Expression of miR-106b-25 induced by salvianolic acid B inhibits epithelial-to-mesenchymal transition in HK-2 cells

European Journal of Pharmacology 741 (2014) 97–103

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

Expression of miR-106b-25 induced by salvianolic acid B inhibits epithelial-to-mesenchymal transition in HK-2 cells Qiong Tang a, Haizhen Zhong a, Fengyan Xie a, Jiayong Xie a, Huimei Chen b,n, Gang Yao a,nn a b

Department of Nephrology, Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, PR China Jiangsu Key Laboratory of Molecular Medicine, School of Medicine, Nanjing University, Nanjing 210093, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 25 February 2014 Received in revised form 22 July 2014 Accepted 23 July 2014 Available online 2 August 2014

Epithelial-to-mesenchymal transition (EMT) is a highly conserved physiological program involved in renal fibrosis. Previous studies have shown that transforming growth factor (TGF)-β1 induces EMT in human kidney proximal tubular epithelial cells (HK-2 cells), whereas salvianolic acid B (Sal B) has a protective effect against EMT. The molecular pathogenesis of such processes is currently not well understood. In this study, a miRCURYTM LNA Array was used to screen HK-2 cells for expression changes of microRNAs (miRNAs) implicated in EMT. After validation by real-time PCR, all three members of the miR-106b-25 cluster (miR-106b, miR-93, and miR-25) were found to be markedly down-regulated during EMT in response to TGF-β1, whereas these miRNAs were up-regulated by Sal B treatment in a dose-dependent manner. Moreover, enhanced expression of miR-106b attenuated EMT by retaining the epithelial morphology of HK-2 cells, reducing the levels of α-smooth muscle actin (α-SMA), and increasing the levels of E-cadherin. To explore the molecular basis underlying the inhibitive effect of the miR-106b-25 cluster against EMT, bioinformatics analysis revealed that TGF-β type II receptor, a regulator of TGF-β signaling, might be a direct target of the miR-106b-25 cluster. In turn, low levels of TGF-β type II receptor in EMT of HK-2 cells were shown under the increase of miR-106b. In conclusion, our data suggest that the miR-106b-25 cluster may contribute to EMT in the kidney, and is involved in the protective effect of Sal B. Targeting of specific miRNAs may be a novel therapeutic approach to treat renal fibrosis. & 2014 Elsevier B.V. All rights reserved.

Chemical compounds studied in this article: Salvianolic acid B (Pubchem CID: 11629084) Keywords: Epithelial-to-mesenchymal transition miR-106b-25 Salvianolic acid B Transforming growth factor-β1

1. Introduction Tubulointerstitial fibrosis is characterized as a main feature, irrespective of the diverse initial causes, contributing to progressive chronic kidney disease and the subsequent functional deterioration and eventual loss of renal function (Zeisberg and Neilson, 2010). Despite fundamental advances in understanding the pathophysiology of renal fibrosis, definitive therapies remain limited. Therapeutic approaches aimed at novel targets are urgently needed to effectively prevent and/or treat this disorder. During renal fibrosis, myofibroblasts can be derived from tubular epithelial cells by epithelial-to-mesenchymal transition (EMT) (Carew et al., 2012). Furthermore, transforming growth factor (TGF)-β1 is a crucial inducer of EMT and the generation of interstitial fibroblasts (Yang et al., 2006). Our previous studies suggest that salvianolic acid B (Sal B), the most abundant bioactive

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Corresponding author. Tel.: þ 86 25 83686043; fax: þ 86 25 83686451. Corresponding author. E-mail addresses: [email protected] (H. Chen), [email protected] (G. Yao).

nn

http://dx.doi.org/10.1016/j.ejphar.2014.07.051 0014-2999/& 2014 Elsevier B.V. All rights reserved.

