of Cardiovasc. Trans. Res. (2012) 5:413–422 415

ischemia associated cell starvation. Blocking miR-503 byeither decoy or antisense oligonucleotides (oligos) improvedfunctional capacities of ECs in vitro [49]. They validatedCCNE1 and cdc25A as direct targets of miR-503 whoselevels were downregulated in HG conditions. The expres-sion levels of miR-503 were also increased in ischemic limbmuscles of STZ diabetic mice. Furthermore, the delivery ofan adenovirus based miR-503 decoy to the ischemicmuscles of diabetic mice in vivo corrected the impairmentof post-ischemic angiogenesis in these mice. Notably, mus-cle samples obtained from human diabetic patients hadelevated levels of miR-503 levels that were inversely corre-lated with cdc25 protein levels. This data suggest that miR-503 could be a potential therapeutic target for diabeticpatients affected with critical limb ischemia.

Diabetic Cardiovascular Complicationsand Cardiomyopathy

Evidence shows that hyperglycemia leads to aberrant cellsignaling by activating several inflammatory and fibroticpathways in vascular cells that can lead to increased cardio-vascular complications which include coronary arterial dis-ease, stroke, hypertension, and atherosclerosis [50–53]. Inaddition, diabetic cardiomyopathy is a complication that canlead to heart failure. Shan et al. have shown that HG treatedrat neonatal cardiomyocytes and rat myocardium itself haveincreased levels of miR-1 and miR-206 through modulatingserum response factor and the MEK1/2 pathway [54]. Theyalso demonstrated that miR-1/miR-206 negatively regulateheat shock protein-60 (Hsp60), thereby contributing to theHG mediated apoptosis in cardiomyocytes. Katare et al.showed that miR-1 was increased in STZ-injected type 1diabetic mice during the progression of cardiomyopathy andcould target Pim-1 which has anti-apoptotic properties [55].In addition, anti-miR-1 upregulated Pim-1 and promotedcardiomyocyte survival under HG conditions. Wang et al.examined the expression of miRNAs in normal and diabeticmyocardial microvascular endothelial cells (MMVEC) fromtype 2 diabetic GK rats and examined their role in impairedangiogenesis associated with diabetic ischemic cardiovascu-lar disease [56]. They found that miR-320 is upregulated inthe MMVEC from GK rats and is associated with impairedangiogenesis while miR-320 inhibitor improved the angio-genesis activity in vitro and also increased levels of IGF-1protein. Hence key miRNAs such as miR-320 can be tar-geted as novel therapeutic approaches role for impairedangiogenesis in diabetes. Care et al. observed decreasedexpression levels of miR-133 in mouse models of cardiachypertrophy and human myocardial hypertrophy [57]. Theyalso demonstrated that miR-133 targets RhoA (a GDP-GTPexchange protein) and Cdc42 (a signal transduction kinase)regulating cardiac hypertrophy. Inhibition of miR-133 in

vitro by decoy sequences or in vivo by infusion of antago-mirs led to marked and sustained cardiac hypertrophy whileoverexpression inhibited cardiac hypertrophy. Feng et al.also showed that miR-133a was decreased in a model ofcardiomyocyte hypertrophy in STZ injected diabetic miceand this was related to hypertrophy since it could target keygrowth related genes MEF2A/C, IGF-1R, SGFK1 [58]. Inanother evaluation of diabetic cardiomyocyte hypertrophy,miR-373 was reported to be decreased in the hearts of STZtreated diabetic mice and in rat cardiomyocytes treated withHG, and could target MEF2C [59]. miR-373 was also down-regulated by HG via p38 mitogen activated protein kinase(MAPK) signaling. Recently, Greco et al. reported a dysre-gulation of several miRNAs ( miR-34b/c, miR-199b, miR-210, miR-650, and miR-223) in left ventricle biopsiesobtained from diabetic heart failure patients relative tonon-diabetic heart failure, while miR-216a was increasedin both groups [60]. Bioinformatic analyses of the predictedtargets of these miRNAs showed enrichment of heart failureand cardiac dysfunction related genes. Studies from variousgroups have demonstrated the role of key miRNAs such asmiR-29, miR-30, and miR-21 in cardiac fibrosis related tomyocardial infarction and heart failure due their regulationof fibrotic genes such as collagens and connective tissuegrowth factor [61–63]. It is possible that these miRNAs mayalso be involved in diabetic heart disease.

