FEBS Letters 583 (2009) 2009–2014

journal homepage: www.FEBSLetters .org

MicroRNA-21 protects from mesangial cell proliferation induced by diabeticnephropathy in db/db mice

Zheng Zhang a,b,*, Huimin Peng a,b,*, Junxia Chen a,b, Xin Chen c, Fei Han a, Xiaoming Xu a,Xiaoyan He a, Ning Yan a

a Department of Cell Biology and Medical Genetics, Chongqing Medical University, No. 1, Medical College Road, Chongqing, Chinab Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chinac Department of Endocrinology, The First Affiliated Hospital, Chongqing Medical University, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 7 January 2009Revised 23 April 2009Accepted 8 May 2009Available online 19 May 2009

Edited by Laszlo Nagy

Keywords:Diabetic nephropathyMesangial cellMicroRNAsPTEN phosphohydrolase

0014-5793/$36.00 � 2009 Federation of European Biodoi:10.1016/j.febslet.2009.05.021

* Corresponding authors. Address: Department oGenetics, Chongqing Medical University, No. 1, MediChina. Fax: +86 23 68485555.

E-mail addresses: zhangzheng92@163.com (Z. Zha(H. Peng).

Diabetic nephropathy (DN) is a major diabetic complication. But the initiating molecular events trig-gering DN are unknown. Recent researches have addressed the role of microRNAs in diabetes and itscomplications. In this study, we looked for microRNAs expression during early DN, and showedmicroRNA-21 (miR-21) expression was downregulated in response to early DN in vitro andin vivo. Over-expression of miR-21 inhibited proliferation of mesangial cells and decreased the24-h urine albumin excretion rate in diabetic db/db mice. Moreover, we identified PTEN as a targetof miR-21. We also found PI3 K and p-Akt increased in miR-21 treated mesangial cells and db/dbmice. Overall, these studies for the first time provide evidence for the potential role of miR-21 inearly DN.� 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

Diabetes is the leading cause of end-stage renal failure, andmuch of the morbidity and mortality of diabetes can be attributedto nephropathy. Diabetic nephropathy (DN) is characterized earlyin its course by glomerular hypertrophy and, importantly, mesan-gial hypertrophy, which is associated with the eventual glomerulo-sclerosis [1]. Despite the great progresses that have been made inrecent decades, the mechanism involved in mesangial hypertrophyis not fully understood.

MicroRNAs (miRNAs) are a group of endogenous, small non-coding RNAs that can modulate protein expression [2]. Recently,it has been reported that miRNAs play a role in diabetes mellitusand its complications [3]. The pancreatic islet-specific miRNA,miR-375, plays a negative role in insulin secretion, and it is alsoa key determinant of blood homeostasis [4]. The over-expressionof miR-29 aggravates insulin resistance [5]. MiR-133 promotes dia-betic cardiopathy by inhibiting kalium ion channel of ERG gene,indicating the participation of miRNAs in diabetes mellitus and

chemical Societies. Published by E

f Cell Biology and Medicalcal College Road, Chongqing,

ng), penghuimin6@sohu.com

its complications [6]. However, the role of miRNAs in mediatingmesangial hypertrophy of DN has never been studied.

In this study, we investigated the potential role of miRNA inmesangial hypertrophy during early DN in vitro and in vivo.Through a screen with miRNA array, we identified miR-21 as a reg-ulator of early DN. First, over-expression of miR-21 inhibited pro-liferation of mesangial cells in high glucose condition. Inaddition, the 24-h urine albumin excretion rate (UAE) of diabeticdb/db mice was decreased after being treated with plasmids con-taining miR-21 (30 mg/kg/d) for 4 weeks. Consistent with its func-tion in vitro and in vivo, miR-21 regulates the expression of theDN-related genes, including PTEN [7], which has a role in organsize [8,9]. This has been well established. Taken together, our re-sults strongly suggest that there is an important regulatory roleof miR-21 in DN mesangial cell proliferation, implying that it mightbe a novel potential target for intervention and prevention of DN.