component in Radix Salviae Miltiorrhizae, inhibits renal interstitial fibrosis by preventing and reversing EMT inducted by TGF-β1 (Yao et al., 2009; Pan et al., 2011). Medications targeting such processes are a current topic of EMT in renal fibrosis. MicroRNAs (miRNAs) are small noncoding RNAs (ncRNAs) of 22 nucleotides in length, which bind to the 30 -untranslated region (UTR) of target mRNA and repress translation and/or induce degradation of target mRNAs (Khvorova et al., 2003; Altuvia et al., 2005). miRNAs have been shown to regulate numerous molecular and cellular processes (Shivdasani, 2006). Aberrant expression of miRNAs is associated with the initiation and progression of pathological processes including diabetes, cancer, and cardiovascular disease (Krupa et al., 2010; Zhang et al., 2012; Qin et al., 2013). A recent report has revealed the role of miRNAs in kidney injury and repair (Chandrasekaran et al., 2012), giving new insights into the mechanism underlying EMT in renal fibrosis. In the present study, we indentified a unique miRNA signature associated with EMT. We found that some miRNAs of human kidney proximal tubular epithelial cells (HK-2 cells) induced by TGF-β1 were alternatively expressed in response to Sal B treatment. Among these miRNAs, the miR-106b-25 cluster, including

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miR-106b, miR-93, and miR-25, demonstrated expression changes coinciding with EMT in vitro. The enhanced expression of the miR106b-25 cluster prevents EMT inducted by TGF-β1, presumably through diminishing the expression of the possible target protein, TGF-β type II receptor.

2. Materials and methods 2.1. Cell culture and treatments The human kidney proximal tubular epithelial cell line HK-2 was cultured in DMEM/F12 (Gibco, Paisley, UK) supplemented with 5% fetal calf serum (FCS; Sijiqing, Hangzhou, China) at 37 1C in a humidified atmosphere with 5% CO2. For experiments, the cells were divided into four groups as follows: a 5% FCS vehicle group, TGF-β1 (5 ng/ml; PeproTech, Rocky Hill, NJ, USA) induced EMT group, and groups in which cells were exposed to both TGFβ1 (5 ng/ml) and Sal B (99% pure; Chinese National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) at different final concentrations (1 or 50 μmol/l). Specifically, HK-2 cells were cultured with DMEM/F12 5% FCS in 6-well plates overnight, and then culture medium was changed to fresh DMEM/F12 5% FCS containing TGF-β1 (5 ng/ml) plus Sal B at the different concentrations (1 or 50 μmol/l), with both TGF-β1 (5% FCS þTGF-β1) and vehicle (5% FCS) groups. Cultures were continued for other 48 h. All treatments were performed in triplicate. 2.2. miRNA microarray analysis HK-2 cells were cultured with 5% FCS in vehicle group, additionally treated with TGF-β1 (5 ng/ml) in EMT-induced group, and treated with TGF-β1 plus Sal B (1 or 50 μmol/l) in treatment group, all of which were subjected to miRNA microarray analysis. Total RNA was extracted at 48 h post-treatment using TRIzol reagent (Takara, Kyoto, Japan). The miRNA microarray was performed by KangChen (Shanghai, China). Total RNA from the samples was labeled with Hy3TM/Hy5TM fluorescent dye using the miRCURYTM LNA Array power labeling kit (Exiqon, Vedbaek, Denmark) following the manufacturer's procedure, and then mixed and hybridized to the miRCURYTM LNA Array version 16.0 (Exiqon). The arrays were scanned with a Genepix 4000B, and the obtained images were analyzed using Genepix Pro 6.0 that employed the RMA (robust multichip analysis) normalization method. Fold changes of gene expression by 2-fold up- or down-regulation were considered as significantly different after normalization.

