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Original Article MicroRNA-22 inhibits apoptosis and promotes migration in human osteoarthritis (OA) chondrocytes by targeting SP1 and activating ERK/NF-κB pathway

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Original Article

MicroRNA-22 inhibits apoptosis and promotes

migration in human osteoarthritis (OA) chondrocytes

by targeting SP1 and activating ERK/NF-κB pathway

Qichun Zhao, Wei Che, Kai Cheng, Puyan Zhang, Chengxiang Zhu, Yingming Wang

Department of Orthopedics, Anhui Provincial Hospital, Anhui Medical University, Hefei 230001, Anhui, China

Received February 15, 2017; Accepted April 24, 2017; Epub June 1, 2017; Published June 15, 2017

Abstract: The current study aimed to elucidate the role and possible mechanism of microRNA-22 (miR-22) in the development of osteoarthritis (OA). The expression of miR-22 and SP1 was observed in human OA chondrocytes. In order to determine the effect of miR-22 dysregulation on cell proliferation, apoptosis, and migration, the chondro-cytes were transfected with a miR-22 mimic, miR-22 inhibitor and scrambled miR-22. Further, it was also analyzed

if SP1 is the direct target of miR-22. An association was established between miR-22 and DNA ERK/NF-κB pathway. Human OA chondrocytes showed significant down-regulation of miR-22 and a marked up-regulation of SP1. The miR-22 suppression significantly promoted proliferation and migration of chondrocytes and inhibited apoptosis. On

the contrary, these observations showed a reverse pattern upon over-expression of miR-940. The co-transfection of

miR-22 inhibitor and si-SP1 showed that SP1 is the direct target of miR-22, as the knockdown of SP1 significantly

affected the miR-22 mediated inhibition of apoptosis and the migration of chondrocytes. Furthermore, the

suppres-sion of miR-22 led to a significant increase in the expressuppres-sion of p-ERK and p-p65 and a decrease in the expressuppres-sion of IκBa. The co-transfection with miR-22 inhibitor and PD98059 or PDTC showed a considerable reversal of the effects of miR-22 inhibition on apoptosis and migration of chondrocytes. Thus, our findings indicate that the sup -pression of miR-22 may lead to the inhibition of apoptosis and induce the migration of OA chondrocytes by targeting

SP1 and activating the ERK/NF-κB pathway.

Keywords: Osteoarthritis, miR-22, SP1, ERK/NF-κB pathway

Introduction

Osteoarthritis (OA) is a common degenerative disease of the joints that results in joint pain, stiffness and the loss of function of the joint tissues [1, 2]. It is characterized by the inflam -mation and an imbalance between the repair and degradation of the articular cartilage in joints [3, 4]. The incidence of OA continues to rapidly increase worldwide, while no effective treatment is available for it till date [5, 6]. Unfortunately, the progression of OA remains elusive. Therefore, it is really important to understand the underlying molecular events that are involved in the development of OA condition.

MicroRNAs (miRNAs), a group of small non-cod-ing RNAs, can regulate the expression of their target mRNAs by binding to the 3’-untranslated

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Roles of miR-22 in human OA chondrocytes

miR-22 has been reported to be involved in cel-lular metabolism and chronic inflammatory dis -eases, including the OA [20]. However, the potential role and possible regulatory mecha-nism of miR-22 in the development of OA is not thoroughly investigated.

In the present study, chondrocytes were isolat-ed from articular cartilage of the OA patients. The expression levels of miR-22 and SP1 were analyzed. The isolated chondrocytes were sub-jected to the transfection with miR-22 mimic, miR-22 inhibitor and a scramble to study the effect of miR-22 dysregulation on cell prolifera-tion, apoptosis, and migration. In addiprolifera-tion, it was also assessed if the SP1 is a direct target of miR-22. Besides, a relationship between the miR-22 and the DNA ERK/NF-κB pathway was also observed. The purpose of our current anal-ysis was to elucidate the potential role and pos-sible mechanism of miR-22 in the development of osteoarthritis.

