MicroRNA21 promotes interstitial fibrosis via targeting DDAH1:a potential role in renal fibrosis

Xiu-Juan Liu1 • Quan Hong2 • Zhen Wang1 •

Yan-yan Yu1 • Xin Zou1 • Li-hong Xu1

Received: 15 August 2015 / Accepted: 26 September 2015 / Published online: 12 October 2015

� Springer Science+Business Media New York 2015

Abstract Scarring of the kidney directly promotes loss of

kidney function. A thorough understanding of renal fibrosis

at the molecular level is urgently needed. One prominent

microRNA, miR-21, was previously reported to be up-

regulated in renal fibrosis, but its mechanism is unclear. In

the present study, an unbiased search for downstream

messenger RNA targets of miR-21 using the HK-2 human

tubular epithelial cell line was performed. Effects of the

target gene in renal fibrosis and underlying mechanism

were explored. Results show that forced expression of

miR-21 significantly increased cell apoptosis, interstitial

deposition, and decreased E-cadherin level of the HK-2

cells. Conversely, inhibition of miR-21 promoted the

opposite effects. We identified that miR-21 directly inter-

acted with the 30-untranslated region of the suppressor of

dimethylarginine dimethylaminohydrolase 1 (DDAH1) by

dual-luciferase assay. Moreover, pcDNA3.1-DDAH1 pre-

treatment could effectively reduce a-SMA, collagen I,

fibronectin expression, and promoted E-cadherin expres-

sion, as well as inhibiting HK-2 cell apoptosis, while all

those effects can be attenuated by pretreatment with the

Wnt/b-catenin signaling activator Licl. Taken together, our

results suggest that miR-21 may regulate renal fibrosis by

the Wnt pathway via directly targeting DDAH1. Therefore,

this study may provide novel strategies for the develop-

ment of renal fibrosis therapy.

Keywords MiR-21 � DDAH1 � Interstitial deposition �Renal fibrosis � Wnt/b-catenin

Abbreviations

DDAH1 Dimethylarginine dimethylaminohydrolase 1

CKD Chronic kidney disease

ESRD End-stage renal disease

miRs microRNAs

RISC RNA-induced silencing complex

UUO Unilateral ureteral obstruction

ADMA Asymmetric dimethylarginine

HK-2 Human tubular epithelial cells

Introduction

Renal fibrosis is the principal pathological process under-

lying the progression of chronic kidney disease (CKD) and

finally leads to end-stage renal disease (ESRD) [1]. The

only possible treatment for patients with ESRD is lifelong

dialysis or a kidney transplant. As a common feature of

CKD, renal interstitial fibrosis, which is involved in vir-

tually all renal cells, is the strongest indicator of disease

progression. Therefore, delay and prevention of renal

fibrosis is a potential target for CKD treatment.

MicroRNAs (miRs) are a family of small, non-coding,

single-stranded RNA molecules of approximately 18–25

nucleotides in length. Nascent miRs can associate with the

Xiu-Juan Liu and Quan Hong contributed equally to this work.