2.4. RNA transfection The HK-2 cells were transfected with miR-106b mimic or its negative control RNA (GenePharma, Shanghai, China) using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) following manufacturer's instructions, and then stimulated with 5 ng/ml TGF-β1. According to the manufacturer's recommendations and our previous study, we found that HK-2 cells were transfected with miR-106b mimic or N.C. with high efficiency at 80 nM final concentration. After transfection, the cells were incubated at 37 1C in a 5% CO2 incubator for 24–48 h prior to analysis by gene expression assays or further treatment. 2.5. Western blot analysis The treated cells were harvested and lysed for 30 min on ice using a protein extraction reagents kit (KeyGene, Nanjing, China). Protein concentrations were quantified by the Bradford method. Equal amounts of protein were separated by 10% SDS-polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked with a solution containing 5% fat-free milk powder at room temperature for 2 h, and then incubated with anti-TGF-β type II receptor (1:1000 dilution; Cell Signaling, Danvers, MA, USA), anti-E-cadherin (1:1000 dilution; Cell Signaling, Danvers, MA, USA), or anti-α-smooth muscle actin (αSMA, 1:500 dilution; Epitomics, Burlingame, CA, USA) antibodies at 4 1C overnight. After six washes for 10 min each in phosphate buffer containing 0.1% Tween 20 (PBST), the membrane was incubated with the secondary antibody (1:5000 dilution; Abmart, Shanghai, China) for 2 h at room temperature. After six washes with PBST, the signals were detected using ECL chemiluminescence reagent. Quantification was performed by measurement of the intensity of the signals with BandScan image-analysis software. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. 2.6. Statistical analysis All data are presented as the mean 7standard error and analyzed using SPSS version 18.0 for Windows (SPSS Inc.). Multiple groups of values were compared by one way-ANOVA and followed by post-test. The P-values of less than 0.05 were considered statistically significant.

3. Results

2.3. Isolation of RNA and real-time PCR

3.1. miRNA expression profiles in TGF-β1-induced and Sal B-treated EMT of HK-2 cells

To detect the expression levels of mature miR-25, miR-93, and miR-106b, total RNA was extracted from the variously treated HK-2 cells using TRIzol reagent, and then reverse transcribed using a TaqMan microRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) following the manufacture's recommendations. The resultant cDNA was amplified by TaqMan microRNA assays with TaqMan Universal PCR master mix (Applied Biosystems). PCR was performed in a total volume of 10 μl containing 0.5 μl diluted cDNA, TaqMan 2  PCR MasterMix, and the TaqMan probe. PCR conditions were 30 s at 95 1C, 30 s at 58 1C, and 40 s at 72 1C for the appropriate number of cycles. All reactions were run in triplicate in a StepOne plus RT-PCR system (Applied Biosystems). Data analysis was performed with the StepOne software package (Applied Biosystems). The relative quantities of mature miRNAs were calculated using the comparative CT method, and all data were normalized to U6 expression.

HK-2 cells showed a typical myofibroblast phenotype induced by TGF-β1 treatment for 48 h, whereas they exhibited restoration of their epithelial morphology after simultaneous treatment with Sal B in a dose-dependent manner (Fig. 1A). Actually α-SMA and E-cadherin immunocytochemistry were made in our previous experiment (Yao et al., 2009), and reduction in α-SMA and increase in E-cadherin in experiment with Sal B were observed; the results consisted with the cell morphology change. Using the Exiqon miRNA microarray, altered expression of miRNAs was found in the various groups of HK-2 cells (Fig. 1B). Compared with the vehicle group, 64 miRNAs were significantly up-regulated in HK-2 cells with EMT inducted by TGF-β1. Among them, expression of 11 miRNAs was significantly inhibited after Sal B treatment at both doses of 1 and 50 μmol/l (Fig. 1C). In contrast, expression of 19 miRNAs was significantly down-regulated following EMT in HK2 cells, and eight of them showed recovered expression at both

