- Email: [email protected]
mTOR signaling pathway
Accepted Manuscript Human umbilical cord-derived mesenchymal stem cells enhanced HK-2 cell autophagy through MicroRNA-145 by inhibiting the PI3K/AKT/mTOR signaling pathway Jin Xiang, Tingting Jiang, Wenying Zhang, Wei Xie, Xun Tang, Jun Zhang PII:
S0014-4827(18)31047-4
DOI:
https://doi.org/10.1016/j.yexcr.2019.03.019
Reference:
YEXCR 11355
To appear in:
Experimental Cell Research
Received Date: 2 October 2018 Revised Date:
10 March 2019
Accepted Date: 11 March 2019
Please cite this article as: J. Xiang, T. Jiang, W. Zhang, W. Xie, X. Tang, J. Zhang, Human umbilical cord-derived mesenchymal stem cells enhanced HK-2 cell autophagy through MicroRNA-145 by inhibiting the PI3K/AKT/mTOR signaling pathway, Experimental Cell Research (2019), doi: https:// doi.org/10.1016/j.yexcr.2019.03.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Human Umbilical Cord-Derived Mesenchymal Stem Cells Enhanced HK-2 Cell Autophagy Through MicroRNA-145 By Inhibiting The
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PI3K/AKT/mTOR Signaling Pathway
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Jin Xiang1, 2#, Tingting Jiang3#, Wenying Zhang1, Wei Xie1, Xun Tang1*, Jun Zhang1*
Guangdong Province, 510282, China
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1 Department of Nephrology, Zhujiang Hospital of Southern Medical University, Guangzhou,
2 Department of Nephrology, People's Hospital of Yuxi City, Yuxi, Yunnan Province, 653100, China
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3 Department of Nephrology, The People’s Hospital of Guangxi Zhuang Autonomous Region,
*
These authors contributed equally to this work.
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#
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Nanning, Guangxi Province, 530021, China
corresponding author: Jun Zhang, Department of Nephrology, Zhujiang Hospital of Southern Medical University, 253 Gongye Road, Guangzhou, Guangdong, 510282 (PR China). E-mail: [email protected]
Xun Tang, Department of Nephrology, Zhujiang Hospital of
ACCEPTED MANUSCRIPT Southern Medical University, 253 Gongye Road,
Guangzhou, Guangdong, 510282 (PR China).
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E-mail: [email protected]
Abstract: Recent studies have shown that autophagy exhibits a protective role in
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acute kidney injury (AKI), and the accumulation of advanced oxidation protein products (AOPP) participates in the progression of kidney diseases. Our previous study indicated that AOPP induced injury in renal tubular epithelial cells (RTECs) through autophagy inhibition. Besides, we found that human umbilical cord-derived
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mesenchymal stem cells (hUC-MSCs) enhanced autophagy in AOPP-treated RTECs, but the underlying mechanism remains unclear. We regulated microRNA-145
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(miR-145) expression in HK-2 cells (a cell line of RTECs), or co-cultured hUC-MSCs with HK-2 cells and studied the autophagic activity in HK-2 cells to explore the
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underlying mechanism. Our data demonstrated that upregulated miR-145 increased LC3 II and Beclin 1 levels, decreased p62 level, three autophagy related proteins, inhibited the phosphorylation of PI3K/AKT/mTOR, and increased LC3B-positive staining and the autophagosome number. Furthermore, hUC-MSCs enhanced autophagy and inhibited phosphorylation of PI3K/AKT/mTOR in AOPP-treated HK-2 cells, which was then partially rescued using miR-145 knockdown in the hUC-MSCs co-culture system. In conclusion, our study showed that hUC-MSCs
ACCEPTED MANUSCRIPT enhanced autophagy in AOPP-treated HK-2 cells mediated by miR-145 via inhibition of the PI3K/AKT/mTOR pathway, which indicated that hUC-MSCs might serve as a
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prospective therapy for AKI.
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Keywords: hUC-MSCs; miR-145; autophagy; HK-2 cells; PI3K/AKT/mTOR
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Introduction
Acute kidney injury (AKI) is a global public health concern and is associated with high morbidity, mortality, and healthcare costs; AKI impacts approximately 13.3 million patients per year [1]. Long-term outcomes may include the development of
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chronic kidney disease (CKD) [2] and end-stage renal disease (ESRD) [3], which require renal replacement therapy and cause a heavy economic burden. Therefore, it is
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important to understand the mechanisms of AKI and develop an effective treatment strategy to reduce the occurrence of AKI or slow the development of CKD and
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ESRD.
A growing number of studies have revealed the role of autophagy in kidney
diseases [4] [5], both in the tubulointerstitial compartment and in glomeruli. Pathologically, AKI is characterized by tubular cell injury and death. In addition, the role of autophagy in tubules, which is the key determinant of kidney prognosis, has been demonstrated in renal tubular epithelial cells (RTECs) [6] and is generally
ACCEPTED MANUSCRIPT accepted as a protective cellular response in AKI under various causes [7] [8]. Advanced oxidation protein products (AOPP) is a harmful protein product that is highly expressed in patients with kidney diseases. Furthermore, phosphatidylinositol
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3-kinase (PI3K)/AKT/mTOR signaling pathway is a crucial negative modulator of autophagy[9]. Our work team have made some researches about the harmful effects of AOPP on kidney diseases. In the latest investigation, our data showed that AOPP
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inhibited autophagy of RTECs trough activation of the PI3K/AKT/mTOR signaling
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pathway. Moreover, we also reported that autophagy inhibition mediated AOPP-induced RTEC injury in this study. [10]. Therefore, enhancing RTEC autophagy may be critical for suppressing the progression of kidney diseases including AKI.
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Currently, stem cell therapy has therapeutic potential to repair the impaired kidney. The mesenchymal stem cell (MSC) is a mesodermal stem cell that has the ability to
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self-renew and differentiate into other mesodermal cell lineages. Compared with MSCs from other sources, human umbilical cord-derived MSCs (hUC-MSCs) have
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received much attention because of their abundance, ease of separation, strong growth capacity, low immunogenicity, and lack of harm to mothers and newborns [11]. It is interesting to note that a systematic review and meta-analysis entering 71 articles showed that MSC therapy could delay the progression of CKD according to subgroup analysis, blood biochemical indexes, different related models and cell related factors [12]. Furthermore, the study of MSCs in the treatment of kidney diseases has become one of the hot topics in international nephrology research, including diabetic
ACCEPTED MANUSCRIPT nephropathy [13], renal transplantation [14], and lupus nephritis [15]. More excitingly, we have found that hUC-MSCs enhanced autophagy in AOPP-injured RTEC, which may perform a protective role in renal tissue and cells (the article is under review.)
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An increasing number of studies have demonstrated that MSCs regulate the microenvironment of renal cells to play a protective role by secreting a variety of
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cytokines, proteins and miRNAs [16] [17]. Notably, the potential therapeutic role of microRNA-145 (miR-145) secreted from MSCs has attracted increasing attention. A
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previous study found that hUC-MSCs inhibited the TGF-β/SMAD2 signaling pathway to protect the body from burn injury by secreting miR-145 [18]. Another study showed that MSCs inhibited the migration and self-renewal of glioma cells and glioma stem cells by secreting miR-145 [19]. More excitingly, it was reported that
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miR-145 repaired myocardial infarction by promoting myocardial cell autophagy [20]. In addition, there was another study showing that upregulation of miR-145 induced
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inhibition of the PI3K/AKT/mTOR pathway in laryngeal squamous cell carcinoma[21]. However, whether miR-145 secreted from hUC-MSCs plays
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protective roles in AKI by enhancing RTEC autophagy and the underlying mechanism need to be explored.
To further investigate the relationship between MSCs and renal autophagy, we
performed the current study to investigate the role of hUC-MSCs on autophagy inhibited by AOPP in HK-2 cells (a cell line of human RTECs) and the underlying mechanism.
