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Author’s Accepted Manuscript
miR-133b and miR-199b knockdown attenuate
TGF-β1-induced epithelial to mesenchymal
transition and renal fibrosis by targeting SIRT1 in
diabetic nephropathy
Zhiqiang Sun, Yali Ma, Fang Chen, Shiying Wang,
Baoping Chen, Jun Shi
www.elsevier.com/locate/ejphar
PII:
DOI:
Reference:
S0014-2999(18)30467-9
https://doi.org/10.1016/j.ejphar.2018.08.022
EJP71947
To appear in: European Journal of Pharmacology
Received date: 10 July 2018
Revised date: 11 August 2018
Accepted date: 15 August 2018
Cite this article as: Zhiqiang Sun, Yali Ma, Fang Chen, Shiying Wang, Baoping
Chen and Jun Shi, miR-133b and miR-199b knockdown attenuate TGF-β1induced epithelial to mesenchymal transition and renal fibrosis by targeting
SIRT1 in diabetic nephropathy, European Journal of Pharmacology,
https://doi.org/10.1016/j.ejphar.2018.08.022
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miR-133b and miR-199b knockdown attenuate TGF-β1-induced epithelial to
mesenchymal transition and renal fibrosis by targeting SIRT1 in diabetic
nephropathy
Zhiqiang Sun1, Yali Ma1, Fang Chen, Shiying Wang, Baoping Chen, Jun Shi*
Department of Nephrology, Huaihe Hospital of Henan University, Kaifeng, 475000,
China
*Corresponding Author: Department of Nephrology, Huaihe Hospital of Henan
University, No. 357 Ximen Street, Kaifeng, 475000, China
Tel: +86-
0371-23906586, Email: mkokohhnui@sina.com
ABSTRACT
Transforming growth factor-β1 (TGF-β1)-induced epithelial to mesenchymal
transition (EMT) and renal fibrosis plays critical role in the development and
progression of diabetic nephropathy (DN). Our study aimed to determine the detailed
roles of miR-133b &miR-199b on TGF-β1-induced EMT &renal fibrosis in DN and
its underlying mechanism. The expressions of miR-133b &miR-199b in OLETF rats,
LETO rats&TGF-β1-treated human proximal tubule cell line (HK-2) were examined
by qRT-PCR. Inhibition of miR-133b or miR-199b was realized in cells by
transfection of lentivirus containing miR-133b inhibit or miR-199b inhibitor. The
expression levels of collagen I (COL I, fibronectin (FN), α-smooth muscle actin
(α-SMA), E-cadherin & sirtuin 1 (SIRT1) were detected by western blot and
immunohistochemistry. Masson staining was conducted to estimate the degree of
renal fibrosis. The interaction between SIRT1 and miR-133b, miR-199b was explored
by luciferase reporter assay and RNA immunoprecipitation (RIP) assay.miR-133b and
miR-199b were highly expressed in the renal cortex of diabetic OLETF rats and
TGF-β1-treated HK-2 cells. EMT and renal fibrosis were induced in diabetic OLETF
rats and TGF-β1-treated HK-2 cells. Inhibition of miR-133b and miR-199b attenuated
EMT and renal fibrosis in diabetic OLETF rats and TGF-β1-treated HK-2 cells. In
addition, SIRT1 was identified as a target of miR-133b &miR-199b in HK-2 cells.
1
These authors contributed equally to this work
SIRT1 knockdown dramatically reversed the suppression on TGF-β1-induced EMT
and renal fibrosis in HK-2 cells mediated by anti-miR-133b or
anti-miR-199.Inhibition of miR-133b &miR-199b attenuated TGF-β1-induced EMT
&renal fibrosis by upregulating SIRT1 shows that using different miRNAs is a
potential strategy for the future treatment of DN.