& Xiu-Juan Liu

xiujuan_liu796@163.com

1 Department of Nephrology, the 94th Hospital of Chinese

People’s Liberation Army, Changcheng Hospital affiliated to

Nanchang University, Jinggangshan Road 1028,

Nanchang 330002, Jiangxi, People’s Republic of China

2 Chinese PLA General Hospital, Chinese PLA Institute of

Nephrology, State Key Laboratory of Kidney Diseases,

National Clinical Research Center for Kidney Diseases,

Beijing 100039, People’s Republic of China

123

Mol Cell Biochem (2016) 411:181–189

DOI 10.1007/s11010-015-2580-2

RNA-induced silencing complex (RISC) after synthesis,

then bind to target mRNAs, and can inhibit translation or

destabilize transcripts [2]. Specific miRNAs regulate bio-

logic processes and are involved in a myriad of pathologic

states, including cancer, cardiovascular diseases, and also

kidney function and CKD [3]. Among previously reported

miRs, miR-21 is one of the anti-apoptotic factors in a

variety of solid tumors and is related to embryonic stem

cell self-renewal capacity [4, 5]. In recent years, several

reports have demonstrated that miR-21 was involved in

TGF-b-mediated renal fibrosis, and miR-21 levels were

positively correlated with the degree of fibrosis [2]. Addi-

tional work confirmed that in a unilateral ureteral

obstruction (UUO) rat model, TGF-b1 up-regulated the

miR-21 expression in renal tubular epithelial cells by

Smad3 signaling, significantly increasing the collagen I,

fibronectin, and a-SMA protein level, resulting in renal

fibrosis [6], indicating a promotion effect of miR -21 in

renal fibrosis. MiR-21 overexpression may promoted

fibrosis, while previous studies have shown that miR-21

silencing could promote apoptosis in tumor cells [7, 8],

whether the down-regulated miR-21 expression also

influence the normal renal cell apoptosis? Therefore, fur-

ther research is required to fully understand the mechanism

of miR-21 in renal fibrosis.

DDAH1 belongs to the dimethylarginine dimethy-

laminohydrolase (DDAH) gene family. The translated

protein, DDAH1, plays a role in nitric oxide generation by

regulating cellular concentrations of asymmetric dimethy-

larginine (ADMA), which in turn inhibits nitric oxide

synthase activity. Numerous studies have shown that

ADMA accumulation decreased NO production, resulting

in peritubular capillary loss, tubule interstitial ischemia,

chronic hypoxia, and renal fibrosis [9–11].

In the present study, we aimed to investigate whether

DDAH1 was a putative target of miR-21 in human tubular

epithelial cells (HK-2). The functional effect of DDAH1 in

miR-21-mediated renal fibrosis was assessed by measuring

cell apoptosis and a-SMA, E-cadherin, collagen I, and

fibronectin protein levels. Furthermore, the underlying

mechanism was also analyzed.

Materials and methods

Antibodies and reagents

All substances were purchased from Gibco (Grand Island,

NY), if not otherwise mentioned. Dulbecco’s modified

Eagle’s medium (DMEM) was purchased from Abcam

(Cambridge, MA). Licl (Wnt/b-catenin signaling pathway

activator) was from Calbiochem (San Diego, CA). Rabbit

anti-collagen I, fibronectin, a-SMA, and TGF-b polyclonal

antibodies were from Santa Cruz Biotechnology (Santa

Cruz, CA). b-actin antibodies were obtained from Upstate

Biotechnology, Inc. (Lake Placid, NY). Anti-b-cateninantibodies were bought from Sigma (St. Louis, MO). The

monoclonal antibody against endothelial cadherin (E-cad-

herin) was obtained from Cell Signaling Technology

(Beverly, MA). Horseradish peroxidase (HRP)-conjugated

goat anti-mouse and anti-rabbit antibodies were from

Abcam (Cambridge, MA).

Cell lines

The present study used the HK-2 human tubular epithelial

cell line and HEK293 cell line, which were obtained from

the American Type Culture Collection (ATCC, Manassas,

VA) and cultured according to ATCC protocols. Both cell

lines were maintained in DMEM supplemented with 10 %

fetal bovine serum, 2 mM L-glutamate, 100 U/ml peni-

cillin, and 100 lg/ml streptomycin. All cells were cultured

at 37 �C in a 5 % CO2 incubator (Life Technologies,

Baltimore, MD).

In silico prediction of miR-21 targets

Three algorithms were used to computationally predict

targets of differentially expressed miRNAs, including

microRNA.org (http://www.microrna.org), TargetScan 5.1

(http://www.targetscan.org), and the EIMMomiRNA target

prediction server (http://www.mirz.unibas.ch/ElMMo3).