Materials and methods

Chondrocytes isolation

This study was approved by the Ethics Co- mmittee of Anhui provincial hospital, Anhui Medical University (Anhui, Hefei, China). Ch- ondrocytes were isolated from the articular car-tilage of patients who were diagnosed with OA and underwent total knee arthroplasty in the hospital. The articular cartilage was minced into small pieces (1 mm × 1 mm × 1 mm), di- gested with 0.15% Type II collagenase (Wor- thington, Lakewood, NJ) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (FBS, Gibco) for incubation of 16 h at 37°C.

Cell transfection and treatment

The isolated chondrocytes were maintained in the culture as a monolayer until confluent, fol -lowed by the transfection with miR-22 mimic, miR-22 inhibitor, scramble, or siRNA-SP1 (si-SP1) using Lipofectamine 2000 (Invitrogen) as per the manufacturer’s protocol. After incuba-tion for 12 h, 10 μmol/L PD98059 (Calbiochem, San Diego, CA), an ERK inhibitor or 10 μmol/L pyrrolidine dithiocarbamate (PDTC) (Sigma, St Louise, CA, USA), an inhibitor of NF-κB, was added to the cell culture medium and the cells

were incubated at appropriate culture condi- tions.

Cell proliferation assay

Chondrocytes (5 × 103 cells/well) were grown in 96-well tissue culture plates and allowed to adhere overnight. The viable cells were estimat-ed at different time points post transfection (24, 48, 72 and 96 h, respectively) using the Cell Counting Kit-8 (Dojindo Laboratory, Ku- mamoto, Japan) as per the instructions of the manufacturer. Briefly, each well was supple -mented with a 10% working solution of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4- disulphophenyl)-2H-tetrazolium monosodium salt (WST-8) and incubated at 37°C for 4 h. The absorbance at the wavelength 450 nm was measured by the microplate reader (SPECTRA- max190; Molecular Devices, Sunnyvale, CA, USA).

Cell apoptosis assay

Apoptosis of the cells was assessed by flow cytometry after dual staining by annexin-V-FITC and propidium iodide (PI) as per the standard protocol of the Annexin V-FITC cell apoptosis kit (Invitrogen, USA). In brief, post 48 h of transfec-tion, cells were harvested, washed thrice with PBS, and re-suspended in the staining buffer. The cells were incubated with 5 μL of annexin-V-FITC and 5 μL of PI for 10 min at room tem -perature and analyzed using a FACS Calibur flow cytometer (Becton-Dickinson, San Jose, CA, UDA) equipped with the Cell Quest software (Becton, Franklin Lakes, NJ, USA). Annexin V-positive and propidium iodide-negative cells indicated apoptotic cells.

Cell migration assay

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Quantitative real-time PCR (qRT-PCR)

Using Trizol (Invitrogen), total RNA was extract-ed from the cells and subjectextract-ed to reverse tran-scription (cDNA synthesis) by M-MLV reverse transcriptase (Promega). The qPCR reaction was conducted using the SYBR ExScript qRT-PCR Kit (Takara, China). The cycling parameters were set as follows: initial activation step at 95°C for 10 min, followed by 40 cycles of dena-turation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 15 s. The melt-ing curve analysis was performed to ensure that only one product was amplified. GAPDH or U6 were used as the endogenous reference controls to normalize the expression of mRNAs and miRNAs. The levels of gene expression were analyzed using the MxPro qPCR system software (Stratagene, La Jolla, CA, USA) and the eventual calculations were done using the 2-ΔΔCt method. The primers that were employed

in the study for amplification of the target genes are shown in Table 1.

Western blotting

Total protein was extracted using the RIA ly- sis buffer (Beyotime Biotechnology, Shanghai, China) containing protease mimics (Roche, Guangzhou, China). The quantification of total protein was done with the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). Western blot was performed using a Bio-Rad Bis-Tris Gel sys -tem as per the manufacturer’s instructions. Briefly, equal amounts of proteins were subject -ed to 12% SDS-PAGE, follow-ed by transfer onto a Polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The primary anti -bodies (Bax, ab51354; Bcl-2, ab119506; SP1, ab13370; caspase-3, ab13586; MMP1,

AB52-631; mmp3, ab53015; COL2A1, ab185430) (Santa Cruz, CA, USA) were diluted as 1:1000 and were incubated with the membrane at 4°C overnight, followed by incubation with horse-radish peroxidase-conjugated secondary anti-body for 1 h at room temperature. Afterward, the membranes were transferred into the Bio-Rad ChemiDoc™ XRS system and incubated with a chemiluminescent HRP substrate (Mi- llipore, MA, USA) to capture the signals. Pho- sphoglyceraldehyde dehydrogenase (GAPDH) was the internal control and the intensity of the bands was quantified by the Image Lab™ Software (Bio-Rad, Shanghai, China).