Key words: miR-133b, miR-199b, TGF-β1, EMT, renal fibrosis, diabetic
nephropathy
1. Introduction
Diabetic nephropathy (DN), one of severe microvascular complications of
diabetes mellitus (DM), is the leading cause of end stage renal disease (ESRD), which
accounts for high morbidity and mortality in diabetic patients (Liu, 2013). It is
well-established that progressive renal fibrosis is one of the hallmark pathological
characteristics of DN (Song et al., 2013). The degree of renal fibrosis is regarded as a
crucial indicator to reflect the deterioration of kidney function, which is mainly
attributed to accumulation of glomerular extracellular matrix (ECM) and mesangial
expansion (Wynn, 2007). Accumulating evidence highlights the critical roles of
epithelial to mesenchymal transition (EMT) in the development of DN (Liu, 2004).
EMT is a reversible process during which the tubular epithelial cells undergo
cytoskeletal remodeling and acquire mesenchymal phenotypes, accompanied by the
loss of epithelial markers such as E-cadherin and the acquisition of mesenchymal
markers such as vimentin, and α-smooth muscle actin (α-SMA) (Hills and Squires,
2011). Hence, EMT has been considered as one of the potential mechanisms of the
renal fibrosis in DN (Simonson, 2007). Substantive studies have suggested
transforming growth factor-β1 (TGF-β1) as the predominant mediator of EMT and
renal fibrosis (Bottinger, 2007).
It has been proposed that TGF-β1 mediates its effects via microRNAs (miRNAs),
a group of endogenous, single-stranded noncoding RNAs with approximately 22
nucleotides in length that regulate multiple physiological biological mechanism (Butz
et al., 2012). miRNAs function as an important regulator of gene expression by
interacting with the 3’-untranslated region (3’-UTR) of target mRNAs to induce
translational inhibition or transcript degradation (Bushati and Cohen, 2007). A
number of miRNAs have been reported to be frequently deregulated in DN, and have
been identified as valuable and early biomarker in various chronic kidney diseases
(Barutta et al., 2013). Cumulative studies have demonstrated that dysregulated
miRNAs contribute to the development and progression of diabetes and diabetic
complications, including DN (Xiao and Rajewsky, 2009). Recent studies showed that
miR-133b was highly expressed in type 2 diabetic nephropathy (T2DN), and
identified as novel urinary exosomal miRNA biomarker of DN (Eissa et al., 2016). In
addition, it was previously discovered that serum miR-199b-5p level was increased in
diabetes patients with abnormal albuminuria and overexpression of miR-199b-5p
enhanced renal function in streptozotocin (STZ)-induced DN mice under the
treatment with atrasentan (Kang and Xu, 2016). Accordingly, the detailed roles of
miR-133b and miR-199b on TGF-β1-induced EMT and renal fibrosis in DN and its
underlying mechanism remain far from addressed.
2. Materials and methods
2.1 Animals and treatment
All experimental procedures were approved by the Animal Experimental
Committee of Huaihe Hospital of Henan University and conducted according to the
NIH guide for the care and use of laboratory animals. The 40-week-old male
Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats were used for a model of
spontaneous type II diabetes mellitus and purchased from Otsuka Pharmaceutical Co.,
Ltd. (Tokyo, Japan). Healthy Long-Evans Tokushima Otsuka (LETO; Otsuka
Pharmaceutical Co., Ltd.) were served as control rats. These rats were maintained in a
specific pathogen-free facility at a constant temperature (23°C ± 2°C) and a relative
humidity (55% ± 5%), under an automated 12-h artificial light/dark cycle and were
allowed free access to standard rat chow and drinking water. Recombinant lentivirus
containing miR-133b inhibitor (LV-anti-miR-133b), miR-199b inhibitor
(LV-anti-miR-199b) and their control (LV-anti-miR-NC) were all from Genechem
(Shanghai, China). To determine the effect of miR-133b and miR-199b on EMT and
renal fibrosis, LV-anti-miR-133b, LV-anti-miR-199b, LV-anti-miR-NC were
peritoneally injected into diabetic rats every day at a multiplicity of infection of 20. At
the end of 12 weeks, the rats were killed by cervical dislocation and rat renal cortices
were removed for histopathological analysis.