Quantitative real-time polymerase chain reaction

(qRT-PCR) for miR and mRNA expression

We performed qRP-PCR to confirm the expression of miR-

21 and DDAH1 mRNA. The RNA of HK-2 cells was

extracted using Unizol Reagent according to the manu-

facturer’s instructions (Biostar, Shanghai, China). About

5 lg of total RNA for each sample was reverse transcribed

into first-strand cDNA for qRT-PCR analysis. The qRT-

PCR was performed in a final volume of 10 ll, which

contained 5 ll of SsoFastTMEvaGreenSupermix (Bio-Rad,

Hercules, CA, USA), 1 astcDNA (1:50 dilution), and 2 s

reverse transcribed into reverse primers (1 mM). b-actinwas used as quantity and quality control to normalize the

gene expression. Data were analyzed using the formula:

R ¼ 2�½ta sample�DCt control� (R: relative expression level; DCt

sample: the difference between the Ct of the gene and the

average b-actin; DCt control: the difference between the Ct

of the gene and the average of the control samples). b-actinand U6 SnRNA were used as internal controls form RNA

and miRNA, respectively. PCR reactions were carried out

in duplicate.

182 Mol Cell Biochem (2016) 411:181–189

123

Luciferase reporter assay

A truncated fragment of the DDAH1 30-untranslated region

(30-UTR) containing the miR-21 predicted binding site

(pGLO-DDAH1-BS) was cloned into the 30-UTR of the

firefly luciferase structural gene in the pmirGLO Dual-

Luciferase miRNA Target Expression Vector (Promega,

Madison, WI). We also constructed separate plasmids

containing the DDAH130-UTR with a mutated seed region

for the predicted miR-21 binding site (pGLO-DDAH1-

Mut) as the negative control. Twenty-four hours before

transfection, HEK293 cells were seeded into a 24-well

plate (8 9 104 cells/well). Cells were co-transfected with

100 ng of the respective plasmid and 100 nmol/l miR-21 or

control mimic using Lipofectamine 2000 (Invitrogen,

Carlsbad, CA). Forty-eight hours after plasmid vector

transfection, the luciferase reporter assay was performed

using a Dual-GLO luciferase assay kit (Promega). Data are

presented as the ratio of firefly to renilla luciferase activity.

Oligonucleotide and siRNA transfection

Cells were seeded into 6-well plates, transfected with miR-

21 mimics, miR-21 inhibitors, or miR controls (50 nM,

GenePharma) using Lipofectamine, and then harvested for

assays 48 h later. DDAH1siRNA (Sigma-Aldrich) trans-

fection was performed according to the manufacture’s

instruction. Briefly, 1 lg of vector was incubated with

50 ll of serum-free medium for 5 min (Solution A), and

2 ll of Lipofectamine 2000 was incubated with serum-free

medium for 5 min (Solution B). Solution A was mixed

with solution B, and incubated for 20 min. After incuba-

tion, HK-2 cells were added to the mixture. Cells were then

harvested at 48 h after transfection for further analysis. The

efficiency of DDAH1siRNA was confirmed by Western

blotting analysis.

Cells apoptosis by flow cytometry (FCM)

Cell apoptosis was analyzed using flowcytometric analysis.

Briefly, 24 h after transient transfection, HK-2 cells were

trypsinized, washed with PBS, and suspended with binding

buffer. Cells were incubated with 10 ll annexin V-FITC

and 5 ll propidium iodide (PI) (Sigma) for 15 min at room

temperature in the dark before being suspended in 300 llbinding buffer. The cells were then analyzed on a FAC

Scan flow cytometer (Becton–Dickinson, NJ) for relative

quantitative apoptosis.

Western blotting analysis

Cells were homogenized and lysed with RIPA lysis buffer

(100 mM NaCl, 50 mM Tris–HCl pH 7.5, 1 % TritonX-

100, 1 mM EDTA, 10 mM b-glycerophosphate, 2 mM

sodium vanadate, and protease inhibitor). Protein concen-

tration was assayed using the micro-BCA protein assay

(Pierce, Rockford, IL). Protein (40 lg/lane) was separatedby 12 % SDS-PAGE, and electroblotted onto nitrocellulose

(Amersham Pharmacia, Germany). Non-specific binding

was blocked by incubating with 5 % non-fat milk in TBST

buffer at room temperature for 1 h. The polyvinylidened-

ifluoride (PVDF) membrane was incubated with anti-TGF-

b, anti-collagen I, anti-fibronectin, anti-a-SMA, anti-b-catenin, anti-E-cadherin, or anti-b-actin antibodies for 1 h

at room temperature. Following three washes with Tris

buffer saline Tween-20 buffer, HRP-conjugated secondary

antibodies were used, and enhanced chemiluminescence

(ECL, Amersham Pharmacia, NJ, USA) was used for

detection.

