Cross talk between miR-214 and PTEN attenuates glomerular hypertrophy under diabetic conditions

Glomerular mesangial cells (MCs) hypertrophy is one of the earliest pathological abnormalities in diabetic nephropathy (DN), which correlates with eventual glomerulosclerosis. This study aimed to investigate the therapeutic role of miRNA in diabetic glomerular MCs hypertrophy and synthesis of extracellular matrix (ECM). Microarray analysis revealed a significant up-regulation of miR-214 in the renal cortex of diabetic db/db mice, which was confirmed by real-time PCR of isolated glomeruli and primary cultured human MCs. In vitro studies showed that inhibition of miR-214 significantly reduced expression of α-SMA, SM22 and collagen IV, and partially restored phosphatase and tensin homolog (PTEN) protein level in high glucose-stimulated human MCs. Furthermore, we identified PTEN as the target of miR-214 by a luciferase assay in HEK293 cells. Moreover, overexpression of PTEN ameliorated miR-214-mediated diabetic MC hypertrophy while knockdown of PTEN mimicked the MC hypertrophy. In vivo study further confirmed that inhibition of miR-214 significantly decreased the expression of SM22, α-SMA and collagen IV, partially restored PTEN level, and attenuated albuminuria and mesangial expansion in db/db mice. In conclusion, cross talk between miR-214 and PTEN attenuated glomerular hypertrophy under diabetic conditions in vivo and in vitro. Therefore, miR-214 may represent a novel therapeutic target for DN.


MiR-214 contributed to diabetic MC hypertrophy in vitro by targeting PTEN. Transfection of
anti-miR-214 reduced the mRNA expression of miR-214, α -SMA, SM22 and collagen IV in high glucose-treated human MCs (Fig. 3A). It also induced a significant decrease in the protein level of α -SMA,SM22 and collagen IV, as well as a significant increase in the protein level of PTEN (Fig. 3B,C). These results indicated that miR-214 contributed to MC hypertrophy, and inhibition of miRNA-214 ameliorated high glucose-induced expression of hypertrophic marker genes and significantly restored PTEN protein level in human MCs.
We performed sequence alignment of PTEN 3′ -untranslated region (UTR) by using five species including human, rat, mouse, cow and dog, and then listed miR-214 target sites region (Fig. 3D). We also used a luciferase assay in HEK293 cells to identify whether PTEN as the target of miR-214. We constructed a wild type (WT) or mutated (MU) PTEN-psi-CHECK-2 vector (the mutated complementary sequences of 3′ UTR of PTEN for the seed sequence of miR-214) (Fig. 3E). miR-214 mimics were co-transfected with either WT or mutant-PTEN-psi-CHECK-2 Vector into HEK 293 cells, respectively. The cells were harvested and luciferase reporter activity was measured 48 hours after the co-transfection. The results showed that co-transfection with miR-214 mimics and WT resulted in a significant reduction in luciferase reporter activity compared with the control cells. After mutating the nucleotides of seeding sequence in the 3′ UTR of PTEN, the inhibitory effect of miR-214 mimics on luciferase reporter activity were largely abolished (Fig. 3F). These results demonstrated a direct binding of miR-214 to the 3′ UTR of PTEN.
More importantly, we transfected human MCs with lentiviral vectors expressing miR-214 and coding sequence (CDS) of PTEN in high glucose-treated human MCs to confirm whether miR-214 regulated mesangial hypertrophy by targeting PTEN. The results demonstrated that overexpression of miR-214 inhibited the expression of PTEN and activated the expression of α -SMA, SM22 and collagen IV. In addition, co-transfection of human MCs with lentiviral vectors expressing miR-214 and CDS of PTEN induced an increase in PTEN expression and a decrease in expression of hypertrophic-related genes. In contrast, knock down of PTEN by short interfering RNA (siRNA) in human MCs led to a significant reduction in PTEN protein expression and significant increase in expression of α -SMA, SM22 and collagen IV (Fig. 3G-I). Taken together, overexpression of PTEN markedly attenuated miR-214-mediated MC hypertrophy while knockdown of PTEN mimicked miR-214-mediated MC hypertrophy. Thus, miR-214 regulated mesangial hypertrophy by targeting PTEN.