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Fig. 1. Upregulation of miRNA expression in HK-2 cells undergoing EMT inducted by TGF-β1 and downregulation by Sal B. (A) Epithelial morphological conversion of HK-2 cells with TGF-β1-induced EMT by Sal B treatment. (B) Hierarchical cluster analysis of differentially expressed miRNAs: red, high expression; green, low expression. (C) miRNAs down-regulated by more than 2-fold in TGF-β1þ Sal B (1 μmol/l) (light purple and black) and TGF-β1þ Sal B (50 μmol/l) groups (black) among the upregulated miRNAs in the TGF-β1 group compared with that in the vehicle. (D) miRNAs up-regulated by more than 2-fold in TGF-β1 þSal B (1 μmol/l) (black) and TGF-β1 þSal B (50 μmol/l) groups (dark purple and black) among the downregulated miRNAs in the TGF-β1 group compared with that in the vehicle. (E) Bar graph depicting the significantly changed expression of miRNAs during EMT induced by TGF-β1 and prevented by Sal B treatment. The average fold changes were log2-transformed for the microarray analysis. miRNAs included in the graph were down- or up-regulated by more than 2-fold (values of less than  1 or more than 1 after log2 transformation).

doses of Sal B (Fig. 1D). The detailed information and fold changes of these 19 miRNAs are shown in Fig. 1E. 3.2. Expression of the miR-106b-25 cluster in EMT Most of the 19 miRNAs have been studied previously (Table 1). Interestingly, miR-106b, miR-93, and miR-25 are included in one cluster, named the miR-106b-25 cluster, and their expression was dramatically decreased during EMT induced by TGF-β1, whereas increases of expression were observed after Sal B treatment. Because of a similar nucleic acid structure, a cluster of miRNAs often demonstrate the same regulatory functions. For the miR106b-25 cluster, it has been intensively implicated as a prooncogenic cluster of miRNAs (Li et al., 2009; Hudson et al., 2013; Smith et al., 2012). However, an association has not demonstrated for the miR-106b-25 cluster in renal fibrosis. On the other hand,

the miR-106b-25 cluster is involved in a key pathway of cell proliferation and apoptosis, which is related to EMT in renal fibrosis (Petrocca et al., 2008). Expression of the miR-106b-25 cluster was confirmed by realtime PCR (Fig. 2). Consistent with miRNA microarray data, expression of miR-106b, miR-93, and miR-25 was significantly lower in the group with EMT induced by TGF-β1 than that in the vehicle. This effect was recovered by Sal B treatment in a dose-dependent manner, which retained the epithelial morphology. 3.3. miR-106b inhibits EMT induced by TGF-β1 Enhanced expression of miR-106b was induced by transfection of a miR-106b mimic to investigate whether miR-106b in HK-2 cells affects TGF-β1-induced EMT. Previously, it has been shown that HK-2 cells exhibit a myofibroblast phenotype induced by

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Table 1 miRNA expression in Sal B-inhibited EMT of HK-2 cells. miRNAs with altered expression, including their target mRNAs and pathways are shown. miRNA

Target mRNA reported

Function and pathway

Reference

Up-regulate in TGF-β1 group while down-regulate in Sal B prevention group 1 hsa-miR-493n PTEN Cell cycle progression and DNA recombination 2 hsa-miR-373n – – 3 hsa-miR-654-5p AR Cell growth and proliferation n 4 hsa-miR-92b – – 5 hsa-miR-564 E2F3/Akt2 mTOR pathway and VEGF pathway 6 hsa-miR-1200 NEIL2 DNA repair 7 hsa-miR-486-5p ARHGAP5 Cell proliferation and angiogenesis 8 hsa-miR-4292 C9orf86 Apoptosis 9 hsa-miR-588 ATXN1 (Ataxin1) Apoptosis 10 hsa-miR-548m PTGS2/PPM1H Cell growth and proliferation 11 hsa-miR-4313 CREB1 Cell cycle progression and apoptosis

(Yang et al., 2008) – (Ostling et al., 2011) – (Rokah et al., 2012) (Wei et al., 2012) (Wang et al., 2013) (Issabekova et al., 2011) (Roshan et al., 2009) (Khella et al., 2012; Wang et al., 2013) (An et al., 2012)