DDAH1 plasmid construction and cell transfection

Total RNA was isolated from HK-2 cells using TRIzol

reagent (Invitrogen, Carlsbad, CA, USA), based on the

manufacturer’s instructions. The cDNA was synthesized by

reverse transcription of total RNA, using the PrimeScript

RT reagent kit (Takara, Dalian, China) with oligo-dT pri-

mers, according to the manufacturer’s protocol. Then, the

open reading frame of DDAH1 cDNA was cloned and

inserted into the pcDNA3.1 vector (Invitrogen, Carlsbad,

CA), namely pcDNA3.1-DDAH1 expression vector, The

pcDNA3.1 vector-alone transfected cells were used as

negative control. For cell transfection, cells were cultured

to 60 % confluence, and cell transfection was performed

using the FuGENE HD transfection reagent (Roche, Indi-

anapolis, IN) methods as suggested by the manufacturer.

Data analysis

The results are presented as the mean ± standard deviation

(SD). Statistical analyses were performed with a paired or

an unpaired Student’s t test for the direct two-group com-

parisons, and the Tukey–Kramer test after a significant

one-way analysis of variance F test for the multiple-group

comparisons. Differences were considered statistically

significant at p\ 0.05.

Results

miR-21 regulates apoptosis and interstitial

deposition in HK-2 cells

To explore the effect of miR-21 on HK-2 cell apoptosis and

interstitial deposition, HK-2 cells were transiently trans-

fected with miR-21 mimics, miR-21 inhibitor, or miR

Mol Cell Biochem (2016) 411:181–189 183

123

controls. qRT-PCR indicated that miR-21 mimics and

inhibitor were successfully transfected (Fig. 1a). Since

previous findings report that miR-21 is involved in TGF-b-mediated renal fibrosis, in order to figure out whether miR-

21 mimic or inhibitor transfection affected TGF-bexpression, the protein levels of TGF-b were detected by

Western blotting (Fig. 1b), and results show that there was

no effect of miR-21 mimic or inhibitor transfection on the

TGF-b levels. The FCM assay demonstrated that the

apoptosis of cells transfected with miR-21 inhibitors was

much lower than those transfected with miR controls

(10.5 ± 1.5 vs. 17.1 ± 1.3 %), while cells transfected with

miR-21 mimics was much higher than the miR controls

(27.3 ± 2.7 vs. 16.9 ± 1.6 %) (Fig. 1c, p\ 0.05).

The qRT-PCR assay of interstitial deposition-related

genes showed that compared with the control group, the

mRNA levels of a-SMA, collagen I, and fibronectin were

increased upon transfection with miR-21 mimics, while

E-cadherin mRNA expression was significantly reduced

(p\ 0.05). Indeed, transfection of miR-21 mimics into

HK-2 cells induced an approximately fourfold increase in

the level of a-SMA mRNA, a fivefold increase in the level

of collagen I mRNA, and a fourfold increase in fibronectin

mRNA (Fig. 1d). The a-SMA, collagen I, and fibronectin

protein levels were also increased upon miR-21 overex-

pression in accordance with the mRNA data (Fig. 1e).

Conversely, inhibition of miR-21 displayed the opposite

effects. After transfection with the miR-21 inhibitor, both

the mRNA and protein levels of a-SMA, collagen I, and

fibronectin were down-regulated, but there was an increase

in E-cadherin.

MiR-21 targets the DDAH1 30-UTR

To identify novel targets for miR-21 that could exert a

biological impact on renal fibrosis, we employed three

search engines (microRNA.org, TargetScan, and the

EIMMomiRNA target prediction server) for the in silico

search for miR-21 targets. Among the miR-21 target gene

transcripts found in all the programs, DDAH1 was the

highest-ranked gene, and the binding of miR-21 with the

30-UTR is shown in Fig. 2a. We therefore selected DDAH1

for further analyses.