Several diabetic vascular complications including athero-sclerosis have been associated with enhanced inflammation.Increased expression of inflammatory genes in vascularcells and inflammatory cells like monocytes has been ob-served under diabetic conditions in vitro and in vivo, andrecent studies have also suggested an involvement of epige-netic mechanisms [5, 6, 64]. Some reports have demonstrat-ed a role for miRNAs in regulating inflammatory genesunder diabetic conditions. Shanmugam et al. demonstratedthe distinct roles of heterogeneous nuclear ribonuclear pro-tein K (hnRNPK) and miR-16 in RNA stability of theproinflammatory cyclo-oxygenase (COX-2) gene in mono-cytes. Based on several lines of experimental evidence, theyreported that miR-16 can bind to the 3′UTR of COX-2 todestabilize it, while S100b, a proinflammatory ligand for thereceptor for advanced glycation end products (RAGE),could enhance COX-2 expression by downregulating miR-16 levels. These studies from our group also showed a cross-talk between the DNA/RNA binding protein hnRNPK andmiR-16 [65]. Additional studies from our group examinedthe role of miRNAs in the increased inflammatory geneexpression and dysfunctional behavior of vascular smoothmuscle cells (VSMC) derived from type 2 diabetic db/dbmice relative to those cultured from genetic control db/+mice [66, 67]. There was a significant increase in the levelsof miR-125b in the diabetic db/db VSMC compared to db/+.Interestingly, miR-125b could target and downregulate


Suv39h1, a histone methyltransferase that can mediate epi-genetic histone H3-lysine-9 trimethylation (H3K9me3), achromatin mark associated with repressed genes. Alongwith the increases in miR-125b levels, there was a reciprocaldecrease of Suv39h1 protein levels in the db/db VSMC aswell as aortas of the db/db mice relative to control db/+mice. Chromatin immunoprecipitation assays showed a par-allel decrease in histone H3K9me3 levels at the promotersof key inflammatory genes interleukin-6 (IL-6) and mono-cyte chemoattractant protein-1 (MCP-1). Furthermore,transfection of miR-125b mimics into normal db/+ MVSMCconferred a diabetic phenotype of decreased SUV39h1 andH3K9me3, increased expression of MCP-1 and IL-6, as wellas enhanced monocyte binding. These studies reveal a rolefor miR-125b in the epigenetic regulation of inflammatorygenes in diabetic db/db mice. They also reveal a novel cross-talk between miRNAs and epigenetic histone lysine meth-ylation that can lead to the de-repression of pathologicalgenes under diabetic conditions and thereby contribute tothe progression of diabetic complications [67]. In anotherrecent study, we showed that miR-200b levels were alsoincreased in the VSMC from type 2 diabetic db/db mice andcould enhance the expression of inflammatory genes by alsotargeting and inhibiting a transcriptional repressor Zeb1,which is a negative regulator of certain inflammatory genesthrough E-boxes in their promoters [68]. A recent reportused small RNA sequencing strategies and expression pro-filing to identify Angiotensin II-regulated miRNAs inVSMC and demonstrated their roles in signaling and vascu-lar dysfunction [69].

Relations Between miRNAs, Genetics, and Epigeneticsof Diabetic Complications

Complications like DN appear to have some element ofgenetic predisposition and numerous efforts includingGenome Wide Association Studies (GWAS) in specific clin-ical cohorts have led to the identification of some candidategenes and sequence variants [70]. However, the functionaland pathological role(s) of these genes and variants have notyet been clearly established and hence the specific involve-ment of inherited susceptibility seems more complicated orrather small [70]. Recent studies have suggested that epige-netics may also play a significant role [5, 6]. Given thatmiRNAs have epigenetic mechanisms of action, they shouldalso be considered in the equation. Importantly, there couldbe novel cross talk mechanisms between these multilayersconsisting of miRNAs, epigenetics, and genetics. As indi-cated earlier, genes with epigenetic functions in chromatinmay be directly targeted and downregulated by miRNAsand this could affect chromatin remodeling and geneexpression. miR-125b was upregulated in VSMC of diabetic

mice relative to control mice, and could target the chromatinhistone H3K9 methyltransferase Suv39h1 leading to in-creased expression of inflammatory gene expression in thediabetic cells [66, 67]. Thus, by downregulating key cellularrepressive factors, miRNAs can act as riboregulators topromote epigenetic de-repression mechanisms in the chro-matin which lead to the induction of genes associated withthe pathology of diabetic complications.

While several single nucleotide polymorphisms (SNPs)have been reported to be associated with various diseases,very few have been directly shown to correlate with diseaseincidence. Furthermore, to-date most genetic studies haveevaluated SNPs in the coding regions of genes. On the otherhand increasing evidence suggests that SNPs in non-codingregions, including promoters and enhancers and miRNAseed regions, might play significant roles. Since miRNAs(non-coding RNAs) can affect gene expression by targetingof the 3′UTRs of target genes, if any sequence variationswere present around the miRNA processing sites, or in themature miRNA seed sequence, this would greatly alter themiRNA biogenesis as well as its functions and target genes[71, 72]. This could alter the repertoire of tissue and cellulargene expression profiles which in turn would affect diseasesusceptibility [71, 72]. Thus it is worth paying more atten-tion to SNPs and variations in miRNA seed sequences asthey could have more functionality.