2.2 Masson and immunohistochemistry examination of the kidney
Renal cortex tissues were fixed in 4% paraformaldehyde for 24 h, embedded in
paraffin and then cut into 3-μm-thick sections for histological analysis. Tissue
sections were stained with a Masson modified IMEB stain kit (K7298, IMEB inc. San
Marcos, CA, USA) to estimate the degree of renal fibrosis. The sections were
visualized at last observed at 400× magnification under a BX51 light microscope
(Olympus, Tokyo, Japan) and obtained with an image analysis system (Image-Pro
Plus 4.0, Media Cybernetics, Silver Spring, MD).
For immunohistochemistry experiments, 3-μm sections were deparaffinized,
hydrated and boiled for 10 min in 10 mM citric acid (pH 6.0) for antigen retrieval.
The endogenous peroxidase activity was quenched with 3% hydrogen peroxide.
Following blocked with 5% skim milk for 10 min, the renal cortex sections were
incubated with primary antibodies against collagen I (COL I; ab34710, Abcam,
Cambridge, UK), fibronectin (FN; ab2413, Abcam), α-SMA (ab5694, Abcam), or
E-cadherin (#3195; Cell Signaling, Beverly, MA, USA) overnight at 4°C, followed by
incubation with horseradish peroxidase (HRP)-conjugated secondary antibody
(ab6728, Abcam) at room temperature for 2 h. The sections were then treated with 3,
3’-diaminobenzidine (DAB) (Zhongshanjinqiao, Beijing, China), and counterstained
with hematoxylin. All sections were photographed at 400× magnification using a
BX51 light microscope (Olympus).
2.3 Cell culture and transfection
Human proximal tubule cell line (HK-2) and 293T cells were purchased from
American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown
in Dulbecco’s modified Eagle medium (DMEM), 10% fetal bovine serum (FBS;
Invitrogen, Carsbad, CA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin
(Gibco BRL, Grand Island, NY, USA) at 37°C in a incubator with 5% CO2. For the
TGF-β1 assay, HK-2 cells were seeded into 6-well plates when grown to 70-80%
confluency and treated with 10 ng/ml recombinant human TGF-β1 (PeproTech,
Rocky Hill, NJ, USA) for 48 h to induce cellular fibrogenesis.
miR-133b mimic (miR-133b), miR-199b mimic (miR-199b) and their negative
control (miR-NC), miR-133b inhibitor (anti-miR-133b), miR-199b inhibitor
(anti-miR-199b) and inhibitor control (anti-miR-NC), siRNA specially against sirtuin
1 (SIRT1) (si-SIRT1), siRNA scrambled control (si-NC) were purchased from
GenePharma (Shanghai, China). HK-2 cells were planted into 6-well plates and
transfected with these above oligonucleotides using Lipofectamine 2000 (Invitrogen)
before 24 h of TGF-β1 inducement.
2.4 Quantitative real-time PCR (qRT-PCR)
Total RNA from renal cortex tissues or treated HK-2 cells was isolated using a
miRNeasy FFPE Kit (Qiagen, Valencia, CA, USA) and quantified using NanoDrop
ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A total
of 1 μg total RNA was reversely transcribed into cDNA using a miRNA First-Strand
cDNA Synthesis Kit (TianGen, China). miR-133b and miR-199b expressions was
detected using TaqMan miRNAs Quantitation Kit (Applied Biosystems, Foster City,
CA, USA) on Step One Plus Real-Time PCR System (Applied Biosystems). The PCR
procedure was set for 95°C for 2 min, followed by 40 cycles of 15 s at 95°C, 60 s at
60 °C, and 30 s at 70°C. The relative expressions of miR-133b and miR-199b were
calculated using the 2−ΔΔCt method, with U6 small nuclear RNA as an internal control.