In order to assess whether DDAH1 was a direct target of

miR-21, as predicted by bioinformatic analysis, we per-

formed two experiments. Depending on complementary

base pairing, binding of miRNA to the 30-UTR of the target

mRNA may result in transcript degradation or translational

repression. In HK-2 cells, when cells were transiently

transfected with the miR-21 mimic, real-time PCR showed a

65 % reduction in DDAH1 mRNA versus cells transfected

with the miRNA mimic-negative control (nc), and when

cells were transfected with the miR-21 inhibitor, a 40 %

increase in DDAH1 mRNA versus the miRNA inhibitor- nc

group was seen. (Figure 2b). Furthermore, the expression of

DDAH1 protein was also decreased, as detected by Western

blotting (Fig. 2c). Next, we used the pmiR GLO Dual-Lu-

ciferase miRNA target expression vector to clarify the effect

of miR-21 on DDAH1 by inserting the miR-21 binding site

(pGLO-DDAH1-BS) in the 30-UTR of the luciferase gene.

Cells were also transfected with an expression plasmid

containing the same DDAH1 30-UTR fragment with a

mutated miR-21 binding site. An approximately 40 %

reduction of luciferase activity in HEK293 cells transfected

with pGLO-DDAH1-BS plasmid and the miR-21 mimic

was observed compared with miR-21 mimic-nc-transfected

cells. However, mutation of the target site prevented down-

regulation of luciferase activity by miR-21 (Fig. 2d).

Therefore, these results demonstrate that DDAH1 is a direct

target of miR-21.

Silencing DDAH1 expression contributes to HK-2

cell apoptosis and interstitial deposition

To determine whether DDAH1 silence influences HK-2 cell

apoptosis and interstitial deposition, we transiently trans-

fected HK-2 cells with DDAH1siRNA. Flow cytometric

analysis showed that DDAH1siRNA significantly increased

cell apoptosis compared with the control group (Fig. 3a).

RT-PCR showed that DDAH1siRNA significantly promoted

the mRNA levels of a-SMA, collagen I, and fibronectin, and

suppressed E-cadherin mRNA expression; protein levels

showed a similar trend in treatment groups compared with

the control group (Fig. 3b, c). Thus, we speculated that miR-

21 regulated HK-2 cells apoptosis and interstitial deposition

by suppressing DDAH1 expression.

DDAH1 inhibits HK-2 cells apoptosis and interstitial

deposition by suppressing the Wnt/b-cateninsignaling activation

Reports showed that activation of the Wnt/b-catenin sig-

naling pathway was associated with enhanced renal fibrosis

[12]. To determine whether DDAH1 inhibited the renal

fibrosis process via the Wnt/b-catenin pathway, we first

transfected the HK-2 cells with pcDNA3.1-DDAH1.

cFig. 1 Effect of miR-21 on cell apoptosis and interstitial deposition

of HK-2 cells. MiR-21 expression after transfection with miR-21

mimic or inhibitor (a). The protein expression of TGF-b was analyzed

by Western blotting (b). Detection of miR-21 mimics and miR-21

inhibitor on cell apoptosis of HK-2 cells by FCM assay (c). Afterpretreatment with miR-21 mimics or miR-21 inhibitor, miRNA levels