Inhibition of miR-214 ameliorated albuminuria and glomerular mesangial expansion in db/db mice.
To examine the effect of miR-214 on DN in vivo, we delivered lentivirus-packed miR-214 inhibitor at a dose of 1 × 10 7 TU into diabetic mice by tail vein injections every 2 weeks. At the end of the 12 weeks, UAE was significantly increased in db/db animals compared with nondiabetic db/m mice. However, treatment with miR-214 inhibitor significantly decreased UAE in db/db mice (Fig. 4A). Treatment with miR-214 inhibitor also markedly reduced the miR-214 level in isolated glomeruli from db/db mice. These results indicated that the lentivirus-packed miR-214 inhibitor significantly knocked down the endogenous miR-214 and the delivery procedure was effective (Fig. 4B). Quantitative analysis showed that mesangial expansion scores were significantly increased in db/db mice compared with db/m control animals. In contrast, treatment with miR-214 inhibitor significantly ameliorated mesangial expansion in db/db mice (Fig. 4C,D). Furthermore, we examined the ultrastructure of mesangial area by electron microscopy and found that inhibition of miR-214 also attenuated ECM Heat map for differential miRNA profiles in renal cortex between db/db and db/m groups. (E) Representative images for the isolated glomeruli (Original magnification × 100).Scale bars: 20 μ m. (F) Quantitative real-time PCR in isolated glomeruli to validate the miRNA data and confirm the gene expression results obtained from microarray analysis. All data were expressed as means ± SD (n = 10/each group). ▲ p < 0.05, ▲▲ p < 0.01. deposition in db/db mice (Fig. 4E). These results have demonstrated that inhibition of miR-214 significantly ameliorates functional (UAE) and morphological glomerular defects in db/db mice.

Inhibition of miR-214 attenuated glomerular hypertrophy via targeting PTEN in db/db mice.
To further explore the mechanisms by which miR-214 regulated diabetic glomerular hypertrophy, we examined the expression of PTEN in the kidneys. Immunohistochemistry staining revealed that expression of PTEN in diabetic kidneys was significantly decreased compared with that from the control animals (Fig. 5A). In contrast, inhibition of miR-214 significantly restored the expression of PTEN in diabetic kidneys (Fig. 5A). The results of western blot analysis and real-time PCR further confirmed these findings. The mRNA and protein expression of PTEN in diabetic glomeruli was significantly decreased compared with that from the nondiabetic db/m controls. However, inhibition of miR-214 significantly restored the mRNA and protein expression of PTEN in diabetic glomeruli ( Fig. 5B-D). Moreover, inhibition of miR-214 also reduced mRNA and protein expression of hypertrophic markers α -SMA, SM22 and collagen IV in diabetic glomeruli ( Fig. 5B-D). These data conclusively demonstrated that inhibition of miR-214 ameliorated glomerular hypertrophy in db/db mice and this effect was associated with the restoration of PTEN expression.

Discussion
Our study demonstrated that cross talk between miR-214 and PTEN attenuated glomerular hypertrophy under diabetic conditions in vivo and in vitro. This conclusion was based upon the following findings: (i) inhibition of miR-214 ameliorated high glucose-induced expression of hypertrophic marker genes in human MCs; (ii) in vivo studies in db/db mice further confirmed that inhibition of miR-214 significantly reduced the expression of SM22, α -SMA and collagen IV, markedly restored PTEN level, and attenuated albuminuria and mesangial expansion; (iii) miR-214 was identified to target PTEN. Overexpression of PTEN could ameliorate miR-214-mediated MC  It has been demonstrated that miRNAs are dysregulated in kidney diseases and are thought to contribute to the pathophysiology of the DN 14 . MC hypertrophy precedes marked over-expression of ECM in the diabetic kidneys 6 . MC hypertrophy also triggers synthesis of ECM proteins, and their subsequent deposition in glomeruli, leading to glomerulosclerosis and loss of renal function in DN 5,7 . A study showed that miRNA-200b/c was involved in the regulation of glomerular mesangial hypertrophy related to DN 22 . Aberrant expression of miR-214 was identified in a wide range of human tumors such as nasopharyngeal carcinoma, breast cancer, ovarian cancer, colorectal cancer etc., which contributes to the pathogenesis and metastasis of these tumors [23][24][25] . However, the potential role of miR-214 in the development of DN has not been fully explored. In this study, we showed that miR-214 was markedly upregulated in isolated glomeruli in db/db mice. We also demonstrated that miR-214 promoted human MC hypertrophy and overexpression of collagen IV proteins in the presence of high glucose. However, inhibition of miR-214 significantly reduced expression of hypertrophic markers α -SMA, SM22 and collagen IV, and partially restored PTEN protein level in high glucose-stimulated human MCs. In vivo study further confirmed that inhibition of miR-214 markedly downregulated the expression of SM22, α -SMA in isolated glomeruli and attenuated the mesangial expansion in db/db mice. These results clearly demonstrated that inhibition of miR-214 attenuated glomerular hypertrophy under diabetic conditions in vivo and in vitro.