Down-regulate in TGF-β1 group while up-regulate in Sal B prevention group 1 hsa-miR-25 DR4/BCL2L11(Bim) Cell proliferation, differentiation, apoptosis and cell cycle 2 hsa-miR-106b P21/CASP7/ TβRII Cell proliferation and apoptosis 3 hsa-miR-196n – – 4 hsa-miR-3653 – – 5 hsa-miR-93 GLUT4/TβRII Insulin resistance, cell cycle progression, and proliferation 6 hsa-miR-19b NTN4/CUL5 Cell proliferation, adhesion, invasion and Wnt/β pathway 7 hsa-miR-34a P53/p21 apoptosis 8 hsa-miR-3618 DGCR8 Cell cycle progression and apoptosis

(Zhang et al., 2012; Razumilava et al., 2012) (Li et al., 2011; Hudson et al., 2012) – – (Li et al., 2011; Chen et al., 2013) (Qin et al., 2013; Xu et al., 2012) (Akao et al., 2011) (Godnic et al., 2013)

signaling and EMT in breast oncogenesis, we selected miR-106b as a vital candidate and further studied the association of miR-106b with TGF-β type II receptor in renal fibrosis. As shown in Fig. 4C and D, TGF-β1 induced expression of TGF-β type II receptor in HK2 cells undergoing EMT, whereas enhancement of miR-106b expression inhibited the expression of TGF-β type II receptor (P o0.05). These data suggest that the inhibitory effects of the miR-106b-25 cluster in TGF-β1-induced EMT of HK-2 cells may be due to the reduced activity of TGF-β signaling and the associated decrease in expression of TGF-β type II receptor. Fig. 2. Variations in miR-106b-25 cluster expression during TGF-β1-induced EMT and Sal B treatment. Real-time PCR analysis of mRNA expression levels in HK-2 cells with TGF-β1 treatment (PANOVA ¼ 0.041(miR-25), 0.017(miR-93), 0.002(miR-106b); n Po 0.05 vs. vehicle, n¼ 3) or Sal B treatment (PANOVA ¼ 0.0001(miR-25), 0.000(miR93), 0.000(miR-106b); nPo 0.05 vs. TGF-β1 group, n¼ 3).

TGF-β1 (Yao et al., 2009). As shown in Fig. 3A, enhancing miR-106b expression prevented the change to myofibroblast phenotype and obviously restained the epithelial morphology of HK-2 cells when simultaneously treated with TGF-β1. In kidney tissues and HK-2 cells, E-cadherin is the epithelial marker and α-smooth muscle actin (α-SMA) is the mesenchymal marker; loss of E-cadherin and acquisition of α-SMA are key features of EMT induced by TGF-β1 (Yao et al., 2009). Compared with the TGF-β1 group, HK-2 cells with enhanced expression of miR-106b showed a dramatic decrease in the protein level of α-SMA and notable upregulation of the E-cadherin protein level (P o0.05; Fig. 3B & C). Thus, miR106b attenuates EMT inducted by TGF-β1 in HK-2 cells. 3.4. miR-106b reduces TGF-β type II receptor expression during EMT Consistent with published data (Brett et al., 2011; Gupta et al., 2012), three members of the miR-106b-25 cluster showed similar seed sequences (Fig. 4A). The Targetscan database revealed that TGF-β type II receptor might be a target of miR-106b and miR-93. The 30 UTR of TGF-β type II receptor mRNA is predicted to harbor the same putative binding sites for both these miRNAs (Fig. 4B). The inhibitory effect of miR-106b and miR-93 on TGF-β type II receptor expression has been reported in SH-SY5Y cells and induced pluripotent stem cells (Wang et al., 2010; Li et al., 2011). Because TGF-β type II receptor is crucial for activation of TGF-β