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Material and Methods
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1. Materials and Reagents Bovine serum albumin (BSA) was purchased from Sigma (St. Louis, USA). Antibodies against LC3B, Beclin 1, p62, p-mTOR, mTOR, p-AKT, AKT, and p-PI3K
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were obtained from Cell Signaling Technology (Beverly, USA). The GAPDH antibody was purchased from Bioworld (Minnesota, USA). Antibodies against CD90,
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CD105, CD73, CD34, CD45, CD11b, CD14, and HLA-DR for flow cytometry were purchased from Becton Dickinson and Company (New Jersey, USA). The U6 and miR-145 primers were obtained from Ribobio Company (Guangzhou, China). Sequencing of miR-145 siRNA, miR-145 mimics, and negative siRNA was
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performed by Shanghai GenePharma Co. Ltd (Shanghai, China).
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2. AOPP Preparation
AOPP was prepared as previously described [22]. Briefly, HOCl (200 mmol/L) was
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mixed with BSA solution for 30 min at room temperature, and then dialyzed against PBS at 4°C to remove free HOCl for 24 h. Native BSA was dissolved in PBS alone as the control. The endotoxin levels were measured and were required to be <0.025 EU/mL. The AOPP content was measured at 340 nm to obtain the absorbance under acidic conditions and calibrated using chloramine-T in the presence of potassium iodide.
3. HK-2 Cell Culture and Treatment
ACCEPTED MANUSCRIPT HK-2 cells were purchased from the American Type Culture Collection (Rockville, USA) and cultured in DMEM/F12 (Gibco, USA) supplemented with 10% heat-inactivated FBS and maintained in a humidified incubator containing a 5% CO2
until they reached approximately 70-80% confluence.
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4. hUC-MSCs Isolation and Co-culture with HK-2 cells
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atmosphere at 37°C. Cells were incubated in BSA (200 µg/mL) or AOPP (200 µg/mL)
We isolated hUC-MSCs using an adherent tissue method[23]. Briefly, a 10 cm
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umbilical cord from a full-term healthy newborn was washed with PBS (containing 1% penicillin-streptomycin double-resistant solution) 3 times. Then, we cut the cord into small pieces, dislodged the umbilical vein and umbilical artery, and left Wharton's
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jelly at last. Wharton's jelly was then cut into 1 mm × 1 mm × 1 mm and cultured in DMEM/F12 medium containing 5% FBS; 3-6 passages (P3-6) were selected for the following experiments. We used a co-culture chamber to block the immediate contact
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between the hUC-MSCs and HK-2 cells to explore the paracrine action of hUC-MSCs
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in the co-culture system.
5. Flow Cytometry and hUC-MSCs Identification hUC-MSCs were trypsinized, and the cell concentration was adjusted to 1×106/mL
in PBS. Then, 200 µl of the suspension was incubated with 5 µl of antibodies against CD90, CD105, CD73, CD34, CD45, CD11b, CD14, and HLA-DR without light for 30 min. Primary antibodies were directly conjugated with FITC and phycoerythrin. For isotype control, non-specific FITC-conjugated IgG was substituted for the
ACCEPTED MANUSCRIPT primary antibodies. Lastly, the samples were analyzed using flow cytometry.
6. RNA Extraction and Quantitative Real-time PCR (RT-qPCR) Analysis
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Total RNA was extracted from treated HK-2 cells using TRIzol reagent (TaKaRa, Japan). 1 µg RNA was reverse transcribed using the MMLV reverse transcriptase kit according to the manufacturer’s instructions (TaKaRa, Japan). The U6 and miR-145
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primers were provided directly by RiboBio Company and the sequences were kept
U6.
7. Western Blotting Analysis
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secreted by RiboBio Company. All data were normalized using the internal control
We extracted total protein using RIPA lysis buffer. Then, 20-50 µg of protein was
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separated using SDS-PAGE and transferred to PVDF membranes. Subsequently, the membranes were blocked in 5% nonfat milk or BSA at room temperature for 2 h,
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followed by incubation of the primary antibodies at 4°C overnight. The primary antibodies were as follows: anti-LC3B, anti-Beclin 1, anti-p62, anti-p-mTOR,
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anti-mTOR, anti-p-AKT, anti-AKT, anti-p-PI3K (dilution, 1:1000) and anti-GAPDH (dilution,
1:5000).
The membranes
were
incubated
with
the
appropriate
HRP-conjugated secondary antibodies at room temperature for 2 h and were finally detected using an enhanced chemiluminescence (ECL) system. GAPDH was used as the internal control and semiquantitative analysis was performed using the ImageJ system.
8. Immunofluorescence Staining
ACCEPTED MANUSCRIPT Cells plated in 96-well plates were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 10 minutes and incubated in a blocking buffer containing 5% BSA for 30 min at room temperature. Then, cells were incubated with LC3B Finally, we incubated cells with
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antibody (1:50 dilution) overnight at 4°C.
fluorescently labeled secondary antibodies (Alexa Fluor 488, 1:400, Gongsi) for 1 h at room temperature in the dark, followed by incubation with 0.1% DAPI for 10 min.
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We used an inverted fluorescence microscope to observe and record the
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LC3B-positive staining.
9. Transmission Electron Microscopy (TEM)
HK-2 cells were gently harvested using trypsin with EDTA and were centrifuged at a speed
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of 1000 r/m for 5 min. Then, the sediment was washed twice with cold PBS and fixed in 2.5% glutaraldehyde for 2 h at 4°C. Cells were conventionally dehydrated, embedded, sliced into 60 nm sections and stained with uranyl acetate for 15 min at room temperature, followed by
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lead citrate for 15 min at room temperature. Autophagosome (AP) and autolysosome (AL)
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formation were observed using TEM at a magnification of low-power field (x6000, x8000, x10000) or high-power field (x40000) operating at 60 kV.
10. HK-2 Cell Transfection And hUC-MSC Transfection
HK-2 cells were transfected with miR-145 siRNA, miR-145 mimics, or negative siRNA, and hUC-MSCs was transfected with miR-145 siRNA, using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions. Targeting sequences were as follows: miR-145 siRNA: sense (5’-
ACCEPTED MANUSCRIPT AGGGAUUCCUGGGAAAACUGGAC-3’); miR-145 mimics: sense (5’GUCCAGUUUUCCCAGGAAUCCCU-3’), antisense (5’GGAUUCCUGGGAAAACUGGACUU-3’); negative siRNA: sense
(5’-ACGUGACACGUUCGGAGAATT-3’).
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11. Statistical Analysis
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(5’-UUCUCCGAACGUGUCACGUTT-3’), antisense
Statistical analyses were performed using SPSS 20.0 software. Continuous
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variables are presented as the mean ± SD. One-way ANOVA was carried out to determine differences among groups. For the comparison of 2 groups, the LSD method was used when the assumption of variance was equal. Otherwise, the Dunnett
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Results
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T3 method was used. P values < 0.05 indicated a statistically significant difference.
1. Identification results of hUC-MSCs
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We chose P3 hUC-MSCs to identify the cell phenotype by flow cytometry.
Inverted phase contrast microscopy showed that P4 hUC-MSCs presented 50% fusion and 100% vortex fusion, as shown in in Figure 1A and 1B. Flow cytometry analysis of the cell phenotype showed that the hUC-MSCs were positive for CD90, CD105, and CD73, but were negative for CD34, CD45, CD11b, CD14, and HLA-DR (Figure 1C-J).
ACCEPTED MANUSCRIPT 2. miR-145 was upregulated in HK-2 cells under hUC-MSCs co-cultured system We co-cultured hUC-MSCs with HK-2 cells and used RT-qPCR to measure the expression of miR-145 in HK-2 cells. The result showed that when HK-2 cells were
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co-cultured with hUC-MSCs, the mRNA level of miR-145 was significantly upregulated. Next, we knocked down miR-145 in the hUC-MSCs and then
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co-cultured it with HK-2 cells. We found that in the later co-culture system, miR-145 did not increased in HK-2 cells (Figure 2). The data indicated that hUC-MSCs
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secreted miR-145 and transferred it to HK-2 cells through paracrine action.
3. miR-145 affected HK-2 cell autophagy
We first downregulated or upregulated the expression of miR-145 in HK-2 cells
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and examined the mRNA level of miR-145. The RT-qPCR results showed that miR-145 siRNA downregulated miR-145; however, miR-145 mimics upregulated miR-145 in HK-2 cells. At the same time, the expression of miR-145 showed no
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difference between negative siRNA and the normal control group (Figure 3A).