2.5 Western blot
Total proteins were extracted from renal cortex tissues or treated HK-2 cells with
RIPA buffer (Thermo Fisher Scientific) supplemented with Protease Inhibitor
Cocktail (Pierce, Rockford, IL, USA). Equal amount of protein samples were
subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and subsequently electrophoretically transferred onto polyvinylidene
difluoride membranes (PVDF; Millipore, Billerica, MA, USA). The PVDF
membranes were blocked with 10% non-fat milk in Tris-buffered saline containing
0.05% Tween 20 (TBST) for 1 h at room temperature, and incubated with primary
antibodies including collagen I (COL I; ab34710, Abcam), fibronectin (FN; ab2413,
Abcam), α-SMA (ab5694, Abcam), or E-cadherin (#3195; Cell Signaling, Beverly,
MA, USA) overnight at 4°C, followed by incubation with HRP-conjugated secondary
antibody (ab6728, Abcam) at room temperature for 2 h. Finally, protein signals were
detected using a chemiluminescence detection kit (Amersham Pharmacia Biotech Inc.,
Piscataway, NJ, USA).
2.6 Dual luciferase reporter assay
The fragments of SIRT1 3’UTR region containing the wild-type binding sites of
miR-133b or miR-199b were amplified using a human cDNA library and cloned into
pGL3-Promoter vector (Promega, Madison, WI, USA) to generate SIRT1-WT. A
Quick-Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) was
performed to construct the mutant SIRT1 3’UTR (SIRT1-MUT). For luciferase
reporter assay, 293T cells were plated in 6-well plates and transfected with 10 pmol
miR-133b, miR-199b, miR-NC, and 2 μg of constructed luciferase reporter plasmids
using Lipofectamine 2000 (Invitrogen). At 24 h of transfection, cells were harvested
and luciferase activity was detected by the dual luciferase reporter assay system
(Promega).
2.7 RNA immunoprecipitation (RIP) assay
RIP assay was performed using the EZ-Magna RIP Kit (Millipore). HK-2 cells
were collected and lysed using NP-40 lysis buffer containing 1 mM PMSF, 1 mM
DTT, 1% Protease Inhibitor Cocktail (Sigma-Aldrich), and RNase inhibitor
(Invitrogen). Subsequently, whole cell extract was then incubated with RIP buffer
containing A + G magnetic beads conjugated with human anti-Argonaute (Ago) 1
(Abcam), negative control IgG (Millipore), and positive control anti-snRNP70
(Millipore) for 1 h at room temperature. The proteins in samples were digested with
Proteinase K buffer, and immunoprecipitated RNA was isolated and subjected to
qRT-PCR analysis.
2.8 Statistical analysis
All data were expressed as mean ± standard deviation (S.D.) from three
independent experiments. Statistical analysis was analyzed using SPSS 19.0 software
(SPSS, Chicago, IL, USA) with Student’s t tests or one-way analysis of variance
(ANOVA). Difference was considered as statistically significant when P < 0.05.
3. Results
3.1 miR-133b and miR-199b were highly expressed in the renal cortex of
diabetic OLETF rats
To determine the association between miR-133b/199b and DN, the expressions
of miR-133b/199b in the renal cortex of diabetic OLETF rats were measured by
qRT-PCR. As shown in Fig. 1A and 1B, miR-133b and miR-199b expressions were
significantly upregulated in diabetic OLETF rats in comparison with non-diabetic
LETO rats. In addition, western blot analysis and immunohistochemical staining both
indicated that the expression levels of myofibroblast markers including COL I and FN
and mesenchymal marker α-SMA were greatly increased, while epithelial marker
E-cadherin was drastically declined in the renal cortex of diabetic OLETF rats with
respect to LETO rats (Fig. 1C–1E), suggesting that the EMT and renal fibrosis were
induced in diabetic OLETF rats. Consistently, Masson staining displayed more
collagen deposition in the tubular interstitial area of the diabetic OLETF rats with
respect to non-diabetic LETO rats (Fig. 1E). Thus, we inferred from these results that
aberrant expressions of miR-133b and miR-199b might participate in the regulation of
EMT and renal fibrosis in diabetic OLETF rats.