2. Materials and methods

2.1. Animals

C57BL/6JLepr background db/db, db/m mice from SLACCAS(Shanghai, China) were used and the guidelines on humane useand care of laboratory animals for biomedical research publishedby NIH (No. 85-23, revised 1996) were strictly complied for all

lsevier B.V. All rights reserved.

2010 Z. Zhang et al. / FEBS Letters 583 (2009) 2009–2014

animal experiments. Nine 8-week-old male db/db mice and ninedb/m controls were killed for microarray and cell culture studieswhen significant elevation of UAE was detected in db/db mice.Glomeruli were taken from mice in both groups and were sievedfrom renal cortical tissue, as described previously [10]. A further48 male mice at 4 weeks of age were randomized into 4 groups:control group (12 normal untreated db/m mice), miR-21-treateddb/db group (12 mice), empty plasmid pGenesil-1-treated db/dbgroup (12 mice), or untreated db/db group (12 mice). Mice were in-jected intraperitoneally with plasmids (30 mg/kg/d of miR-21 or30 mg/kg/d of pGenesil-1) until UAE was significantly higher inthe untreated db/db mice than the db/m normal control mice.Whole blood was obtained from the retro-orbital sinus of fed mice,and glucose and UAE were measured using an automatic biochem-istry analyzer (Hitachi Ltd., Tokyo, Japan). After mice were killed,cortical sections of both kidneys from each mouse were pooled.A slice of renal cortical tissue from each mouse was also immedi-ately frozen in liquid nitrogen for later use.

2.2. Cell culture, cloning and expression of miR-21

Primary mouse mesangial cells isolated and cultured as describedpreviously [7,10], cultured in RPMI 1640 medium with 5 mmol/l glu-cose and 10% fetal bovine serum (FBS). Mmu-miR-21 gene sequenceswere selected from the miRBase at http://microrna.sanger.ac.uk/se-quences/. Mir-21 (mmu-miR-21, MIMAT0000530) was chemicallysynthesized (Takara Shuzo, Shiga, Japan), and contained a SacIrestriction enzyme cut site: mmu-miR-21-A 50-CACCTAGCTTATCAGACTGATGTTGATTTTTTG-30; mmu-miR-21-B 50-AGCTCAAAAAATCAACATCAGTCTGATAAGCTA-30. The DNA segments were clonedinto pGenesil-1 expression vectors (Wuhan Genesil BiotechnologyCo. Ltd., Wuhan, China) and confirmed by DNA sequencing. Theexpression of miR-21 was induced by transfection of the plasmidinto the cells using Lipofectamine 2000 (Invitrogen, CA, USA) accord-ing to the manufacturer’s instructions. Cells were maintained in ser-um-free medium with 5 mmol/l glucose for 24 h, which wasfollowed by incubation with fresh serum-free medium with 25mmol/l glucose (high glucose) for 48 h. Control cells were incubatedwith 5 mmol/l glucose plus 20 mmol/l mannitol (low glucose) for48 h. Mesangial cells in this study were divided into four groups:cells in low glucose as the normal control group, a miR-21-transfec-ted high glucose group, empty plasmid pGenesil-1-transfected highglucose group, and untransfected high glucose group.

2.3. Isolation of total RNA

RNA was extracted by Trizol reagent (Invitrogen) as standardmethod.

2.4. MiRNA microarray

Small RNA separating, quality control, labeling, hybridizationand scanning were performed by LC Sciences (Houston, TX, USA)using miRMouse_10.0_071620 miRNA array chip, based on SangermiRBase Release 10.0. The array was repeated twice. Preliminarystatistical analysis was performed by LC sciences on mean valuenormalized by Locally-weighted Regression (LOWESS) method onthe background-subtracted data. Then, Student’s test was per-formed to identify the different miRNA expression. MiRNAs withP < 0.01 was considered as having significant difference betweenDN and control.

2.5. Real time reverse transcription (RT)-PCR

MiR-21 expression was measured by real time RT-PCR (detailsshown in Supplementary data).

2.6. Growth inhibition test

MTT Cell Proliferation Assay Kit (Invitrogen) was employed toanalyze cell proliferation (details shown in Supplementary data).