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We then assayed the autophagy-related proteins LC3B, Beclin 1, and p62 by
western blotting. The results in figure 3(B-C) showed that when we inhibited the expression of miR-145 by siRNA, the protein levels of LC3 II and Beclin 1 decreased and the protein level of p62 increased. However, when we upregulated the expression of miR-145, the protein levels of those markers mentioned above had the opposite effect. In addition, there was no difference between negative siRNA and the normal control group.
ACCEPTED MANUSCRIPT Furthermore, the autophagic activity of HK-2 cells was assessed using immunofluorescence technology and TEM. LC3B-positive staining was significantly decreased in cells transfected with miR-145 siRNA but increased in cells transfected
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with miR-145 mimics compared with that of the normal control group (Figure 3D). Similarly, the TEM results demonstrated a decrease in the number of typical Aps (double membrane vacuoles engulfing cytoplasmic structures) and ALs (single
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membrane vacuole structures containing high density materials) in miR-145 siRNA
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transfected cells but revealed an increase in the number of Aps and ALs in miR-145 transfected cells compared with those in the normal control group (Figure 3E). Collectively, these results suggested that overexpression of miR-145 activated HK-2 cell autophagy.
cells
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4. miR-145 mediated hUC-MSCs enhanced-autophagy in AOPP-treated HK-2
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To further confirm that hUC-MSCs-enhanced autophagy was mediated by the
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secretion of miR-145, we transfected HK-2 cells with miR-145 siRNA after AOPP-treated HK-2 cells were co-cultured with hUC-MSCs to investigate the effect of hUC-MSCs on HK-2 cells. We first measured the miR-145 expression in transfected and co-culture system. RT-qPCR analysis showed that the level of miR-145 was increased in the hUC-MSCs and AOPP co-culture group compared with that in the normal control group, but the miR-145 level was decreased in the transfected and co-culture system compared with the hUC-MSCs and AOPP co-culture group (Figure 4A).
ACCEPTED MANUSCRIPT Next, we detected the autophagous activity after transfection with miR-145 siRNA in the hUC-MSCs and AOPP co-culture system. The western blotting results showed that when we decreased the expression of miR-145 by siRNA, the protein levels of
those in
the hUC-MSCs
co-culture system
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LC3 II and Beclin 1 decreased and the p62 protein level increased compared with (Figure 4B-C).
Furthermore,
LC3B-positive staining was significantly decreased in cells transfected with miR-145
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siRNA compared with that in the hUC-MSCs co-culture system (Figure 4D).
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Similarly, the TEM results demonstrated a decrease in the numbers of typical Aps and ALs in HK-2 cells in siRNA transfected with the hUC-MSCs co-culture system compared with those in the hUC-MSCs co-culture system alone (Figure 4E). Taken together, these results indicated that miR-145 secreted from hUC-MSCs enhanced
5.
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HK-2 cell autophagy.
miR-145 mediated hUC-MSCs enhanced-autophagy in HK-2 cells by
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inhibiting the PI3K/AKT/mTOR signaling pathway
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To verify the underlying pathway, we detected PI3K/AKT/mTOR signaling pathway related proteins after regulating miR-145 expression in HK-2 cells or transfection with miR-145 siRNA in the hUC-MSCs co-culture system. The western blotting results showed that when we decreased the expression of miR-145 by siRNA, the phosphorylation of PI3K, AKT, and mTOR was induced. However, when we upregulated the expression of miR-145 by mimics, the phosphorylation of PI3K, AKT, and mTOR was inhibited. In addition, there was no difference between negative siRNA and the normal control group (Figure 5A-B). Finally, transfection with
ACCEPTED MANUSCRIPT miR-145 siRNA in the hUC-MSCs co-culture system promoted the phosphorylation of PI3K, AKT, and mTOR, which was inhibited by hUC-MSCs (Figure 5C-D). We concluded that miR-145 mediated hUC-MSCs enhanced-autophagy in HK-2 cells via
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inhibiting the PI3K/AKT/mTOR signaling pathway.
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Discussion
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The present study demonstrated the significant role of hUC-MSCs in HK-2 cell autophagy in AKI. We provided evidence that hUC-MSCs increased HK-2 cell autophagy via secretion of miR-145 through inhibition of the PI3K/AKT/mTOR signaling pathway. The current results implicate hUC-MSCs as a promising
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therapeutic strategy in protecting AKI through its paracrine action.
A considerable number of studies have focused on the role of autophagy in renal
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diseases. An increasing number of findings have highlighted that autophagy is rapidly induced during AKI to protect tubular cells from injury and death, and
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autophagy-related gene deficiency leads to impaired renal function and tubular cell apoptosis and necrosis in conditional knockout mouse models [24] [25]. However, our study found that AOPP inhibited HK-2 cell autophagy and induced HK-2 cell injury instead of inducing autophagy to protect cells [10]. Whether the noxious stimuli induce or inhibit cell autophagy may depend on the tissue, magnitude, time or other factors that cells suffer. Livingston and colleagues [26] summarized a similar opinion, proposing that it was very important to monitor autophagy in the correct tissue at the
ACCEPTED MANUSCRIPT correct time to determine the optimal conditions and therapeutic window, thus ensuring that autophagy induction would yield protective effects in renal diseases. Moreover, given the evidence that autophagy plays an important role in the
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development and severity of AKI, targeting autophagy could be a novel and potential strategy for the treatment of AKI and even other renal diseases. In this study, we discovered a new mechanism under autophagy regulation by hUC-MSCs in HK-2
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cells.
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The first key finding of this study was that miR-145 was upregulated in hUC-MSCs and HK-2 cells in a co-culture system. It is general knowledge that MSCs demonstrate paracrine mechanisms, including the secretion of various nutrition factors, proteins, mRNA, or miRNA. In this way, MSCs directly or indirectly induce regeneration,
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rescuing injured cells and accelerating organ repair [27]. We co-cultured hUC-MSCs with HK-2 cells and found that miR-145 expression was upregulated in HK-2 cells in
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this system. We then knocked down miR-145 in the hUC-MSCs before it co-cultured with HK-2 cells and found that miR-145 did not increased in HK-2 cells, which
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indicated that hUC-MSCs secreted miR-145 and transported it to HK-2 cells through paracrine action. Previous studies showed that MSCs delivered exogenous miRNAs to neural cells [28] or myofibroblasts [18]. In addition, it has been proposed that MSC-derived extracellular vesicles protect against renal injury [29] [30]. However, the underlying mechanism of MSC treatment on renal diseases and whether the upregulated miR-145 in HK-2 cells derived from hUC-MSCs contribute to the renal protection are unclear.
ACCEPTED MANUSCRIPT The second crucial finding of this study was that regulation of miR-145 affected HK-2 cell autophagy. Currently, may studies have indicated that abnormal expression of miRNA participates in the regulation and progression of various renal diseases,
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such as AKI[31], diabetic nephropathy[32], and IgA nephropathy [33]. Furthermore, multiple studies have previously demonstrated that miRNA affects renal function or the pathology and physiological of renal cells through various mechanisms. Zou et al.
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[34] demonstrated that miRNA mediated epithelial mesenchymal transition in fibrotic
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diseases, including renal fibrosis. Dewanjee et al. [35] concluded in their review that miRNA regulated various molecular pathways in diabetic nephropathy, including the TGF-β signaling pathway, PI3K/AKT signaling pathway, and collagen gene expression. To investigate whether miR-145 influences HK-2 cell autophagy, we first
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downregulated or upregulated the expression of miR-145 in HK-2 cells. The results showed that when we inhibited the expression of miR-145 by siRNA, the protein levels of LC3 II and Beclin 1 decreased and the protein level of p62 increased and
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phosphorylation of PI3K, AKT, and mTOR increased, but the expression of these
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markers showed the opposite change when we upregulated the overexpressed miR-145 with mimics. In addition, LC3B-positive staining and the numbers of typical Aps and ALs were significantly decreased in cells transfected with miR-145 siRNA but increased in cells transfected with miR-145 mimics compared with those in the normal control group. Collectively, these results suggested that overexpression of miR-145 activated HK-2 cell autophagy and that miR-145 knockdown inhibited HK-2 cell autophagy. It was reported that miR-145 repaired myocardium by accelerating
ACCEPTED MANUSCRIPT cardiomyocyte autophagy [20]. There was also another investigation showing that miRNA-145 promoted osteosarcoma cell autophagy by targeting HDAC4 [36]. In addition, there was a study showing that upregulation of miR-145 induced inhibition
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of the PI3K/AKT/mTOR pathway in laryngeal squamous cell carcinoma[21]. Excitingly, our study was the first time to show that miR-145 affects RTEC
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autophagy by inhibiting PI3K/AKT/mTOR signaling pathway.