Apart from genetic predisposition, additional factors andespecially environment and epigenetics may provide that“second chromatin hit” to confer functionality to convertdisease associated SNPs to disease causing SNPs. This wasillustrated in a recent study comparing epigenomic histonemodification profiles in blood monocytes of type 1 diabeticpatients versus normal volunteers which demonstrated sig-nificant differences in the levels of histone H3K9 acetyla-tion at enhancer regions of two MHC locus genes DRB1and DQB1 known to be highly associated with type 1diabetes [73]. Another study compared DNA methylationin saliva samples from diabetic patients with end stage renaldisease versus those without diabetic nephropathy. Interest-ingly, some of the differentially methylated candidate geneswere previously reported to harbor variants associated withkidney disease [74]. Several efforts are currently ongoing toevaluate epigenetic changes (such as DNA methylation) inarchived genomic DNA, as well as miRNA profiles in tissueand blood cell RNA from large clinical cohorts of diabeticpatients with various complications. Since genetic data fromthese patients are already available in most cases, integrationof datasets from these multiple layers of the genome usinghigh throughout sequencing, experimental validation as wellas in silico/systems biology approaches aided by advancedgenomic, transcriptomic, epigenomic, and computationalmethods, will no doubt yield significant new information.These new ventures will greatly advance our knowledge to


accelerate the discovery of much needed new therapies fordiabetic complications.

miRNAs as Biomarkers of Diabetic Complications

There have been several recent advances in technologies formiRNA detection in cells, tissues and biofluids, includingsensitive quantitative PCRs, microarrays, and high through-put deep sequencing. As such, there is tremendous potentialand interest in developing miRNAs as sensitive biomarkersfor human diseases and tissue injury [75–79]. Since miR-NAs are relatively stable and easily quantified in a non-invasive manner in plasma and urine, they might fulfill thecritical need for biomarkers for the early detection of

diabetic complications which could greatly facilitate theclinical management of long term outcomes. Recent reportsshowed that circulating miRNAs in blood can be sensitivebiomarkers for cancer, tissue injury, and heart failure[75–79]. miRNA levels have been studied in the urinarysediment of patients with IgA nephropathy [80]. CirculatingmiRNA levels were also evaluated in chronic kidney dis-eases [81, 82]. Furthermore, since there are reports ofchanges in miRNAs such as miR-1, miR-133, miR-125b,miR-200b, miR-206, and miR-503 in models of diabeticcardiovascular and heart diseases, miR-146 and miR-29bin DR, and miR-192, miR-200b/c, miR-216a, miR-217,miR-29b/c, and miR-377 in DN, as shown in Table 1, thesemiRNAs may potentially be worth examining as biomarkersto detect early stages of the associated diabetic complications.

Table 1 miRNAs implicated in diabetic complications

miRNAs in complications Targets Cell types and animal models Expression Ref.

Diabetic retinopathy

miR-29b Rax STZ rat Increase [28]

miR-146 Irak1, Traf6 STZ rat, Retinal EC Increase [25]

miR-146a Fibronectin STZ rat, db/db mice, HUVEC Decrease [26]

miR-200b VEGF STZ rat, HUVEC Decrease [27]

Diabetic nephropathy

miR-21 Pten OVE26 mice Increase [46]

miR-21 Pten db/db mice, MC Decrease [47]

miR-25 Nox4 STZ rat, MC Decrease [48]

miR-29c Spry1 db/db mice, EC, podocytes Increase [41]

miR-29 Collagens ApoE−/− mice, PTEC Decrease [42]

miR-93 Vegf STZ mice, db/db mice, EC, podocytes Increase [40]

miR-192 Zeb1/2 STZ mice, db/db mice, MC Increase [31, 34–36, 38, 39, 41, 85, 89]

miR-192 Zeb1/2 ApoE−/− mice, PTEC Decrease [43, 44]

miR-200a Tgfb2 ApoE−/− mice, PTEC Decrease [45]

miR-200b/c Zeb1 STZ mice, db/db mice, MC Increase [35, 41]

miR-216a Ybx1 STZ mice, db/db mice, MC Increase [34]

miR-216a/217 Pten STZ mice, db/db mice, MC increase [36]

miR-377 Pak1, Sod1/2 STZ mice, MC Increase [38]