We further revealed the mechanisms underlying the regulation of miR-214 on MC hypertrophy. First, we listed candidate miRNA genes and target genes on degree level, and used the degree to take miR-214 as the strongest candidate gene and take PTEN as the strongest target gene (data shown in supplementary Tables S1 and S2). Second, we examined the role of miR-214 in the pathogenesis of MC hypertrophy in vitro. We found that inhibition of miR-214 reduced the expression of α -SMA and SM22, accompanied by an increase in the protein level of PTEN. Finally, in vivo studies further confirmed that treatment with miR-214 inhibitor restored protein and mRNA expression of PTEN in kidney tissue from db/db mice. In addition, our data showed that miR-214 targeted the same sites as previously reported 26 , however, we found that inhibition of miR-214 reduced the expression of SM22, α -SMA, restored PTEN level, as well as attenuated albuminuria and mesangial expansion in db/db mice. We further demonstrated that exogenous PTEN markedly ameliorated miR-214-mediated MC hypertrophy while knockdown of PTEN mimicked the MC hypertrophy. These results suggest that inhibition of miR-214 might ameliorate glomerular hypertrophy under diabetic condition by targeting PTEN.
Inhibition of PTEN contributes to enlargement of cell size in Drosophila and cardiomyocyte hypertrophy in mice [27][28][29] , whereas hypertrophy of skeletal muscle cells is not detected, suggesting that PTEN functions in a tissue-specific manner 30 . Previous study has demonstrated that expression of PTEN significantly inhibits high glucose-induced protein synthesis and expression of dominant-negative PTEN is sufficient to induce hypertrophy 31 . These results conclusively indicate that PTEN is a key regulator of diabetic mesangial hypertrophy. Our data showed that the expression of PTEN in diabetic glomeruli was significantly decreased compared with that from the control animals. These results demonstrated that hyperglycemia-induced glomerular hypertrophy was associated with a reduction in PTEN expression. Previous study reported that miR-216 and miR-217 promoted TGF β -induced MC hypertrophy in vitro by regulating PTEN 32 . We found that miR-214 directly targeted PTEN, which was consistent with PTEN being a target of miR-214 in monocytes in vitro as previously reported 26 . To further support our findings, in vivo experiments were performed to investigate the role of PTEN as a downstream target of miR-214 in diabetic MC hypertrophy. Our results showed that knockdown of miR-214 significantly attenuated UAE and glomerular mesangial expansion, which was accompanied by restoration of the PTEN level in diabetic db/db mice. Recent study has demonstrated that deletion of miR-214 inhibits tubulointerstitial lesions in a unilateral ureteral obstruction (UUO) mouse model 33 . Our study showed that miR-214 promoted diabetic MC hypertrophy via PTEN and provided an understanding of the role of miRNA in the pathophysiology of DN. In summary, these results suggest that the regulatory effects of miR-214 on PTEN are likely to be accountable for its action against diabetic MC hypertrophy.