Finally, we confirmed that hUC-MSCs enhanced HK-2 cell autophagy mediated by
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the secretion of miR-145 through inhibiting the PI3K/AKT/mTOR signaling pathway. In our previous investigation, we proposed that hUC-MSCs enhanced autophagy in AOPP-treated HK-2 cells, but the underlying mechanism requires further investigation. Accumulating evidence suggests that MSCs enhance autophagy through
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various classical pathways or molecules. Li et al. [37] showed that bone marrow-derived MSCs enhanced autophagy via PI3K/AKT signaling to reduce the
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severity of ischaemia/reperfusion-induced lung injury. Liu et al. [38] suggested that MSC induced cardiomyocyte autophagy via the AMPK and AKT signaling pathways
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to rescue myocardial ischaemia/reperfusion injury. To our knowledge, MSCs primarily possess paracrine or endocrine functions, which allow them to play a protective role. Wang et al. [39] indicated that hUC-MSCs-derived exosomes increased the expression of the autophagic marker protein LC3B and the autophagy-related genes ATG5 and ATG7 in cisplatin-treated NRK-52E cells to relieve the nephrotoxicity of cisplatin. In addition, Jia et al. [40] presented a similar conclusion in another study, showing that hUC-MSCs-derived exosomes enhanced
ACCEPTED MANUSCRIPT autophagy via transmission of 14-3-3zeta and modulation of ATG16L in preventing cisplatin-induced AKI. Since paracrine secretion of miR-145 from hUC-MSCs is delivered to HK-2 cells, to further uncover whether hUC-MSCs enhance autophagy
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through secretion of miR-145, we transfected HK-2 cells with miR-145 siRNA after AOPP-treated HK-2 cells were co-cultured with hUC-MSCs. We found that when we decreased the expression of miR-145 by siRNA, the protein levels of LC3 II and
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Beclin 1 decreased, the p62 protein level increased, the LC3B-positive staining and
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numbers of typical Aps and ALs decreased, and the phosphorylation of PI3K, AKT, and mTOR increased compared with those in hUC-MSCs co-culture system. Taken together, in this study, we presented for the first time a new mechanism of hUC-MSCs-enhanced cell autophagy by secreting miR-145 through inhibition of the
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PI3K/AKT/mTOR signaling pathway.
We did not explore how hUC-MSCs transported secreted miR-145 to HK-2 to
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enhance cell autophagy. Previous studies found that MSCs transported RNA or protein to target cells through MSCs-derived exosomes to repair damaged cells and
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tissues[41]. Future in-depth studies will explore whether exosomes act as the mechanism of hUC-MSCs on HK-2 cells. Additionally, the amount of miR-145 expressed in transfection is not the same with that secreted by hUC-MSCs, and we will further regulate and investigate the effects of different dosage of miR-145 secreted by hUC-MSCs on autophagy in HK-2 cells. Finally, further in vitro studies are warranted to validate the significance of hUC-MSCs and secreted miR-145 in the enhancement of autophagy in HK-2 cells and primary cells and even in AKI models.
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Conclusion
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In conclusion, these results indicated that hUC-MSCs played an autophagy enhancement role via overexpression of miR-145, which provided a novel mechanism for the investigation of hUC-MSCs in AKI treatment. MSCs represent an amazing
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therapeutic tool in regenerative medicine; therefore, developing a better understanding
kidney diseases.
Conflict of interest statement
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None.
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of the mechanism of MSCs in renal protection is a promising therapeutic strategy for
Acknowledgements
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The present study was supported by the Natural Science Foundation of Guangdong Province (grant no. 2018A030313557).
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Figure legends
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Fig. 1 hUC-MSCs identification
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A: Inverted phase contrast microscope images (× 50) showing that P4 hUC-MSCs presented 50%
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fusion; B: Inverted phase contrast microscope images (× 50) showing that P4 hUC-MSCs presented 100% vortex fusion; C-J: Flow cytometry showed that hUC-MSCs were positive for CD90, CD105,
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and CD73, but were negative for CD34, CD45, CD11b, CD14, and HLA-DR.
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Fig. 2 Detection of miR-145 in HK-2 cells in the hUC-MSCs co-culture system
The RT-qPCR results showed that miR-145 expression was upregulated in HK-2 cells in the hUC-MSCs co-culture system (hUC-MSCs group). Next, we knocked down miR-145 in the hUC-MSCs and then co-cultured it with HK-2 cells. We found that in the later co-culture system
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(hUC-MSCs siRNA group), miR-145 did not increased in HK-2 cells. Data are expressed as the means
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± SD from 3 independent experiments; *P < 0.05 vs. Ctrl group. Ctrl: normal control.
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Fig. 3 miR-145 affected HK-2 cell autophagy.
We inhibited or promoted the expression of miR-145 in HK-2 cells and detected autophagy related
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markers. A: The RT-qPCR results showed that miR-145 siRNA downregulated miR-145 but miR-145 mimics upregulated miR-145 in HK-2 cells. B-C: Western blotting results showed that LC3 II and
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Beclin 1 were decreased and p62 was increased in the miR-145 siRNA group, but the opposite trend was found in the miR-145 mimics group. D: Immunofluorescence result showed that LC3B-positive
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staining decreased in cells transfected with miR-145 siRNA but increased in cells transfected with miR-145 mimics. E: TEM experiments revealed that typical Aps and ALs were significantly decreased in cells transfected with miR-145 siRNA but increased in cells transfected with miR-145 mimics (the above is the low-power field, x8000, or x10000, and the below is the high-power field, x40000). Data are expressed as the means ± SD from 3 independent experiments; *P < 0.05 vs. Ctrl group. Ctrl: normal control.
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Fig. 4 hUC-MSCs enhanced autophagy in AOPP-treated HK-2 cells by secretion of miR-145.
We transfected HK-2 cells with miR-145 siRNA in the hUC-MSCs and AOPP co-culture system. A: RT-qPCR analysis showed that the increased miR-145 secreted from hUC-MSCs was partially decreased in the miR-145 siRNA transfected and co-culture system compared with that in the
ACCEPTED MANUSCRIPT hUC-MSCs and AOPP co-culture group. B-C: Western blotting showed that LC3 II and Beclin 1 was decreased and p62 was increased when HK-2 cells were transfected with miR-145 siRNA compared with those in the hUC-MSCs co-culture system. D: Immunofluorescence showed that LC3B-positive
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staining was decreased in cells transfected with miR-145 siRNA. E: TEM experiments presented that typical Aps and ALs were significantly decreased in cells transfected with miR-145 siRNA (the above is the low-power field, x6000, or x8000, and the below is the high-power field, x40000). Data are
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expressed as the means ± SD from 3 independent experiments; *P < 0.05 vs. AOPP group; # P < 0.05
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vs. AOPP+hUC-MSCs group.
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Fig. 5 miR-145 mediated hUC-MSCs enhanced-autophagy in HK-2 cells by inhibiting the
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PI3K/AKT/mTOR signaling pathway.
We regulated miR-145 expression in HK-2 cells and measured PI3K/AKT/mTOR signaling pathway
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related proteins. A-B: Western blotting results showed that when we decreased the expression of miR-145 by siRNA, phosphorylation of PI3K, AKT, and mTOR was induced, but was inhibited when we upregulated the expression of miR-145 by mimics. C-D; Western blotting results indicated that transfection with miR-145 siRNA in the hUC-MSCs co-culture system partially rescued the
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phosphorylation of PI3K, AKT, and mTOR, which was inhibited by hUC-MSCs. Data are expressed as the means ± SD from 3 independent experiments; *P < 0.05 vs. Ctrl group (A, B) or AOPP group (C,
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D); # P < 0.05 vs. AOPP+hUC-MSCs group.