2.7. Glomerular morphological observation

Sieved renal glomeruli taken from mice were observed andmeasured under the microscope with a micrometer. The averagediameter of 100 glomeruli in a group was counted in three differ-ent visual fields.

2.8. Bioinformatics analysis

Three programs, PicTar, miRanda, and TargetScan [11–13], wereused to predict the targets of miR-21.

2.9. Western blot analysis

PTEN, PI3K p85a and phospho-Akt (Ser473) proteins were mea-sured by Western blot using mouse anti-PTEN (Santa Cruz, CA),mouse anti-PI3K (Santa Cruz, CA) and rabbit anti- phospho-Akt(Cell Signaling Technology, Beverly, MA), respectively (detailsshown in Supplementary data).

2.10. Immunocytochemistry and immunohistochemistry

PTEN protein of mesangial cells was analyzed by immunocyto-chemistry, and PTEN protein of renal tissues was tested by immu-nohistochemistry using mouse anti-PTEN (Santa Cruz, CA) (detailsshown in Supplementary data). The average gray value of 50 sec-tions in each group was measured by Beihang CM-2000 BiomedicalImage Analysis System software (Beihang, Beijing, China).

2.11. Immunofluorescence staining

PI3K p85a and phospho-Akt (Ser473) proteins were detected byimmunofluorescence using mouse anti-PI3K (Santa Cruz, CA) andrabbit anti-phospho-Akt antibody (Cell Signaling Technology),respectively (details shown in Supplementary data).

2.12. Statistical analysis

All statistical tests were performed using SPSS software (SPSS,Chicago, IL). Comparisons between groups were carried out by AN-OVA or Kruskal–Wallis tests, as appropriate, according to theGaussian or non-Gaussian distribution of the data. Changes in eachgroup were analyzed using one-way ANOVA or non-parametrictests. P-values < 0.05 were considered statistically significant. Alldata are presented as means ± standard deviation (S.D.).

3. Results and discussion

3.1. Reduced expression of miR-21 in db/db DN mice

To study the potential role of miRNA in DN, we used the miR-Mouse_10.0_071620 miRNA array, which contained 568 probes,to scan miRNAs that were differentially expressed between db/dbDN mice and controls. We used db/db mice with hyperglycemiaand significant elevation of urinary albumin excretion were usedas animal model of the early stage of DN because db/db mice de-velop kidney disease with similarities to human diabetic nephrop-athy [14]. Among 568 miRNAs screened, 66 miRNAs exhibitedsignificantly different expression level between glomerular tissuesof db/db DN mice and db/m controls (Supplementary Table 1). Of

Table 1Body weight, blood glucose and UAE of mice.

Groups Body weight (g) Blood glucose (mmol/l) 24-h urine albumin excretion (UAE; lg/24 h)

4 weeksThe miR-21-treated db/db group 18.5 ± 0.3** 10.1 ± 1.2* 11.7 ± 1.1The pGenesil-1-treated db/db group 18.9 ± 0.6** 10.1 ± 1.0* 11.5 ± 0.7The untreated db/db group 19.5 ± 0.2** 10.2 ± 0.5* 11.0 ± 1.2The control db/m group 13.6 ± 0.1 8.8 ± 0.6 10.4 ± 0.7

8 weeksThe miR-21-treated db/db group 29.0 ± 1.4** 20.1 ± 3.7** 22.5 ± 7.1The pGenesil-1-treated db/db group 29.9 ± 1.5** 27.6 ± 4.7** 88.0 ± 13.8**,***

The untreated db/db group 31.2 ± 1.5** 24.4 ± 3.8** 91.8 ± 9.9**,***

The control db/m group 18.6 ± 1.2 9.9 ± 0.5 11.5 ± 0.9

* P < 0.05 vs. controls.** P < 0.01 vs. controls.*** P < 0.01 vs. the miR-21-treated group.

Z. Zhang et al. / FEBS Letters 583 (2009) 2009–2014 2011

these potential candidates, we focused on miR-21 because its oneof the most obviously altered miRNAs and inhibited cell growthin many pathological changes [15–17]. Consistent with the arrayresults, real-time RT-PCR further validated that down-regulationof miR-21 in DN was a recurrent event (Supplementary Fig. 1A).Together, it suggested that expression level of miR-21 was inver-sely related to DN.