of interstitial deposition-related genes (a-SMA, collagen I, and

fibronectin) and endothelial markers (E-cadherin) were analyzed by

qRT-PCR (d). Protein expression was detected by Western blotting

(e). Data are expressed as the mean ± SD, *Compared with

corresponding controls, p\ 0.05

184 Mol Cell Biochem (2016) 411:181–189

123

Mol Cell Biochem (2016) 411:181–189 185

123

Protein expression analysis showed that after transfection,

the DDAH1 level was significantly increased; meanwhile,

pcDNA3.1-DDAH1 pretreatment led to an decrease in the

protein levels of b-catenin compared with the control

(Fig. 4a), suggesting that DDAH1 may inhibit the activa-

tion of the Wnt/b-catenin pathway. For further mechanism

analysis, the Wnt activator Licl was applied in the medium

for 24 h. Western blotting analysis showed that DDAH1

overexpression tended to decrease cleavage caspase-3

expression compared with the control, while the Licl pre-

treatment effectively promoted cleavage caspase-3 levels

(Fig. 4b), suggesting that DDAH1 may inhibit cell apop-

tosis which was restored by Wnt/b-catenin signaling acti-

vation. Further analysis of interstitial deposition showed

that DDAH1 overexpression significantly brought down

both the mRNA and the protein levels of a-SMA, collagen

I, fibronectin, as well as increased the expression of

E-cadherin compared with the control group. However,

compared with the pcDNA3.1-DDAH1 group, Licl pre-

treatment markedly increased mRNA and protein expres-

sion of a-SMA, collagen I, and fibronectin, and decreased

the E-cadherin levels (Fig. 4c, d). All of these results

indicate that DDAH1 extracellularly inhibited HK-2 cells

apoptosis and interstitial deposition by suppressing the

Wnt/b-catenin signaling activation.

Discussion

The prevalence of ESRD requiring chronic dialysis or renal

transplantation continues to increase [13]. ESRD is char-

acterized by progressive fibrosis leading to sclerosis, irre-

spective of its primary etiology [14]. Accordingly, the

extent of tubule interstitial fibrosis was found to be the best

predictor for kidney survival, irrespective of the underlying

disease [15]. Fibrosis is difficult to treat and is irreversible

in the more-advanced states. Thus, improved understanding

of renal fibrosis mechanisms at the gene and molecular

level is greatly needed. The current study provides insight

into a potential novel regulatory mechanism involved in

renal fibrosis. Our data suggest that miR-21, which is

known to be over-expressed in renal fibrosis, significantly

up-regulates apoptosis of HK-2 cells, as well as increasing

interstitial deposition-related genes a-SMA, type I colla-

gen, and fibronectin, and decreases endothelial cell marker

endothelial cadherin (E-cadherin), and we also identified

that miR-21 directly interacts with the 30-untranslatedregion of DDAH1, while DDAH1 effectively inhibits the

HK-2 cells apoptosis and interstitial deposition by sup-

pressing the Wnt/b-catenin signaling activation. These data

suggest that DDHA1 may be a novel and promising

downstream target of miR-21 for therapeutic intervention

in renal fibrosis and ESRD.

Recently, miRNAs have been shown to be important in

the maintenance of normal cellular function, and dysreg-

ulation of miRNAs can result in a myriad of pathological

states [16]. Several studies reported that miRNAs were

dysregulated in renal fibrosis [17, 18], and the dysregulated

miRNAs could regulate tubular epithelial cell growth,

apoptosis, and interstitial deposition [19, 20]. Our data

show that the administration of miR-21 mimics markedly

increased apoptosis of HK-2 cell lines, and enhanced

expression of interstitial deposition-related genes. How-

ever, transfection of a miR-21 inhibitor into HK-2 cells

Fig. 2 DDAH1 is a direct target gene of miR-21. The predicted

binding sequences for the DDAH1 3CSUTR with miR-21 (a). HK-2cells transiently transfected with miR-21 mimic/inhibitor showed a

significant reduction/increase in DDAH1 mRNA (b) and protein

(c) expression. Following transfection with a plasmid containing a

DDAH1 30-UTR binding site (pGLO-Prep-BS), or a mutated binding

site (pGLO-Prep-Mut), the luciferase activity was measured using the

dual-luciferase reporter assay (d). *compared with pGLO-Prep-BS

(miR-21 mimic-nc) p\ 0.05, #compared with pGLO-Prep-Mut (miR-

21 mimic) p\ 0.05

186 Mol Cell Biochem (2016) 411:181–189

123

decreased cell apoptosis, suppressed interstitial deposition

by down-regulating expression of the related genes, and

promoted the E-cadherin level.