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S0014-4827(18)31047-4
DOI:
https://doi.org/10.1016/j.yexcr.2019.03.019
Reference:
YEXCR 11355
To appear in:
Experimental Cell Research
Received Date: 2 October 2018 Revised Date:
10 March 2019
Accepted Date: 11 March 2019
Please cite this article as: J. Xiang, T. Jiang, W. Zhang, W. Xie, X. Tang, J. Zhang, Human umbilical cord-derived mesenchymal stem cells enhanced HK-2 cell autophagy through MicroRNA-145 by inhibiting the PI3K/AKT/mTOR signaling pathway, Experimental Cell Research (2019), doi: https:// doi.org/10.1016/j.yexcr.2019.03.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Human Umbilical Cord-Derived Mesenchymal Stem Cells Enhanced HK-2 Cell Autophagy Through MicroRNA-145 By Inhibiting The
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PI3K/AKT/mTOR Signaling Pathway
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Jin Xiang1, 2#, Tingting Jiang3#, Wenying Zhang1, Wei Xie1, Xun Tang1*, Jun Zhang1*
Guangdong Province, 510282, China
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1 Department of Nephrology, Zhujiang Hospital of Southern Medical University, Guangzhou,
2 Department of Nephrology, People's Hospital of Yuxi City, Yuxi, Yunnan Province, 653100, China
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3 Department of Nephrology, The People’s Hospital of Guangxi Zhuang Autonomous Region,
*
These authors contributed equally to this work.
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#
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Nanning, Guangxi Province, 530021, China
corresponding author: Jun Zhang, Department of Nephrology, Zhujiang Hospital of Southern Medical University, 253 Gongye Road, Guangzhou, Guangdong, 510282 (PR China). E-mail: [email protected]
Xun Tang, Department of Nephrology, Zhujiang Hospital of
ACCEPTED MANUSCRIPT Southern Medical University, 253 Gongye Road,
Guangzhou, Guangdong, 510282 (PR China).
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E-mail: [email protected]
Abstract: Recent studies have shown that autophagy exhibits a protective role in
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acute kidney injury (AKI), and the accumulation of advanced oxidation protein products (AOPP) participates in the progression of kidney diseases. Our previous study indicated that AOPP induced injury in renal tubular epithelial cells (RTECs) through autophagy inhibition. Besides, we found that human umbilical cord-derived
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mesenchymal stem cells (hUC-MSCs) enhanced autophagy in AOPP-treated RTECs, but the underlying mechanism remains unclear. We regulated microRNA-145
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(miR-145) expression in HK-2 cells (a cell line of RTECs), or co-cultured hUC-MSCs with HK-2 cells and studied the autophagic activity in HK-2 cells to explore the
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underlying mechanism. Our data demonstrated that upregulated miR-145 increased LC3 II and Beclin 1 levels, decreased p62 level, three autophagy related proteins, inhibited the phosphorylation of PI3K/AKT/mTOR, and increased LC3B-positive staining and the autophagosome number. Furthermore, hUC-MSCs enhanced autophagy and inhibited phosphorylation of PI3K/AKT/mTOR in AOPP-treated HK-2 cells, which was then partially rescued using miR-145 knockdown in the hUC-MSCs co-culture system. In conclusion, our study showed that hUC-MSCs
ACCEPTED MANUSCRIPT enhanced autophagy in AOPP-treated HK-2 cells mediated by miR-145 via inhibition of the PI3K/AKT/mTOR pathway, which indicated that hUC-MSCs might serve as a
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prospective therapy for AKI.
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Keywords: hUC-MSCs; miR-145; autophagy; HK-2 cells; PI3K/AKT/mTOR
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Introduction
Acute kidney injury (AKI) is a global public health concern and is associated with high morbidity, mortality, and healthcare costs; AKI impacts approximately 13.3 million patients per year [1]. Long-term outcomes may include the development of
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chronic kidney disease (CKD) [2] and end-stage renal disease (ESRD) [3], which require renal replacement therapy and cause a heavy economic burden. Therefore, it is
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important to understand the mechanisms of AKI and develop an effective treatment strategy to reduce the occurrence of AKI or slow the development of CKD and
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ESRD.
A growing number of studies have revealed the role of autophagy in kidney
diseases [4] [5], both in the tubulointerstitial compartment and in glomeruli. Pathologically, AKI is characterized by tubular cell injury and death. In addition, the role of autophagy in tubules, which is the key determinant of kidney prognosis, has been demonstrated in renal tubular epithelial cells (RTECs) [6] and is generally
ACCEPTED MANUSCRIPT accepted as a protective cellular response in AKI under various causes [7] [8]. Advanced oxidation protein products (AOPP) is a harmful protein product that is highly expressed in patients with kidney diseases. Furthermore, phosphatidylinositol
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3-kinase (PI3K)/AKT/mTOR signaling pathway is a crucial negative modulator of autophagy[9]. Our work team have made some researches about the harmful effects of AOPP on kidney diseases. In the latest investigation, our data showed that AOPP
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inhibited autophagy of RTECs trough activation of the PI3K/AKT/mTOR signaling
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pathway. Moreover, we also reported that autophagy inhibition mediated AOPP-induced RTEC injury in this study. [10]. Therefore, enhancing RTEC autophagy may be critical for suppressing the progression of kidney diseases including AKI.
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Currently, stem cell therapy has therapeutic potential to repair the impaired kidney. The mesenchymal stem cell (MSC) is a mesodermal stem cell that has the ability to
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self-renew and differentiate into other mesodermal cell lineages. Compared with MSCs from other sources, human umbilical cord-derived MSCs (hUC-MSCs) have
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received much attention because of their abundance, ease of separation, strong growth capacity, low immunogenicity, and lack of harm to mothers and newborns [11]. It is interesting to note that a systematic review and meta-analysis entering 71 articles showed that MSC therapy could delay the progression of CKD according to subgroup analysis, blood biochemical indexes, different related models and cell related factors [12]. Furthermore, the study of MSCs in the treatment of kidney diseases has become one of the hot topics in international nephrology research, including diabetic
ACCEPTED MANUSCRIPT nephropathy [13], renal transplantation [14], and lupus nephritis [15]. More excitingly, we have found that hUC-MSCs enhanced autophagy in AOPP-injured RTEC, which may perform a protective role in renal tissue and cells (the article is under review.)
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An increasing number of studies have demonstrated that MSCs regulate the microenvironment of renal cells to play a protective role by secreting a variety of
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cytokines, proteins and miRNAs [16] [17]. Notably, the potential therapeutic role of microRNA-145 (miR-145) secreted from MSCs has attracted increasing attention. A
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previous study found that hUC-MSCs inhibited the TGF-β/SMAD2 signaling pathway to protect the body from burn injury by secreting miR-145 [18]. Another study showed that MSCs inhibited the migration and self-renewal of glioma cells and glioma stem cells by secreting miR-145 [19]. More excitingly, it was reported that
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miR-145 repaired myocardial infarction by promoting myocardial cell autophagy [20]. In addition, there was another study showing that upregulation of miR-145 induced
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inhibition of the PI3K/AKT/mTOR pathway in laryngeal squamous cell carcinoma[21]. However, whether miR-145 secreted from hUC-MSCs plays
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protective roles in AKI by enhancing RTEC autophagy and the underlying mechanism need to be explored.
To further investigate the relationship between MSCs and renal autophagy, we
performed the current study to investigate the role of hUC-MSCs on autophagy inhibited by AOPP in HK-2 cells (a cell line of human RTECs) and the underlying mechanism.
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Material and Methods
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1. Materials and Reagents Bovine serum albumin (BSA) was purchased from Sigma (St. Louis, USA). Antibodies against LC3B, Beclin 1, p62, p-mTOR, mTOR, p-AKT, AKT, and p-PI3K
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were obtained from Cell Signaling Technology (Beverly, USA). The GAPDH antibody was purchased from Bioworld (Minnesota, USA). Antibodies against CD90,
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CD105, CD73, CD34, CD45, CD11b, CD14, and HLA-DR for flow cytometry were purchased from Becton Dickinson and Company (New Jersey, USA). The U6 and miR-145 primers were obtained from Ribobio Company (Guangzhou, China). Sequencing of miR-145 siRNA, miR-145 mimics, and negative siRNA was
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performed by Shanghai GenePharma Co. Ltd (Shanghai, China).