3.2. Over-expression of miR-21 in miR-21-transfected cells and miR-21-treated mice kidneys

Results of real-time RT-PCR showed that miR-21 was signifi-cantly increased in the miR-21-transfected cells compared withthe empty plasmid pGenesil-1-transfected cells (P < 0.01), suggest-ing miR-21 can be over-expressed by Lipofectamine 2000 transfec-tion in the cells (Supplementary Fig. 1B). Also, the expression ofmiR-21 was significantly enhanced in the kidneys of miR-21-trea-ted db/db mice compared with the pGenesil-1-treated mice(P < 0.01). It suggested that miR-21 was over-expressed in the kid-neys of miR-21-treated db/db mice by injection plasmids contain-ing miR-21 (30 mg/kg/d) for 4 weeks (Supplementary Fig. 1C).

3.3. MiR-21 prevents mesangial cells proliferation in vitro

We cultured mesangial cells under high glucose conditions tomimics diabetic pathology [7]. The cloning of miR-21 is shown inSupplementary Fig. 2. The cells proliferation of cells in the miR-21-transfected group was significantly inhibited compared withthe empty plasmid pGenesil-1-transfected and untransfectedgroups (all: P < 0.01). Thus, our results showed that miR-21 inhib-ited mesangial cells proliferation in high glucose conditions (Sup-plementary Fig. 3).

3.4. Effects of miR-21 on biochemical parameters and glomerularmorphology in vivo

At 4 weeks of age, db/db mice already showed significanthyperglycemia, whereas UAE did not increase compared with db/m mice (Table 1). At 8 weeks of age, db/db mice showed significantelevation of UAE (Table 1). Thus, 4-week-old db/db mice exhibitedfeatures similar to the human normoalbuminuric stage, and 8-week-old mice showed features similar to the human early stageof diabetic nephropathy.

Plasmids containing miR-21 (30 mg/kg/d) were injected into 4-week-old db/db mice for 4 weeks. Body weight and blood glucosewere significantly higher in the three diabetic groups comparedwith the normal control group between 4 and 8 weeks of age,but there were no significant differences between the three dia-betic groups. Additionally, UAE did not differ significantly between

the four groups of mice at 4 weeks of age. However, in the 8-week-old mice (i.e., 4 weeks after starting injection of plasmids), UAEwas significantly decreased in miR-21 treated group among thethree diabetic groups (Table 1).

The diameter of glomeruli of 8-week-old mice was significantlygreater in the pGenesil-1-treated and untreated db/db groups thanthat in the control group. Interestingly, it was smaller in miR-21-treated group than in the pGenesil-1-treated and untreated groups(Supplementary Fig. 4). Together, our results demonstrated thatmiR-21 played a protective role in glomerular hypertrophy andthe onset of early DN.

3.5. PTEN was a target of miR-21 in DN

All known mRNA 30-UTRs were scanned as potential targetsfor miR-21 by in silico analysis. Of all the miR-21 targets pre-dicted, PTEN was found to be most appropriate. It was predictedPTEN, which contained the binding site of miR-21 in its 30 UTR,could be regulated by miR-21 (Supplementary Fig. 5). And it wasreported PTEN gene was a direct target of miR-21 by cloning afragment of the 30-UTR of PTEN mRNA containing the putativemiR-21-binding sequence into a firefly luciferase reporter con-struct using the dual-luciferase reporter assay system (Promega)in other diseases [18,19]. Interestingly, previous works havedemonstrated that PTEN expression was associated with DN[7]. Therefore, it is tempting to speculate that over-expressionof miR-21 might prevent mesangial hypertrophy via down-regu-lation of PTEN in DN.