A recent study has demonstrated that miR-130 family negatively regulates PTEN protein expression in bladder cancer cells 34 . Our previous study 35 has demonstrated that miR-196a acts as an important molecular regulator in high glucose-induced MC hypertrophy by targeting p27 kip1 . In this study, we showed that inhibition of miR-214 significantly ameliorated glomerular hypertrophy under diabetic conditions by targeting PTEN in vivo and in vitro.
There were limitations in this study. First, we only showed changes in hypertrophy markers and did not present the morphology of human MCs at different conditions. Second, podocyte injury is a key event in the initiation and progression of DN. We did not examine the effects of miR-214 inhibition on diabetic podocytes. Thus, the effects of miR-214 inhibition on the morphology of human MCs and podocyte injury in diabetes need to be further investigated in future studies. after co-transfection of miR-214 mimics (MiR-214) with wild type (WT) or mutant (MU) of psi-CHECK-2-PTEN-3′ UTR vector, respectively, in high glucose-stimulated MCs. ▲ p < 0.05 vs. pGL4.10 Vector, ★ p < 0.05 vs. All experiments were performed in triplicate. All data were expressed as means ± SD, ▲ p < 0.05 vs. control, ★ p < 0.05 vs. miR-214+ PTEN.
In conclusion, cross talk between miR-214 and PTEN attenuated glomerular hypertrophy under diabetic conditions in vivo and in vitro. These findings suggest that miR-214 may represent a novel therapeutic approach for DN.  ScRNA, db/db mice treated with miR-214 scramble; anti-miR-214, db/db mice treated with miR-214 inhibitor. All data were expressed as means ± SD, ▲ p < 0.05 vs. db/db group, ★ p < 0.05 vs. ScRNA group.
Scientific RepoRts | 6:31506 | DOI: 10.1038/srep31506 Histological analysis. Renal tissues were fixed in 10% formalin in PBS at room temperature overnight, embedded in paraffin and sliced into 5 μ m sections. Periodic acid-Schiff (PAS) staining was performed to assess parameters for glomerular hypertrophy using an Olympus BX51, DP2-BSW microscope as described previously 35 . Briefly, normal glomerulus was scored 0, mild mesangial expansion was scored 1, and moderate mesangial expansion was scored 2. The sections were then examined independently by two blinded investigators.

Electron microscopy.
To determine the morphological changes in glomerular mesangial area, electron microscopic morphometric evaluation was performed by routine procedures. Renal cortex samples were cut into 1 mm 3 pieces on ice, immediately fixed in 2.5% glutaraldehyde, and then embedded. Ultrathin sections were examined by electron microscopy (Philips CM-120) in a blind fashion. The morphologic assessment was performed.
Immunohistochemical analysis. Sections of kidneys from the experimental mice were immunostained for PTEN (1:100, Cell Signaling). Immunostaining procedures were performed according to the manufacturer's instructions. Paraffin-embedded sections (5 μ m-thick) were deparaffinized with xylene and rehydrated through a descending ethanol gradient. Primary antibodies were diluted in PBS containing 1% bovine serum albumin (BSA). All antibodies were incubated for 45 minutes at room temperature. Sections incubated with PBS, instead of the primary antibody, served as the negative controls. In each glomerulus, the percentage of positive area within the glomerular area was calculated. All slides were observed independently by two blinded investigators.
Cell Culture and lentivirus infection. Primary human cell culture was performed as described previously 36 .