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2. AOPP Preparation
AOPP was prepared as previously described [22]. Briefly, HOCl (200 mmol/L) was
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mixed with BSA solution for 30 min at room temperature, and then dialyzed against PBS at 4°C to remove free HOCl for 24 h. Native BSA was dissolved in PBS alone as the control. The endotoxin levels were measured and were required to be <0.025 EU/mL. The AOPP content was measured at 340 nm to obtain the absorbance under acidic conditions and calibrated using chloramine-T in the presence of potassium iodide.
3. HK-2 Cell Culture and Treatment
ACCEPTED MANUSCRIPT HK-2 cells were purchased from the American Type Culture Collection (Rockville, USA) and cultured in DMEM/F12 (Gibco, USA) supplemented with 10% heat-inactivated FBS and maintained in a humidified incubator containing a 5% CO2
until they reached approximately 70-80% confluence.
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4. hUC-MSCs Isolation and Co-culture with HK-2 cells
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atmosphere at 37°C. Cells were incubated in BSA (200 µg/mL) or AOPP (200 µg/mL)
We isolated hUC-MSCs using an adherent tissue method[23]. Briefly, a 10 cm
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umbilical cord from a full-term healthy newborn was washed with PBS (containing 1% penicillin-streptomycin double-resistant solution) 3 times. Then, we cut the cord into small pieces, dislodged the umbilical vein and umbilical artery, and left Wharton's
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jelly at last. Wharton's jelly was then cut into 1 mm × 1 mm × 1 mm and cultured in DMEM/F12 medium containing 5% FBS; 3-6 passages (P3-6) were selected for the following experiments. We used a co-culture chamber to block the immediate contact
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between the hUC-MSCs and HK-2 cells to explore the paracrine action of hUC-MSCs
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in the co-culture system.
5. Flow Cytometry and hUC-MSCs Identification hUC-MSCs were trypsinized, and the cell concentration was adjusted to 1×106/mL
in PBS. Then, 200 µl of the suspension was incubated with 5 µl of antibodies against CD90, CD105, CD73, CD34, CD45, CD11b, CD14, and HLA-DR without light for 30 min. Primary antibodies were directly conjugated with FITC and phycoerythrin. For isotype control, non-specific FITC-conjugated IgG was substituted for the
ACCEPTED MANUSCRIPT primary antibodies. Lastly, the samples were analyzed using flow cytometry.
6. RNA Extraction and Quantitative Real-time PCR (RT-qPCR) Analysis
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Total RNA was extracted from treated HK-2 cells using TRIzol reagent (TaKaRa, Japan). 1 µg RNA was reverse transcribed using the MMLV reverse transcriptase kit according to the manufacturer’s instructions (TaKaRa, Japan). The U6 and miR-145
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primers were provided directly by RiboBio Company and the sequences were kept
U6.
7. Western Blotting Analysis
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secreted by RiboBio Company. All data were normalized using the internal control
We extracted total protein using RIPA lysis buffer. Then, 20-50 µg of protein was
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separated using SDS-PAGE and transferred to PVDF membranes. Subsequently, the membranes were blocked in 5% nonfat milk or BSA at room temperature for 2 h,
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followed by incubation of the primary antibodies at 4°C overnight. The primary antibodies were as follows: anti-LC3B, anti-Beclin 1, anti-p62, anti-p-mTOR,
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anti-mTOR, anti-p-AKT, anti-AKT, anti-p-PI3K (dilution, 1:1000) and anti-GAPDH (dilution,
1:5000).
The membranes
were
incubated
with
the
appropriate
HRP-conjugated secondary antibodies at room temperature for 2 h and were finally detected using an enhanced chemiluminescence (ECL) system. GAPDH was used as the internal control and semiquantitative analysis was performed using the ImageJ system.
8. Immunofluorescence Staining
ACCEPTED MANUSCRIPT Cells plated in 96-well plates were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 10 minutes and incubated in a blocking buffer containing 5% BSA for 30 min at room temperature. Then, cells were incubated with LC3B Finally, we incubated cells with
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antibody (1:50 dilution) overnight at 4°C.
fluorescently labeled secondary antibodies (Alexa Fluor 488, 1:400, Gongsi) for 1 h at room temperature in the dark, followed by incubation with 0.1% DAPI for 10 min.
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We used an inverted fluorescence microscope to observe and record the
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LC3B-positive staining.
9. Transmission Electron Microscopy (TEM)
HK-2 cells were gently harvested using trypsin with EDTA and were centrifuged at a speed
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of 1000 r/m for 5 min. Then, the sediment was washed twice with cold PBS and fixed in 2.5% glutaraldehyde for 2 h at 4°C. Cells were conventionally dehydrated, embedded, sliced into 60 nm sections and stained with uranyl acetate for 15 min at room temperature, followed by
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lead citrate for 15 min at room temperature. Autophagosome (AP) and autolysosome (AL)
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formation were observed using TEM at a magnification of low-power field (x6000, x8000, x10000) or high-power field (x40000) operating at 60 kV.
10. HK-2 Cell Transfection And hUC-MSC Transfection
HK-2 cells were transfected with miR-145 siRNA, miR-145 mimics, or negative siRNA, and hUC-MSCs was transfected with miR-145 siRNA, using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions. Targeting sequences were as follows: miR-145 siRNA: sense (5’-
ACCEPTED MANUSCRIPT AGGGAUUCCUGGGAAAACUGGAC-3’); miR-145 mimics: sense (5’GUCCAGUUUUCCCAGGAAUCCCU-3’), antisense (5’GGAUUCCUGGGAAAACUGGACUU-3’); negative siRNA: sense
(5’-ACGUGACACGUUCGGAGAATT-3’).
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11. Statistical Analysis
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(5’-UUCUCCGAACGUGUCACGUTT-3’), antisense
Statistical analyses were performed using SPSS 20.0 software. Continuous
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variables are presented as the mean ± SD. One-way ANOVA was carried out to determine differences among groups. For the comparison of 2 groups, the LSD method was used when the assumption of variance was equal. Otherwise, the Dunnett
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Results
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T3 method was used. P values < 0.05 indicated a statistically significant difference.
1. Identification results of hUC-MSCs
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We chose P3 hUC-MSCs to identify the cell phenotype by flow cytometry.
Inverted phase contrast microscopy showed that P4 hUC-MSCs presented 50% fusion and 100% vortex fusion, as shown in in Figure 1A and 1B. Flow cytometry analysis of the cell phenotype showed that the hUC-MSCs were positive for CD90, CD105, and CD73, but were negative for CD34, CD45, CD11b, CD14, and HLA-DR (Figure 1C-J).
ACCEPTED MANUSCRIPT 2. miR-145 was upregulated in HK-2 cells under hUC-MSCs co-cultured system We co-cultured hUC-MSCs with HK-2 cells and used RT-qPCR to measure the expression of miR-145 in HK-2 cells. The result showed that when HK-2 cells were
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co-cultured with hUC-MSCs, the mRNA level of miR-145 was significantly upregulated. Next, we knocked down miR-145 in the hUC-MSCs and then
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co-cultured it with HK-2 cells. We found that in the later co-culture system, miR-145 did not increased in HK-2 cells (Figure 2). The data indicated that hUC-MSCs
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secreted miR-145 and transferred it to HK-2 cells through paracrine action.
3. miR-145 affected HK-2 cell autophagy
We first downregulated or upregulated the expression of miR-145 in HK-2 cells
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and examined the mRNA level of miR-145. The RT-qPCR results showed that miR-145 siRNA downregulated miR-145; however, miR-145 mimics upregulated miR-145 in HK-2 cells. At the same time, the expression of miR-145 showed no
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difference between negative siRNA and the normal control group (Figure 3A).