3.2 Inhibition of miR-133b/199b attenuated EMT and renal fibrosis in diabetic
OLETF rats
To further address the effects of miR-133b/199b on EMT and renal fibrosis in
diabetic OLETF rats, LV-anti-miR-133b, LV-anti-miR-199b, or LV-anti-miR-NC
were peritoneally injected into diabetic OLETF rats. As a result, miR-133b and
miR-199b expressions were notably reduced following the injection of
LV-anti-miR-133b and LV-anti-miR-199b in diabetic OLETF rats versus
LV-anti-miR-NC (Fig. 2A and 2B), respectively. In addition, western blot analysis
revealed that injection of LV-anti-miR-133b and LV-anti-miR-199b triggered a
marked decline of the protein levels of COL I, FN, α-SMA and a substantial elevation
of E-cadherin level in diabetic OLETF rats relative to control group (Fig. 2C and 2D).
Similar results were observed in immunohistochemistry analysis of renal cortex in the
diabetic OLETF rats (Fig. 2E and 2F). Consistently, Masson staining implicated that
tubulointerstitial fibrosis was apparently suppressed after inhibiting miR-133b/199b
expression in the diabetic OLETF rats (Fig. 2E and 2F). These results indicated that
inhibition of miR-133b/199b attenuated EMT and renal fibrosis in diabetic OLETF
rats.
3.3 miR-133b and miR-199b were upregulated in TGF-β1-treated HK-2 cells
HK-2 cells were treated with 10 ng/ml recombinant human TGF-β1 for 48 h to
induce cellular fibrogenesis. The expressions of miR-133b (Fig. 3A) and miR-199b
(Fig. 3B) were found to be higher in TGF-β1-treated HK-2 cells than that in control
cells. Moreover, we found that TGF-β1 prominently induced the increase of the
protein levels of COL I, FN, α-SMA and the decrease of E-cadherin level in HK-2
cells (Fig 3C), suggesting that TGF-β1 induced EMT and renal fibrosis in HK-2 cells.
3.4 Suppression of miR-133b and miR-199b alleviated TGF-β1-induced EMT
and renal fibrosis in HK-2 cells
To characterize the role of miR-133b and miR-199b in TGF-β1-induced EMT
and renal fibrosis, HK-2 cells were transfected with anti-miR-133b, anti-miR-199b, or
anti-miR-NC before 24 h of TGF-β1 inducement. qRT-PCR results showed that
miR-133b and miR-199b expressions were substantially decreased following
introduction with anti-miR-133b or anti-miR-199b in TGF-β1-treated HK-2 cells (Fig.
4A and 4B), respectively. Furthermore, western blot analysis revealed that
transfection with anti-miR-133b or anti-miR-199b dramatically impeded the protein
levels of COL I, FN, and α-SMA and enhanced E-cadherin level in TGF-β1-treated
HK-2 cells versus anti-miR-NC group (Fig. 4C and 4D). Therefore, we concluded that
suppression of miR-133b and miR-199b alleviated TGF-β1-induced EMT and renal
fibrosis in HK-2 cells.
3.5 SIRT1 was identified as a target of miR-133b and miR-199b in HK-2 cells
To clarify the potential mechanism by which miR-133b and miR-199b regulated
TGF-β1-induced EMT and renal fibrosis in HK-2 cells, we searched for the putative
targets of miR-133b and miR-199b using miRNA target prediction algorithms.
Interestingly, SIRT1 was found to be a potential target of miR-133b and miR-199b.