Experiments involving samples of human origin were approved by the Ethic Committee of Shanghai Jiao Tong University Affiliated Sixth People's Hospital with informed consent. All experiments were carried out in accordance with relevant guidelines and regulations. Primary human MCs were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum. The third passage of primary human mesangial cells were used in this study. A total of 2 × 10 5 human mesangial cells were transferred into a six-well dish. At 50% confluency, the cells were serum starved for 24 hours. To test the role of miR-214 in the pathogenesis of MC hypertrophy, we transfected a miR-214 inhibitor (System Bioscience) into cultured human MCs. The cells were infected with miR-214 inhibitor-expressing lentivirus at a dose of 5 × 10 6 TU as published previously 37 , while the cells cultured in serum-free DMEM with 5 mmol/L of glucose and 20 mmol/L of mannitol served as a control as previously described 38  Luciferase activity measurement. The 3′ UTR of human PTEN (Gene ID: 5728) containing complementary sequences for the seed sequence of miR-214 was amplified by PCR and cloned into the psi-CHECK-2 Vector (Promega, WI) (a wild type of psi-CHECK-2-PTEN-3′ UTR, WT). A mutant of the 3′ UTR with a mutation of complementary sequences for the seed sequence of miR-214 was developed by the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, CA). The sequence alignment of PTEN 3′ UTR was perform by using 5 species in mammalian, including human, mouse, rat, cow and dog. For Luciferase reporter measurement, miR-214 mimics (Invitrogen, CA) were co-transfected with WT or mutant of psi-CHECK-2-PTEN-3′ UTR vector into HEK293 cell lines (generous gift from Professor Cijiang John He. Mount Sinai School of Medicine, USA) respectively using lipofectamine 2000 (Invitrogen, CA). We detected firefly luciferase activity by dual-luciferase assays kit (Promega, WI) with renilla luciferase activity as an internal control after 48 hours of transfection 39 .
Quantitative real-time PCR. Total RNA was extracted in isolated glomeruli and human MCs using TRIZOL reagent (Invitrogen, CA) and quantified with ultraviolet spectrophotometer (SmartSpec plus). 2 μ g of total RNA was applied to a reverse transcription reaction using reverse transcription kit (Qiagen, Germany ), or TaqMan microRNA reverse transcription kit (Exiqon, Denmark). Real-time PCR was performed by SYBR Premix (Takara, Japan) in a LightCycler (Roche, Switzerland). Primer sequences for miRNAs, PTEN, SM22, α -SMA and collage IV were listed in Table 1. Each reaction was amplified in triplicate and ratio results were calculated based on the 2 −ΔΔCT method. Gene expression was normalized to β -actin mRNA levels as an endogenous control.
Western blot analysis. Isolated glomeruli and transfected human MCs were harvested and subjected to a lysis buffer. The lysates were centrifuged at 12,500 × g at 4 °C for 25 minutes. The protein concentration of supernatants was measured using protein analysis kit (Bio-rad, CA). Equal amounts of proteins were immunoblotted with primary rabbit anti-PTEN(1:500, Cell Signaling, MA), SM22, α -SMA and collage IV (1:200, Santa Cruz, TE).The blots were incubated in HRP-conjugated goat anti-rabbit secondary antibody respectively (1:2000, Santa Cruz, TE). The protein signals were visualized by X-rays exposures. All the experiments were performed in triplicate. Protein expression was quantified as the ratio of specific band to Tubulin. Relative protein expression was described as the fold change from the control group.
Analysis of mi-RNAs microarray and bioinformatics. The miRNA expression profiles of renal cortex from DN animals of db/db mice and controls of db/m mice (n = 3 respectively) was obtained by μ Paraflo MicroRNA Microarray Assay as described previously 40 . Reported and predicted targets of filtered miRNAs from the Targetscan database (http://www.Targetscan.org/) and miRnaDa database (http://www.microrna.org) were pooled and subjected to GO analysis (http://david.abcc.ncifcrf.gov/). This analysis allows genes to be organized Scientific RepoRts | 6:31506 | DOI: 10.1038/srep31506 into hierarchical categories, uncovering the miRNA-gene regulatory network on the basis of biological processes as previously established 41 . The interactions between miRNAs and mRNAs were analyzed by their differential expression values, and the network was established according to miRNA-(messenger RNA) mRNA target interactions in Sanger MicroRNA database. The key miRNAs and genes in the network always have the biggest degrees. Therefore, the degree of miRNAs and the target genes was used to get the network of miRNA-mRNA interaction. GO analysis was performed to indicate the miRNA-gene regulatory network. GOs with a p value < 0.001 and a false discovery rate (FDR) < 0.05 were chosen to calculate the enrichment degree.
Statistical Analysis. All the data were expressed as means ± standard deviation (SD). Student's t-test was applied to find whether there was significant difference between the two groups. The significance of the data was determined by ANOVA followed by Dunnett's multiple range test when necessary. A P value < 0.05 was considered statistically significant.