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We then assayed the autophagy-related proteins LC3B, Beclin 1, and p62 by
western blotting. The results in figure 3(B-C) showed that when we inhibited the expression of miR-145 by siRNA, the protein levels of LC3 II and Beclin 1 decreased and the protein level of p62 increased. However, when we upregulated the expression of miR-145, the protein levels of those markers mentioned above had the opposite effect. In addition, there was no difference between negative siRNA and the normal control group.
ACCEPTED MANUSCRIPT Furthermore, the autophagic activity of HK-2 cells was assessed using immunofluorescence technology and TEM. LC3B-positive staining was significantly decreased in cells transfected with miR-145 siRNA but increased in cells transfected
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with miR-145 mimics compared with that of the normal control group (Figure 3D). Similarly, the TEM results demonstrated a decrease in the number of typical Aps (double membrane vacuoles engulfing cytoplasmic structures) and ALs (single
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membrane vacuole structures containing high density materials) in miR-145 siRNA
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transfected cells but revealed an increase in the number of Aps and ALs in miR-145 transfected cells compared with those in the normal control group (Figure 3E). Collectively, these results suggested that overexpression of miR-145 activated HK-2 cell autophagy.
cells
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4. miR-145 mediated hUC-MSCs enhanced-autophagy in AOPP-treated HK-2
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To further confirm that hUC-MSCs-enhanced autophagy was mediated by the
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secretion of miR-145, we transfected HK-2 cells with miR-145 siRNA after AOPP-treated HK-2 cells were co-cultured with hUC-MSCs to investigate the effect of hUC-MSCs on HK-2 cells. We first measured the miR-145 expression in transfected and co-culture system. RT-qPCR analysis showed that the level of miR-145 was increased in the hUC-MSCs and AOPP co-culture group compared with that in the normal control group, but the miR-145 level was decreased in the transfected and co-culture system compared with the hUC-MSCs and AOPP co-culture group (Figure 4A).
ACCEPTED MANUSCRIPT Next, we detected the autophagous activity after transfection with miR-145 siRNA in the hUC-MSCs and AOPP co-culture system. The western blotting results showed that when we decreased the expression of miR-145 by siRNA, the protein levels of
those in
the hUC-MSCs
co-culture system
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LC3 II and Beclin 1 decreased and the p62 protein level increased compared with (Figure 4B-C).
Furthermore,
LC3B-positive staining was significantly decreased in cells transfected with miR-145
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siRNA compared with that in the hUC-MSCs co-culture system (Figure 4D).
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Similarly, the TEM results demonstrated a decrease in the numbers of typical Aps and ALs in HK-2 cells in siRNA transfected with the hUC-MSCs co-culture system compared with those in the hUC-MSCs co-culture system alone (Figure 4E). Taken together, these results indicated that miR-145 secreted from hUC-MSCs enhanced
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HK-2 cell autophagy.
miR-145 mediated hUC-MSCs enhanced-autophagy in HK-2 cells by
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inhibiting the PI3K/AKT/mTOR signaling pathway
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To verify the underlying pathway, we detected PI3K/AKT/mTOR signaling pathway related proteins after regulating miR-145 expression in HK-2 cells or transfection with miR-145 siRNA in the hUC-MSCs co-culture system. The western blotting results showed that when we decreased the expression of miR-145 by siRNA, the phosphorylation of PI3K, AKT, and mTOR was induced. However, when we upregulated the expression of miR-145 by mimics, the phosphorylation of PI3K, AKT, and mTOR was inhibited. In addition, there was no difference between negative siRNA and the normal control group (Figure 5A-B). Finally, transfection with
ACCEPTED MANUSCRIPT miR-145 siRNA in the hUC-MSCs co-culture system promoted the phosphorylation of PI3K, AKT, and mTOR, which was inhibited by hUC-MSCs (Figure 5C-D). We concluded that miR-145 mediated hUC-MSCs enhanced-autophagy in HK-2 cells via
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inhibiting the PI3K/AKT/mTOR signaling pathway.
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Discussion
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The present study demonstrated the significant role of hUC-MSCs in HK-2 cell autophagy in AKI. We provided evidence that hUC-MSCs increased HK-2 cell autophagy via secretion of miR-145 through inhibition of the PI3K/AKT/mTOR signaling pathway. The current results implicate hUC-MSCs as a promising
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therapeutic strategy in protecting AKI through its paracrine action.
A considerable number of studies have focused on the role of autophagy in renal
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diseases. An increasing number of findings have highlighted that autophagy is rapidly induced during AKI to protect tubular cells from injury and death, and
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autophagy-related gene deficiency leads to impaired renal function and tubular cell apoptosis and necrosis in conditional knockout mouse models [24] [25]. However, our study found that AOPP inhibited HK-2 cell autophagy and induced HK-2 cell injury instead of inducing autophagy to protect cells [10]. Whether the noxious stimuli induce or inhibit cell autophagy may depend on the tissue, magnitude, time or other factors that cells suffer. Livingston and colleagues [26] summarized a similar opinion, proposing that it was very important to monitor autophagy in the correct tissue at the
ACCEPTED MANUSCRIPT correct time to determine the optimal conditions and therapeutic window, thus ensuring that autophagy induction would yield protective effects in renal diseases. Moreover, given the evidence that autophagy plays an important role in the
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development and severity of AKI, targeting autophagy could be a novel and potential strategy for the treatment of AKI and even other renal diseases. In this study, we discovered a new mechanism under autophagy regulation by hUC-MSCs in HK-2
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cells.
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The first key finding of this study was that miR-145 was upregulated in hUC-MSCs and HK-2 cells in a co-culture system. It is general knowledge that MSCs demonstrate paracrine mechanisms, including the secretion of various nutrition factors, proteins, mRNA, or miRNA. In this way, MSCs directly or indirectly induce regeneration,
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rescuing injured cells and accelerating organ repair [27]. We co-cultured hUC-MSCs with HK-2 cells and found that miR-145 expression was upregulated in HK-2 cells in
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this system. We then knocked down miR-145 in the hUC-MSCs before it co-cultured with HK-2 cells and found that miR-145 did not increased in HK-2 cells, which
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indicated that hUC-MSCs secreted miR-145 and transported it to HK-2 cells through paracrine action. Previous studies showed that MSCs delivered exogenous miRNAs to neural cells [28] or myofibroblasts [18]. In addition, it has been proposed that MSC-derived extracellular vesicles protect against renal injury [29] [30]. However, the underlying mechanism of MSC treatment on renal diseases and whether the upregulated miR-145 in HK-2 cells derived from hUC-MSCs contribute to the renal protection are unclear.
ACCEPTED MANUSCRIPT The second crucial finding of this study was that regulation of miR-145 affected HK-2 cell autophagy. Currently, may studies have indicated that abnormal expression of miRNA participates in the regulation and progression of various renal diseases,
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such as AKI[31], diabetic nephropathy[32], and IgA nephropathy [33]. Furthermore, multiple studies have previously demonstrated that miRNA affects renal function or the pathology and physiological of renal cells through various mechanisms. Zou et al.
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[34] demonstrated that miRNA mediated epithelial mesenchymal transition in fibrotic
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diseases, including renal fibrosis. Dewanjee et al. [35] concluded in their review that miRNA regulated various molecular pathways in diabetic nephropathy, including the TGF-β signaling pathway, PI3K/AKT signaling pathway, and collagen gene expression. To investigate whether miR-145 influences HK-2 cell autophagy, we first
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downregulated or upregulated the expression of miR-145 in HK-2 cells. The results showed that when we inhibited the expression of miR-145 by siRNA, the protein levels of LC3 II and Beclin 1 decreased and the protein level of p62 increased and
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phosphorylation of PI3K, AKT, and mTOR increased, but the expression of these
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markers showed the opposite change when we upregulated the overexpressed miR-145 with mimics. In addition, LC3B-positive staining and the numbers of typical Aps and ALs were significantly decreased in cells transfected with miR-145 siRNA but increased in cells transfected with miR-145 mimics compared with those in the normal control group. Collectively, these results suggested that overexpression of miR-145 activated HK-2 cell autophagy and that miR-145 knockdown inhibited HK-2 cell autophagy. It was reported that miR-145 repaired myocardium by accelerating
ACCEPTED MANUSCRIPT cardiomyocyte autophagy [20]. There was also another investigation showing that miRNA-145 promoted osteosarcoma cell autophagy by targeting HDAC4 [36]. In addition, there was a study showing that upregulation of miR-145 induced inhibition
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of the PI3K/AKT/mTOR pathway in laryngeal squamous cell carcinoma[21]. Excitingly, our study was the first time to show that miR-145 affects RTEC
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autophagy by inhibiting PI3K/AKT/mTOR signaling pathway.