Cardiovascular diseases

miR-1 Pim1 STZ mice, cardiomyocytes Increase [55]

miR-1, miR-206 Hsp60 STZ rat, cardiomyocytes Increase [54]

miR-16 Cox-2 THP-1 monocytes Decrease [65]

miR-125b Suv39h1 db/db mice, VSMC Increase [67]

miR-133 Rho-A,Cdc42 Cardiac hypertrophy Decrease [57]

miR-133a Mef2A/C, Sgk1, Igf1R STZ mice Decrease [58]

miR-200b Zeb1 db/db mice, VSMC Increase [68]

miR-320 Igf-1 GK rat, MMVEC Increase [56]

miR-373 Mef2C STZ mice, cardiomyocytes Decrease [59]

miR-503 Ccne1, Cdc25A STZ mice, HUVEC, HMVEC Increase [49]

EC endothelial cells, HUVEC human umbilical vein endothelial cells, VSMC vascular smooth muscle cells, STZ streptozotocin, MVEC microvas-cular endothelial cells, MMVEC myocardial MVEC, HMVEC human MVEC, MC mesangial cells, PTEC proximal tubular epithelial cells


In particular, it is likely that renal, vascular, and blood cell-associated miRNAs can be detected in urine and serumsamples due to various transport mechanisms. However, itshould be noted that circulating miRNAs do not alwayscorrelate with tissue levels. Notwithstanding, overall, thefuture holds great promise for the evaluation of circulatingmiRNAs in biofluids as diagnostic biomarkers of diabeticcomplications.

Translational Approaches: miRNAs as TherapeuticTargets for Diabetic Complications

Preventing or slowing down the progression of complica-tions is a major goal in the clinical management of diabetes.miRNA targeting alone or in combination with conventionaland currently used drugs could be a new opportunity in thisconnection. Recent advances in the synthesis and chemistryof nucleic acids have allowed us to establish more efficientmethods to inhibit specific miRNAs in vitro and in vivo.Locked nucleic acid (LNA) modified anti-miRNAs (anti-miR) have been shown to be very effective for inhibitingmiRNAs [36, 83, 84]. We recently demonstrated specificand efficient reduction of miR-192 in vivo in mouse kidneysinjected with LNA-modified antimiR-192 (LNA-antimiR-192) [36]. This miR-192 inhibitor also reduced downstreammiRNAs (miR-216a, miR-217, and miR-200 family) andfunctional indices of renal fibrosis and hypertrophy, namelycollagens and TGF-β1 and Akt activation in these mice,similar to the effects in cultured MMC [35, 36]. Further-more, we recently demonstrated that injection of LNA-antimiR-192 into STZ-injected type 1 diabetic mice amelio-rated renal fibrosis [85] demonstrating the translational po-tential of such anti-miRNA therapies for diabetic renaldisease. Interestingly, low doses paclitaxel (a cancer drug)ameliorated fibrosis in the remnant kidney model by down-regulating miR-192 [86]. Reduced rates of DN progressionwere also noted in db/db mice injected with 2′-O-methylantisense oligos targeting miR-29c [41]. Adeno-associatedvirus vectors and miRNA sponges have been considered asin vivo delivery systems for miRNAs [55, 87, 88]. Takentogether, along with the discussion above and the informa-tion in Table 1, several miRNAs appear to be upregulated intarget cells and tissues related to the development of diabeticcomplications. Thus, inhibitor antisense LNA oligos orantagomirs (in other appropriate delivery vehicles) againstkey miRNAs that are increased in diabetic complicationscould be developed as new therapeutic approaches for theprevention or treatment of such complications. As Table 1shows, several miRNAs are also reported to be downregu-lated under diabetic conditions. Injections with mimics ofthese miRNAs would be an option for the associated com-plications, although it is technically more complicated due

to the inherent instability of functional mature miRNAs.Importantly, since the up- or downregulation of miRNAsin vivo could also be related to defensive or other adaptivemechanisms, miRNA based therapies should be carefullydesigned only after documenting the in vivo functional roleof the specific miRNA being targeted.

Closing Remarks

In this review, we have discussed not only the significanceof miRNAs in diabetic complications but also the complex-ity in the tissue and cell-specific expression and functions ofspecific miRNAs in the same complication. This could berelated to the cell-type specific patterns, different modelsystems and animals studied, time of sampling, or theseverity of the complications in the models studied. Further-more, one miRNA can potentially target several genes.These discrepancies and facts reflect the challenges ofmiRNA-based therapeutics. However, this is a dynamicand rapidly advancing field and some clinical trials withanti-miRNAs are already ongoing indicating that miRNAtargeting holds much promise for the future. As we learnmore about the molecular mechanisms by which thesesmall, yet powerful, gene regulatory molecules operate invitro and in vivo, we will be able to device better ways toexploit them as non-invasive biomarkers, as well as better invivo delivery methods for miRNA based therapies for diabeticcomplications.

Acknowledgments The authors gratefully acknowledge fundingfrom the National Institutes of Health (NIDDK and NHLBI), theJuvenile Diabetes Research Foundation and the American DiabetesAssociation.

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