To validate this prediction, SIRT1 3’UTR fragments containing wild-type or mutated
type with miR-133b or miR-199b binding sites were inserted into luciferase reporter
vectors (Fig. 5A) and luciferase reporter assay was conducted. The results
demonstrated that cotransfection with miR-133b or miR-199b and SIRT1-WT
strikingly suppressed the luciferase activity with respect to control group, while
cotransfection with miR-133b or miR-199b and SIRT1-MUT showed no significant
effect on luciferase activity (Fig. 5B). Moreover, RIP assay revealed that SIRT1
mRNA can be specifically recruited to the miRNA ribonucleoprotein complex
(miRNP) complexes isolated using Ago1 antibody after miR-133b or miR-199b
overexpression (Fig 5C). These results demonstrated that miR-133b and miR-199b
could directly target 3’UTR of SIRT1.
3.6 Suppression of miR-133b and miR-199b alleviated TGF-β1-induced EMT
and renal fibrosis by upregulating SIRT1 in HK-2 cells
We examined the regulatory effects of miR-133b and miR-199b on SIRT1
expression in TGF-β1-induced HK-2 cells by western blot and the results
demonstrated that transfection with miR-133b or miR-199b successfully suppressed
SIRT1 level while introduction with anti-miR-133b or anti-miR-199b evidently
elevated SIRT1 level in TGF-β1-induced HK-2 cells (Fig. 6A and 6B), suggesting
that miR-133b and miR-199b negatively regulated SIRT1 expression in
TGF-β1-induced HK-2 cells. To explore whether SIRT1 was a functional target to
regulate the biological functions of miR-133b and miR-199b in TGF-β1-treated HK-2
cells, siRNA-mediated knockdown of SIRT1 was performed in anti-miR-133b or
anti-miR-199b-transfected HK-2 cells prior to TGF-β1 inducement. As shown in Fig.
6C and 6D, anti-miR-133b or anti-miR-199b dramatically suppressed the protein
levels of COL I, FN, and α-SMA, and promoted E-cadherin level in TGF-β1-treated
HK-2 cells, while these effects were remarkably reversed following SIRT1
knockdown. Collectively, these results demonstrated that suppression of miR-133b
and miR-199b alleviated TGF-β1-induced EMT and renal fibrosis by upregulating
SIRT1 in HK-2 cells.
4. Discussion
Increasing studies have focused on the functional role of miRNAs in the
development of multiple kidney diseases including DN. In the present study, we
investigated the roles of miR-133b and miR-199b in regulating TGF-β1-induced EMT
and renal fibrosis in DN. We provided the first evidence that inhibition of miR-133b
and miR-199b effectively inhibited TGF-β1-induced EMT and renal fibrosis in DN in
vivo and in vitro by targeting SIRT1, suggesting that multi-target therapy using
different classes of miRNAs is a potential strategy for the future treatment of DN.
Accumulating evidence demonstrated that EMT of tubular epithelial cells in the
kidney plays a functional role in the renal accumulation of matrix protein associated
with DN and is a crucial event in initiating organ fibrosis including renal fibrosis
(Kalluri and Weinberg, 2009). Moreover, it has been demonstrated that inhibiting
specific process of EMT in tubular epithelial cells of kidney significantly weakens
renal fibrosis in multiple models including DN, highlighting the function of EMT in
DN (Hills and Squires, 2011). Extensive researches have suggested that TGF-β1,
which is known as one of the most predominant inducers of EMT process, induces
EMT process and ultimately contributes to the progression of renal fibrosis
(Lamouille et al., 2014). Our study showed that that the expression levels of
myofibroblast markers including COL I and FN and mesenchymal marker α-SMA
were greatly increased, while epithelial marker E-cadherin was drastically declined in
the renal cortex of diabetic OLETF rats and TGF-β1-stimulated HK-2 cells. Moreover,
Masson staining displayed obvious renal fibrosis in the renal cortex of diabetic
OLETF rats. These results suggested the EMT and renal fibrosis were induced in
diabetic OLETF rats and TGF-β1-stimulated HK-2 cells.