Most miRNAs carry out their biological function by

binding to their target molecule [21]. However, how miR-

21 affects renal fibrosis is unclear. Thus, we used

bioinformatic tools (microRNA.org, EIMMo miRNA

target prediction, and TargetScan 5.1) to search for

potential targets for miR-21. Among miR-21 target gene

transcripts found in all programs, DDAH1 mRNA ranked

within the 10 top-ranked transcripts using microRNA.org

and may associated with the development of renal

fibrosis. Several studies in recent years have reported that

DDAH1 plays an important role in the catabolism of the

uremic toxin ADMA, and that the expression of DDAH1

was down-regulated in CKD patients [22–24], and cor-

related with ADMA aggregation. Studies have also

demonstrated that increased expression of ADMA was

associated with increased loss of peritubular capillaries,

tubule interstitial ischemia, chronic hypoxia, and renal

fibrosis [25, 26]. In this study, up-regulating miR-21

inhibited the mRNA and protein expression of DDAH1.

Furthermore, we identified DDAH1 as a direct target

gene of miR-21 using the dual-luciferase assay, indicating

that DDAH1 was a direct target of miR-21. Further

functional analysis showed that DDAH1 siRNA could

significantly up-regulate a-SMA, collagen I, and fibro-

nectin expression, and suppressed E-cadherin expression,

at both the mRNA and protein level. The results also

demonstrated that DDAH1 siRNA promoted HK-2 cell

apoptosis. Here, we also found other potential targets for

miR-21, such as THBS1, which has been reported to

affect the interstitial fibrosis in rats with unilateral uret-

eral obstruction [27], and GRHL2, PBRM1 which was

reported to be correlated with epithelial phenotype and

confirmed to take part in the clear cell renal cell carci-

noma progression [28, 29]. These genes will be further

explored in our future research.

Fig. 3 Silence DDAH1 expression contributes to HK-2 cell apoptosis

and interstitial deposition. After pretreatment with DDAH1 siRNA

for 48 h, cell apoptosis (a), mRNA (b), and protein (c) expression of

interstitial deposition-related genes (a-SMA, collagen I, and fibro-

nectin) and E-cadherin expression were analyzed by Western blotting

and qRT-PCR. *compared with control group p\ 0.05

Mol Cell Biochem (2016) 411:181–189 187

123

The Wnt/b-catenin signaling is an evolutionarily con-

served, highly complex, critical developmental pathway of

cell fate, organ development, and tissue homeostasis, as

well as injury and repair. Although silent in the normal

adult kidney, the Wnt/b-catenin signaling is re-activated

after renal injury in various animal models and human

kidney disease [30]. To clarify the mechanism underlying

the suppression effects of DDAH1 in cell apoptosis and

interstitial deposition, the expression of the Wnt/b-cateninsignaling was investigated. In accordance with our

hypothesis, pretreatment with pcDNA3.1-DDAH1

decreased b-catenin expression and suppressed Wnt/b-catenin signaling pathway activity. Further analysis of the

mechanism showed that pretreatment with Wnt signaling

activator Licl could significantly attenuate the inhibitory

effect on cell apoptosis and interstitial deposition-related

protein levels induced by DDAH1 up-regulation. These

results revealed that DDAH1 extracellularly triggered the

apoptosis and interstitial deposition of HK-2 cell by

inhibiting the Wnt/b-catenin signaling activity.

In conclusion, the data presented herein identified that

miR-21 functions as a promoter of renal fibrosis by

enhancing interstitial fibrosis and cell apoptosis. Moreover,

luciferase assays demonstrated direct interaction between

miR-21 and the DDAH1 30-UTR. Furthermore, mechanis-

tic analysis demonstrated that DDAH1 contributes to a

reduction in interstitial fibrosis and cell apoptosis via the

Wnt/b-catenin signaling pathway. Taken together, our data

suggest that miR-21 may regulate renal fibrosis by the Wnt

pathway via directly targeting DDAH1. Therefore, this

study may provide novel strategies for the development of

renal fibrosis therapy.

Acknowledgments This work was supported by grants from the

Natural Science Foundation of China (No. 81260114) and the Natural

Science Foundation Project of Jiangxi Province (No.

20142BAB205007).

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict

of interests.

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