To test this hypothesis, we examined PTEN protein expressionby Western blot in vitro and in vivo. Our data showed PTEN proteinexpression was reduced in the cells in the miR-21-transfectedgroup compared with the cells in the pGenesil-1-transfected andthe untransfected groups (Fig. 1A). Similarly, in the renal tissuesof 8-week-old db/db mice, PTEN protein levels were significantlydecreased in the miR-21-treated group compared with the pGene-sil-1-treated group and the untreated group (Fig. 1B).

Furthermore, the expression profile of PTEN protein was re-duced in cells (Fig. 1C and E) or animals (Fig. 1D and F) treated withplasmid miR-21, as tested by immunohistochemistry. Our resultssuggested that over-expression of PTEN may act as an originatoror promoter of DN, which is consistent with the report of Mahimai-nathan [7]. Moreover, the proliferation of mesangial cells may bedelayed when PTEN was specifically inhibited by miR-21 in earlyDN.

3.6. MiR-21 regulates the PI3K/Akt signal pathway

Western blotting revealed that PI3K p85a and phospho-Akt (Ser473) were increased in the cells in miR-21-transfected group than

Fig. 1. MiR-21 can reduce PTEN protein expression in mesangial cells incubated in high glucose conditions and in db/db mice. (A) The protein expression of PTEN wasdecreased in the miR-21-transfected cells compared with that in the pGenesil-1-transfected and untransfected cells. (B) Protein expression of PTEN was significantly lower inthe kidneys of the miR-21-treated db/db mice group than that in the pGenesil-1-treated and untreated db/db mice groups. (C) Immunohistochemical analysis of PTEN proteinexpression in mesangial cells showed PTEN was decreased in the miR-21-transfected cells compared with that in the pGenesil-1-transfected and untransfected cells. Arrowsindicate mesangial cells. Figures are shown at �200 magnification. (D) Immunohistochemical analysis of PTEN protein expression in the glomeruli of mice showed PTEN wasdecreased in the miR-21-treated group compared with that in the pGenesil-1-treated and untreated groups. Arrows indicate renal glomerular tissue. Figures are shown at�400 magnification. (E) Bar graphs show the average gray value of PTEN protein level in cells of each group. Results are showed as means ± S.D. **P < 0.01 vs. controls,NNP < 0.01 vs. the miR-21-transfected group. (F) Bar graphs show the average gray value of PTEN protein level in glomeruli of each group. Results are showed as means ± S.D.**P < 0.01 vs. controls, NNP < 0.01 vs. the miR-21-treated group.

Fig. 2. Effect of miR-21 on protein levels of p-Akt (Ser 473) and PI3K p85a in DN. (A) In mesangial cells, p-Akt (Ser 473) and PI3K protein levels were significantly higher in themiR-21-transfected and control groups than that in the pGenesil-1-transfected and untransfected groups. (B) In db/db DN mice, p-Akt (Ser 473) and PI3K protein levels weresignificantly higher in the miR-21-treated and control groups than that in the pGenesil-1-treated and untreated groups.

2012 Z. Zhang et al. / FEBS Letters 583 (2009) 2009–2014

the pGenesil-1-transfected and the untransfected groups.Moreover, in the renal tissues of 8-week-old db/db mice, PI3Kp85a and phospho-Akt (Ser 473) were increased in the miR-21-treated group than those in the pGenesil-1-treated and the un-treated groups (Fig. 2)

Dual-fluorescence staining was used to assess the localiza-tions of PI3K p85a and phospho-Akt (Ser 473) in renal tissuesof 8-week-old mice. PI3K protein was fluorescently labeled withFITC green and p-Akt (Ser 473) with labeled with TRITC red,which were visualized with confocal laser scanning fluorescencemicroscopy. Merging both images (yellow-color) for the co-expression of PI3K p85a and p-Akt (Ser 473) demonstrated that

they were co-increased in the renal tissues in the miR-21-treatedgroup compared with those in the pGenesil-1-treated and theuntreated groups (Fig. 3). Interestingly, previous works have sug-gested that the PI3K pathway is a crucial pathway involved inthe metabolic responses to insulin. Binding of insulin to theinsulin receptor activates the PI3K pathway. Defects in PI3K sig-naling have been demonstrated in type 2 diabetes mellitus[20,21]. Additionally, the PI3K pathway is altered in renal mes-angial cells by advanced glycation end products (AGEs), whichcontribute to diabetic complications, including DN [22]. Themain biological function of PTEN is to block PI3K signaling[23]. These results link mesangial hypertrophy to miR-21 in