A number of miRNAs have been proposed to be associated with DN in a
TGF-β1-dependent manner (Butz et al., 2012). For example, TGF-β1 stimulation
significantly reduced let-7d expression and overexpression of let-7d suppressed
TGF-β1-induced EMT and renal fibrosis both in vivo and in vitro (Wang et al., 2016).
Additionally, miR-21 overexpression contributed to TGF-β1-induced EMT and
aggravated renal damage in DN by inhibiting target smad7 (Wang et al., 2014).
Moreover, overexpression of miR-26a and miR-30c coordinately ameliorated
TGF-β1-induced EMT in NRK-52E cells (Zheng et al., 2016). In our study, we found
that miR-133b and miR-199b were significantly upregulated in the renal cortex of
diabetic OLETF rats and TGF-β1-stimulated HK-2 cells, consistently with the
previous studies (Eissa et al., 2016; Kang and Xu, 2016). Moreover, loss-of-function
approaches demonstrated that inhibition of miR-133b and miR-199b attenuated EMT
and renal fibrosis in diabetic OLETF rats and TGF-β1-stimulated HK-2 cells.
SIRT1, a member of a conserved family of NAD+-dependent class III
deacetylases, is widely expressed throughout nearly all mammalian organs and
functions as a key regulator of various physiological processes like apoptosis,
autophagy, cellular metabolism, and inflammation through the deacetylation of target
proteins (Kume et al., 2013). Recent studies have proposed that SIRT1 suppresses
EMT in cancer metastasis and organ fibrosis by inhibiting TGF-β1 signaling (Simic et
al., 2013). It was documented that SIRT1 activation alleviated renal fibrosis by
inhibiting the TGF-β1/Smad3 pathway (Huang et al., 2014). Moreover, SIRT1 was
reported to reduce TGF-β-induced mesangial cell apoptosis through acceleration of
Smad7 degradation (Kume et al., 2007). In our study, luciferase reporter assay and
RIP assay demonstrated that SIRT1 was identified as a target of miR-133b and
miR-199b in HK-2 cells. Similar results were observed in hepatocellular carcinoma
(Tian et al., 2016), osteosarcoma (Ying et al., 2017), glioma (Li et al., 2016) and
colorectal cancer (Shen et al., 2016). Moreover, rescue experiments demonstrated that
SIRT1 knockdown dramatically reversed the suppression on TGF-β1-induced EMT
and renal fibrosis in HK-2 cells mediated by anti-miR-133b or anti-miR-199b,
suggesting that suppression of miR-133b and miR-199b alleviated TGF-β1-induced
EMT and renal fibrosis by upregulating SIRT1 in HK-2 cells.
5. Conclusion
In summary, our study demonstrated the profound effects of miR-133b and
miR-199b on EMT and renal fibrosis in diabetic OLETF rats and TGF-β1-stimulated
HK-2 cells. We provided the first evidence that inhibition of miR-133b and miR-199b
attenuated TGF-β1-induced EMT and renal fibrosis by upregulating SIRT1, providing
viable therapeutic avenues for preventing renal fibrosis in DN. These results
suggested that suppression of miR-133b and miR-199b may be an effective target for
the prevention strategies of renal fibrosis in DN.
Conflicts of interest
There is no conflict of interest regarding the publication of this paper.
Funding
This work was supported by the Project of Science and Technology of Henan
Provincial Science and Technology Department (Grant No. 182102310267).
Acknowledgment
None
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Figure 1. Diabetic OLETF rats showed high expressions of miR-133b/199b and
developed EMT and renal fibrosis. (A and B) The expressions of miR-133b and
miR-199b in the diabetic OLETF rats and non-diabetic LETO rats were determined
by qRT-PCR. (C and D) Western blot was conducted to analyze the protein levels of
COL I, FN, α-SMA and E-cadherin in the diabetic OLETF rats and non-diabetic
LETO rats. (E) Representative images of Masson staining of the renal cortex from the
diabetic OLETF rats and non-diabetic LETO rats. Representative images of
immunohistochemical staining of COL I, FN, α-SMA and E-cadherin in the diabetic
OLETF rats and non-diabetic LETO rats. *P < 0.05.