4. Discussion It is clear that the well-described phenomenon of EMT plays a pivotal role in renal fibrosis. There is currently no definitive treatment for renal fibrosis, which reflects the incomplete understanding of the molecular pathogenesis of this disease. miRNAs have emerged as crucial players in many pathophysiological processes and participate in the progression of EMT. Recently, the miR-200 family, miR-192, and miR-29 have been reported to be involved in the EMT of renal fibrosis (Krupa et al., 2010; Chandrasekaran et al., 2012). In the present study, we performed miRNA expression profiling in HK-2 cells, and found a unique miRNA cluster signature associated with EMT. Induction of EMT in HK-2 cells by TGF-β1 in vitro is a classic pathological model employed in studies on renal fibrosis. Previously, we have reported that Sal B exerts significant inhibitory effects on TGF-β1-induced EMT of HK-2 cells (Yao et al., 2009). Here, we found that a cohort of miRNAs demonstrated altered expression in response to EMT induction by TGF-β1 stimulation. More importantly, these miRNAs showed a reversal of the altered expression by Sal B treatment and consistent variations in expression at different concentrations. Among the miRNAs, expression of the miR-106b-25 cluster, including miR-106b, miR-93, and miR-25, was inhibited by TGF-β1 and restored by Sal B. Therefore, the members of the miR-106b-25 cluster may participate in EMT involved in renal fibrosis, which can be prevented by Sal B. Most miRNAs are encoded in intergenic regions, but some miRNAs are hosted within introns of premRNAs or encoded within ncRNA genes. Interestingly, there is clustering of both hosted and non-hosted miRNA genes. In fact, the clustering propensity of

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Fig. 3. Effect of miR-106b on EMT induced by TGF-β1 in HK-2 cells. (A) HK-2 cells were transfected with 80 nM negative control (N.C.) or miR-106b mimic for 6 h, and then stimulated with TGF-β1 for other 48 h. Epithelial phenotypic conversion was observed in the vehicle, TGF-β1þ N.C., and TGF-β1 þmiR-106b mimic groups. (B) E-cadherin and α-SMA protein levels were detected by western blot analysis. C) Graphical representation of relative α-SMA normalized to GAPDH (PANOVA ¼ 0.005; nPo 0.05 vs. vehicle, n¼ 3) (PANOVA ¼ 0.003; nPo 0.05 vs. TGF-β1 þmiR-106b, n¼ 3) and E-cadherin levels (PANOVA ¼0.000; nPo 0.05 vs. vehicle, n ¼3) (PANOVA ¼0.000; n P o0.05 vs. TGF-β1þ miR106b, n¼ 3).

miRNA genes was observed in the initial direct cloning of short ncRNAs. Usually, there are between two to three miRNA genes in a cluster. Clustered miRNA genes may show high sequence similarities, but they can also differ. Accumulating evidence suggests that clustered miRNAs are transcribed as polycistrons and have similar expression patterns and functions (Altuvia et al., 2005; Kan et al., 2009). The miR-106b-25 cluster is highly conserved in vertebrates. The three members, miR-106b, miR-93, and miR-25, are located in a 515 bp region on chromosome 7q22 in intron 13 of the host gene MCM7, where they are co-transcribed with the MCM7 primary transcript (Smith et al., 2012; Brett et al., 2011). miR-106b and miR-93 have the same seed sequence, while miR-25 has a similar seed sequence. Consequently, miR-106b and miR-93 have been predicted to bind to the same target mRNAs including TGF-β type II receptor (Li et al., 2011). Moreover, the miR-106b-25 cluster has been suggested to be a potential oncogenic cluster that is accumulated in various types of cancer including gastric, prostate, pancreatic neuroendocrine tumors, neuroblastoma, and multiple myeloma (Li et al., 2009; Hudson et al., 2013; Brett et al., 2011; Pichiorri et al., 2008). Our results confirmed co-regulation of the expression of miRNAs in a cluster, and that they play a role in a common molecular process. Furthermore, in addition to oncogenesis, the miR-106b-25 cluster might be responsible for EMT in renal fibrosis. TGF-β signaling has been reported as the link between the miR106b-25 cluster and tumor proliferation (Petrocca et al., 2008). Li