Fig. 3. Immunofluorescence staining of PI3K (green) and p-Akt (red) by dual-fluorescence staining. Merging of both images (yellow) showed co-localization of PI3K and p-Akt(Ser473) in renal tissues of 8-week-old mice. Representative photographs were taken from renal tissues of the normal control (A), miR-21-treated (B), pGenesil-1-treated (C)and untreated (D) groups. PI3K and p-Akt were increased in the renal tissues in the miR-21-treated group compared with those in the pGenesil-1-treated and untreatedgroups. Arrows indicate renal glomerular tissue. Figures are shown at �400 magnification.

Z. Zhang et al. / FEBS Letters 583 (2009) 2009–2014 2013

response to PTEN/Akt signaling, miR-21 could be a causal factorof the down-regulation of PTEN and activation of PI3K/Aktpathway in renal mesangial cell hypertrophy in early period ofDN.

In summary, miR-21 prevented mesangial hypertrophy by tar-geting the PTEN/PI3K/Akt pathway in vivo and in vitro. Therefore,miR-21 might play an important role in the pathogenesis of DNand offers a potential target for intervention and prevention ofDN. Further investigations are required, including a larger numberof specimens, and more studies on the ‘‘crosstalk” of the networkover the deregulated miRNAs in DN are also needed.

Acknowledgments

This work was supported by grants from the National NaturalScience Foundation of China (No. 30800534) and Scientific Re-search Foundation of Chongqing Medical University of China (No.NSFYY200719).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.febslet.2009.05.021.

2014 Z. Zhang et al. / FEBS Letters 583 (2009) 2009–2014

References

[1] Mason, R.M. and Wahab, N.A. (2003) Extracellular matrix metabolism indiabetic nephropathy. J. Am. Soc. Nephrol. 14, 1358–1373.

[2] Bartel, D.P. (2004) MicroRNAs: genomics, biogenesis, mechanism, andfunction. Cell 116, 281–297.

[3] Lagos-Quintana, M., Rauhut, R., Lendeckel, W. and Tuschl, T. (2001)Identification of novel genes coding for small expressed RNAs. Science 294,853–858.

[4] Poy, M.N., Eliasson, L., Krutzfeldt, J., Kuwajima, S., Ma, X., Macdonald, P.E.,Pfeffer, S., Tuschl, T., Rajewsky, N., Rorsman, P. and Stoffel, M. (2004) Apancreatic islet-specific microRNA regulates insulin secretion. Nature 432,226–230.

[5] He, A., Zhu, L., Gupta, N., Chang, Y. and Fang, F. (2007) Overexpression of microribonucleic acid 29, highly up-regulated in diabetic rats, leads to insulinresistance in 3T3-L1 adipocytes. Mol. Endocrinol. 21, 2785–2794.

[6] Xiao, J., Luo, X., Lin, H., Zhang, Y., Lu, Y., Wang, N., Zhang, Y., Yang, B. and Wang,Z. (2007) MicroRNA miR-133 represses HERG K+ channel expressioncontributing to QT prolongation in diabetic hearts. J. Biol. Chem. 282,12363–12367.

[7] Mahimainathan, L., Das, F., Venkatesan, B. and Choudhury, G.G. (2006)Mesangial cell hypertrophy by high glucose is mediated by downregulationof the tumor suppressor PTEN. Diabetes 55, 2115–2125.

[8] Crackower, M.A., Oudit, G.Y., Kozieradzki, I., Sarao, R., Sun, H., Sasaki, T., Hirsch,E., Suzuki, A., Shioi, T., Irie-Sasaki, J., Sah, R., Cheng, H.Y., Rybin, V.O., Lembo, G.,Fratta, L., Oliveira-dos-Santos, A.J., Benovic, J.L., Kahn, C.R., Izumo, S., Steinberg,S.F., Wymann, M.P., Backx, P.H. and Penninger, J.M. (2002) Regulation ofmyocardial contractility and cell size by distinct PI3K-PTEN signalingpathways. Cell 110, 737–749.