Figure 2. The effects of inhibition of miR-133b/199b on EMT and renal fibrosis in
diabetic OLETF rats. (A and B) After LV-anti-miR-133b, LV-anti-miR-199b, or
LV-anti-miR-NC was peritoneally injected into diabetic OLETF rats, the expressions
of miR-133b and miR-199b were evaluated by qRT-PCR. (C and D) The protein
levels of COL I, FN, α-SMA, and E-cadherin in the diabetic OLETF rats after the
injection of LV-anti-miR-133b or LV-anti-miR-199b were detected by western blot.
(E and F) Masson staining was applied to detect the tubulointerstitial fibrosis changes
in the renal cortex of diabetic OLETF rats after the lentivirus injection.
Immunohistochemistry analysis was employed to evaluate the expressions of COL I,
FN, α-SMA and E-cadherin in the renal cortex of diabetic OLETF rats after the
lentivirus injection. *P < 0.05.
Figure 3. miR-133b and miR-199b were upregulated in TGF-β1-treated HK-2 cells.
(A and B) qRT-PCR analysis of the expressions of miR-133b and miR-199b in HK-2
cells treated with or without TGF-β1. (C) The protein levels of COL I, FN, α-SMA,
and E-cadherin in HK-2 cells treated with or without TGF-β1 were checked by
western blot. *P < 0.05.
Figure 4. Suppression of miR-133b and miR-199b alleviated TGF-β1-induced EMT
and renal fibrosis in HK-2 cells. HK-2 cells were transfected with anti-miR-133b,
anti-miR-199b, or anti-miR-NC before 24 h of TGF-β1 inducement. (A and B) The
expressions of miR-133b and miR-199b in the treated HK-2 cells were determined by
qRT-PCR. (C and D) The protein levels of COL I, FN, α-SMA, and E-cadherin in the
treated HK-2 cells were analyzed by western blot. *P < 0.05.
Figure 5. The interaction between miR-133b or miR-199b and SIRT1 in HK-2 cells.
(A) The predicted binding sites between miR-133b or miR-199b and SIRT1 3’UTR.
(B) Luciferase reporter assay was performed to measure the luciferase activity in
HK-2 cells cotransfected with constructed luciferase reporter plasmids containing the
wild-type or mutated SIRT1 3’UTR and miR-133b, miR-199b or miR-NC. (C) RIP
with Ago1 antibody was applied to verify the binding status between miR-133b or
miR-199b and SIRT1. *P < 0.05.
Figure 6. Suppression of miR-133b and miR-199b alleviated TGF-β1-induced EMT
and renal fibrosis by upregulating SIRT1 in HK-2 cells. (A) The protein level of
SIRT1 was examined by western blot in HK-2 cells transfected with miR-133b,
anti-miR-133b, or respective controls, followed by TGF-β1 inducement. (B) The
protein level of SIRT1 was examined by western blot in HK-2 cells introduced with
miR-199b, anti-miR-199b, or matched controls, followed by TGF-β1 inducement. (C)
The protein levels of COL I, FN, α-SMA, and E-cadherin in HK-2 cells were
determined by western blot in HK-2 cells after transfection with anti-miR-133b,
anti-miR-NC, or combined with si-SIRT1 or si-NC, and TGF-β1 inducement. (D) The
protein levels of COL I, FN, α-SMA, and E-cadherin in HK-2 cells were evaluated by
western blot in HK-2 cells after transfection with anti-miR-199b, anti-miR-NC, or
combined with si-SIRT1 or si-NC, and TGF-β1 inducement. *P < 0.05.
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