et al. (2009) identified overexpression of the miR-106b-25 cluster in gastrointestinal cancer cells, and suggested that the miR-106b25 cluster is a key modulator of TGF-β signaling, which interferes with cell cycle arrest, apoptosis, and tumor development. Smith et al. (2012) confirmed an increase of miR-106b-25 cluster expression in TGF-β signaling activation involved in human breast cancer. On the other hand, TGF-β1 is widely accepted as a fibrogenic cytokine. As shown in previous studies, TGF-β signaling plays an important role in regulating EMT and renal fibrosis (Lan and Chung, 2012; Meng et al., 2012). Therefore, we hypothesized that the miR-106b-25 cluster might modulate TGF-β signaling in the EMT of HK-2 cells. Further studies using a miR-106b mimic would be required to confirm such a hypothesis. Considering that enhanced expression of miR-106b showed beneficial effects on attenuating EMT in vitro, our findings may shed new light on its role in renal fibrosis. In addition, targetscan database screening revealed that TGF-β type II receptor might be a target mRNA of the miR-106b-25 cluster. It has been reported that miR-106b directly inhibits translation of TGF-β type II receptor in Alzheimer’s disease (Wang et al., 2010). A study of induced pluripotent stem cells has established that miR-106b and miR-93 can induce characteristics of mesenchymal-to-epithelial transition, resulting in suppression of EMT-like changes by directly targeting TGF-β type II receptor (Li et al., 2011). Of note, we found that enforced stable expression of miR-106b resulted in a remarkable decrease of TGFβ type II receptor protein in HK-2 cells exposed to TGF-β1.

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Fig. 4. Effect of miR-106b on TGF-β type II receptor protein expression during TGF-β1-induced EMT. (A) Sequence comparison of miR-106b-25 cluster members. Red indicates similar seed sequences. (B) Two putative binding sites for miR-106b and miR-93 in the 30 UTR of TβRII mRNA in the Targetscan database. (C) Western blot analysis of TGF-β type II receptor protein in HK-2 cells treated with 5% FCS, TGF-β1, or TGF-β1 plus miR-106b by transfection of the mimic. (D) Graphical representation of relative TGF-β type II receptor levels normalized to GAPDH (PANOVA ¼0.005; nP o 0.05 vs. vehicle, n¼ 3) (PANOVA ¼ 0.011; nP o 0.05 vs. TGF-β1þ miR-106b, n¼ 3).

However, as the members of this cluster, the detection of miR-93 and miR-25 may be also essential in further investigation, through which we could confirm their critical role in EMT of renal fibrosis. Disruption of TGF-β type II receptor has recently been demonstrated to inhibit renal fibrosis through impairing TGF-β signaling (Meng et al., 2012). Therefore, deciphering the target molecules of the miR-106b-25 cluster in EMT is essential, and we will elucidate them in future studies. In summary, the present study provides evidence that the miR106b-25 cluster might play a vital role in the pathogenesis of renal fibrosis. We found that expression of these miRNAs during TGFβ1-induced EMT was inhibited by Sal B in vitro. Moreover, we found miR-106b attenuates EMT in renal fibrosis via inhibitory effects on TGF-β type II receptor expression and the TGF-β signaling pathway. However, it should be noted that the efficiency of the miR-106b-25 cluster to inhibit EMT was assessed in an in vitro model. Evidence obtained in vivo would be needed to validate targeting of these miRNAs, simultaneously or sequentially, for treatment of renal fibrosis.

Acknowledgments This study was supported by Scientific Research Foundation of Health Department of Jiangsu Province (Grant no. Z201204), Grant of Jiangs”Six Major Talent Summit” (Grant no. WSN-071) and National Natural Science Foundation of China (Grant no. 81070579).

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