[9] Backman, S.A., Stambolic, V., Suzuki, A., Haight, J., Elia, A., Pretorius, J., Tsao,M.S., Shannon, P., Bolon, B., Ivy, G.O. and Mak, T.W. (2001) Deletion of Pten inmouse brain causes seizures, ataxia and defects in soma size resemblingLhermitte–Duclos disease. Nat. Genet. 29, 396–403.

[10] Shultz, P.J., DiCorleto, P.E., Silver, B.J. and Abboud, H.E. (1988) Mesangial cellsexpress PDGF mRNAs and proliferate in response to PDGF. Am. J. Physiol. 255,F674–684.

[11] Krek, A., Grun, D., Poy, M.N., Wolf, R., Rosenberg, L., Epstein, E.J., Macmenamin,P., da Piedade, I., Gunsalus, K.C., Stoffel, M. and Rajewsky, N. (2005)Combinatorial microRNA target predictions. Nat. Genet. 37, 495–500.

[12] John, B., Enright, A.J., Aravin, A., Tuschl, T., Sander, C. and Marks, D.S. (2004)Human MicroRNA targets. PLoS Biol. 2, e363.

[13] Lewis, B.P., Shih, I.H., Jones-Rhoades, M.W., Bartel, D.P. and Burge, C.B. (2003)Prediction of mammalian microRNA targets. Cell 115, 787–798.

[14] Sharma, K., McCue, P. and Dunn, S.R. (2003) Diabetic kidney disease in the db/db mouse. Am. J. Physiol. 284, F1138–F1144.

[15] Dahiya, N., Sherman-Baust, C.A., Wang, T.L., Davidson, B., Shih, I.M., Zhang, Y.,Wood III, W., Becker, K.G. and Morin, P.J. (2008) MicroRNA expression andidentification of putative miRNA targets in ovarian cancer. PLoS ONE 3, e2436.

[16] Ladeiro, Y., Couchy, G., Balabaud, C., Sage, P.B., Pelletier, L., Rebouissou, S. andZucman-Rossi, J. (2008) MicroRNA profiling in hepatocellular tumors isassociated with clinical features and oncogene/tumor suppressor genemutations. Hepatology 47, 1955–1963.

[17] Tatsuguchi, M., Seok, H.Y., Callis, T.E., Thomson, J.M., Chen, J.F., Newman, M.,Rojas, M., Hammond, S.M. and Wang, D.Z. (2007) Expression of microRNAs isdynamically regulated during cardiomyocyte hypertrophy. J. Mol. Cell Cardiol.42, 1137–1141.

[18] Meng, F., Henson, R., Wehbe-Janek, H., Ghoshal, K., Jacob, S.T. and Patel, T.(2007) MicroRNA-21 regulates expression of the PTEN tumor suppressor genein human hepatocellular cancer. Gastroenterology 133, 647–658.

[19] Roy, S., Khanna, S., Hussain, S.R., Biswas, S., Azad, A., Rink, C., Gnyawali, S.,Shilo, S., Nuovo, G.J. and Sen, C.K. (2009) MicroRNA expression in response tomurine myocardial infarction: miR-21 regulates fibroblast metalloprotease-2via phosphatase, tensin homologue. Cardiovasc. Res. 82, 21–29.

[20] Cantley, L.C. (2002) The phosphoinositide 3-kinase pathway. Science 296,1655–1657.

[21] Jiang, G. and Zhang, B.B. (2002) Pi 3-kinase and its up- and down-streammodulators as potential targets for the treatment of type II diabetes. Front.Biosci. 7, 903–907.

[22] Xu, D. and Kyriakis, J.M. (2003) Phosphatidylinositol 30-kinase-dependentactivation of renal mesangial cell Ki-Ras and ERK by advanced glycation endproducts. J. Biol. Chem. 278, 39349–39355.

[23] Sulis, M.L. and Parsons, R. (2003) PTEN: from pathology to biology. Trends CellBiol. 13, 478–483.