Finally, we confirmed that hUC-MSCs enhanced HK-2 cell autophagy mediated by
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the secretion of miR-145 through inhibiting the PI3K/AKT/mTOR signaling pathway. In our previous investigation, we proposed that hUC-MSCs enhanced autophagy in AOPP-treated HK-2 cells, but the underlying mechanism requires further investigation. Accumulating evidence suggests that MSCs enhance autophagy through
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various classical pathways or molecules. Li et al. [37] showed that bone marrow-derived MSCs enhanced autophagy via PI3K/AKT signaling to reduce the
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severity of ischaemia/reperfusion-induced lung injury. Liu et al. [38] suggested that MSC induced cardiomyocyte autophagy via the AMPK and AKT signaling pathways
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to rescue myocardial ischaemia/reperfusion injury. To our knowledge, MSCs primarily possess paracrine or endocrine functions, which allow them to play a protective role. Wang et al. [39] indicated that hUC-MSCs-derived exosomes increased the expression of the autophagic marker protein LC3B and the autophagy-related genes ATG5 and ATG7 in cisplatin-treated NRK-52E cells to relieve the nephrotoxicity of cisplatin. In addition, Jia et al. [40] presented a similar conclusion in another study, showing that hUC-MSCs-derived exosomes enhanced
ACCEPTED MANUSCRIPT autophagy via transmission of 14-3-3zeta and modulation of ATG16L in preventing cisplatin-induced AKI. Since paracrine secretion of miR-145 from hUC-MSCs is delivered to HK-2 cells, to further uncover whether hUC-MSCs enhance autophagy
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through secretion of miR-145, we transfected HK-2 cells with miR-145 siRNA after AOPP-treated HK-2 cells were co-cultured with hUC-MSCs. We found that when we decreased the expression of miR-145 by siRNA, the protein levels of LC3 II and
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Beclin 1 decreased, the p62 protein level increased, the LC3B-positive staining and
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numbers of typical Aps and ALs decreased, and the phosphorylation of PI3K, AKT, and mTOR increased compared with those in hUC-MSCs co-culture system. Taken together, in this study, we presented for the first time a new mechanism of hUC-MSCs-enhanced cell autophagy by secreting miR-145 through inhibition of the
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PI3K/AKT/mTOR signaling pathway.
We did not explore how hUC-MSCs transported secreted miR-145 to HK-2 to
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enhance cell autophagy. Previous studies found that MSCs transported RNA or protein to target cells through MSCs-derived exosomes to repair damaged cells and
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tissues[41]. Future in-depth studies will explore whether exosomes act as the mechanism of hUC-MSCs on HK-2 cells. Additionally, the amount of miR-145 expressed in transfection is not the same with that secreted by hUC-MSCs, and we will further regulate and investigate the effects of different dosage of miR-145 secreted by hUC-MSCs on autophagy in HK-2 cells. Finally, further in vitro studies are warranted to validate the significance of hUC-MSCs and secreted miR-145 in the enhancement of autophagy in HK-2 cells and primary cells and even in AKI models.
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Conclusion
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In conclusion, these results indicated that hUC-MSCs played an autophagy enhancement role via overexpression of miR-145, which provided a novel mechanism for the investigation of hUC-MSCs in AKI treatment. MSCs represent an amazing
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therapeutic tool in regenerative medicine; therefore, developing a better understanding
kidney diseases.
Conflict of interest statement
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None.
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of the mechanism of MSCs in renal protection is a promising therapeutic strategy for
Acknowledgements
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The present study was supported by the Natural Science Foundation of Guangdong Province (grant no. 2018A030313557).
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Figure legends
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Fig. 1 hUC-MSCs identification
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A: Inverted phase contrast microscope images (× 50) showing that P4 hUC-MSCs presented 50%
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fusion; B: Inverted phase contrast microscope images (× 50) showing that P4 hUC-MSCs presented 100% vortex fusion; C-J: Flow cytometry showed that hUC-MSCs were positive for CD90, CD105,
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and CD73, but were negative for CD34, CD45, CD11b, CD14, and HLA-DR.
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Fig. 2 Detection of miR-145 in HK-2 cells in the hUC-MSCs co-culture system
The RT-qPCR results showed that miR-145 expression was upregulated in HK-2 cells in the hUC-MSCs co-culture system (hUC-MSCs group). Next, we knocked down miR-145 in the hUC-MSCs and then co-cultured it with HK-2 cells. We found that in the later co-culture system
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(hUC-MSCs siRNA group), miR-145 did not increased in HK-2 cells. Data are expressed as the means
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± SD from 3 independent experiments; *P < 0.05 vs. Ctrl group. Ctrl: normal control.
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Fig. 3 miR-145 affected HK-2 cell autophagy.
We inhibited or promoted the expression of miR-145 in HK-2 cells and detected autophagy related
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markers. A: The RT-qPCR results showed that miR-145 siRNA downregulated miR-145 but miR-145 mimics upregulated miR-145 in HK-2 cells. B-C: Western blotting results showed that LC3 II and
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Beclin 1 were decreased and p62 was increased in the miR-145 siRNA group, but the opposite trend was found in the miR-145 mimics group. D: Immunofluorescence result showed that LC3B-positive
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staining decreased in cells transfected with miR-145 siRNA but increased in cells transfected with miR-145 mimics. E: TEM experiments revealed that typical Aps and ALs were significantly decreased in cells transfected with miR-145 siRNA but increased in cells transfected with miR-145 mimics (the above is the low-power field, x8000, or x10000, and the below is the high-power field, x40000). Data are expressed as the means ± SD from 3 independent experiments; *P < 0.05 vs. Ctrl group. Ctrl: normal control.
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Fig. 4 hUC-MSCs enhanced autophagy in AOPP-treated HK-2 cells by secretion of miR-145.
We transfected HK-2 cells with miR-145 siRNA in the hUC-MSCs and AOPP co-culture system. A: RT-qPCR analysis showed that the increased miR-145 secreted from hUC-MSCs was partially decreased in the miR-145 siRNA transfected and co-culture system compared with that in the
ACCEPTED MANUSCRIPT hUC-MSCs and AOPP co-culture group. B-C: Western blotting showed that LC3 II and Beclin 1 was decreased and p62 was increased when HK-2 cells were transfected with miR-145 siRNA compared with those in the hUC-MSCs co-culture system. D: Immunofluorescence showed that LC3B-positive
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staining was decreased in cells transfected with miR-145 siRNA. E: TEM experiments presented that typical Aps and ALs were significantly decreased in cells transfected with miR-145 siRNA (the above is the low-power field, x6000, or x8000, and the below is the high-power field, x40000). Data are
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expressed as the means ± SD from 3 independent experiments; *P < 0.05 vs. AOPP group; # P < 0.05
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vs. AOPP+hUC-MSCs group.
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Fig. 5 miR-145 mediated hUC-MSCs enhanced-autophagy in HK-2 cells by inhibiting the
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PI3K/AKT/mTOR signaling pathway.
We regulated miR-145 expression in HK-2 cells and measured PI3K/AKT/mTOR signaling pathway
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related proteins. A-B: Western blotting results showed that when we decreased the expression of miR-145 by siRNA, phosphorylation of PI3K, AKT, and mTOR was induced, but was inhibited when we upregulated the expression of miR-145 by mimics. C-D; Western blotting results indicated that transfection with miR-145 siRNA in the hUC-MSCs co-culture system partially rescued the
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phosphorylation of PI3K, AKT, and mTOR, which was inhibited by hUC-MSCs. Data are expressed as the means ± SD from 3 independent experiments; *P < 0.05 vs. Ctrl group (A, B) or AOPP group (C,
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D); # P < 0.05 vs. AOPP+hUC-MSCs group.
AC C
EP
TE D
M AN U
SC
RI PT
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