Vector construction and luciferase reporter assay

[image:3.612.90.524.84.217.2]

The target of miR-22 was predicted using the predictive software program TargetScan (http:// www.targetscan.org/). In order to validate if SP1 was a target of miR-22, 3’-UTR of wild-type (WT) SP1, containing the putative binding sites of miR-22, was inserted downstream of the fire -fly luciferase in pGL3 vector as a luciferase reporter. Mutant (MUT) 3’-UTR of SP1 contain-ing the mutated sequence in the complemen-tary site of the miR-22 was also inserted down-stream of the firefly luciferase in pGL3 to serve as a control. Concisely, the chondrocytes were seeded into a 48-well plate and then co-trans-fected with the miR-22 mimic and the lucifer-ase reporter comprising the WT or the MUT 3’- UTR of SP1 gene. The pRL-TK was used as the control vector. Post 48 h of transfection, lucifer-ase activity was measured on a beta-counter luminometer using the Dual Luciferase Assay kit (Promega) and Renilla luciferase activity served as an internal control.

Table 1. The primers used for the amplification of targets

Gene Forward primer (5’-3’) Reverse primer (5’-3’)

miR-22 AAGCTGCCAGTTGAAGAACTGTA CTAGCTAGCGTCATGAGGGCGAACGACGC

SP1 AATTTGCCTGCCCTGAGTGC TTGGACCCATGCTACCTTGC

Bax CTGAGCTGACCTTGGAGC GACTCCAGCCACAAAGATG

Bcl-2 CTGGTGGACAACATCGCTCTG GGTCTGCTGACCTCACTTGTG

Caspase-3 TGTCATCTCGCTCTGGTACG AAATGACCCCTTCATCACCA

MMP1 CTGGGAAGCCATCACTACCTTGC GTTTCTAGAGTCGCTGGGAAGCTG

MMP3 CCTCTATGGACCTCCCACAGAATC GTGCCAATGCCTGGAAAGTTC

COL2A1 TTCAGCTATGGAGATGACAATC AGAGTCCTAGAGTGACTGAG

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Roles of miR-22 in human OA chondrocytes

Statistical analysis

All the experiments were repeated thrice and the data from multiple experiments were expressed as mean ± SD (standard deviation). The differences between two groups were mea-sured using a one-way analysis of variance (ANOVA) by SPSS 19.0 statistical software. A value of P<0.05 indicated a statistically signifi -cant result.

Results

Inverse expression of miR-22 and SP1 in chondrocytes

In this study, chondrocytes were isolated from the articular cartilage of the patients who were undergoing the total knee arthroplasty. We

[image:4.612.94.516.72.424.2]

detected the expression of miR-22 and SP1 in the chondrocytes. Our observations indicated that in comparison to the normal chondrocytes, miR-22 was significantly down-regulated in the chondrocytes isolated from the articular carti-lage of the OA patients (P<0.05, Figure 1A), while the expression of SP1 was markedly increased (P<0.05, Figure 1B). To explore the role of miR-22 in OA development, chondro-cytes were transfected with miR-22 mimic, inhibitor, and scramble. As shown in the Figure 1C, miR-22 expression in the chondrocytes transfected with miR-22 mimic significantly increased in contrast to the scramble or the control groups, while it was markedly decreased in the miR-22 inhibitor group (P<0.05). No sig-nificant difference in the expression of miR-22 between the scramble and the control groups Figure 1. The inverse expression of miR-22 and SP1 in human OA chondrocytes. A: The expression of miR-22 in

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[image:5.792.94.702.74.477.2]

Figure 2. The suppression of miR-22 promoted proliferation, inhibited apoptosis, and enhanced migration of chondrocytes. A: MTT assay showed the effects of

miR-22 dysregulation on cell viability. B: Flow cytometry showed the effects of miR-22 dysregulation on cell apoptosis. C: The expression levels of apoptosis-related proteins, including Bax, Bcl-2, and caspase-3 in different transfected groups. D: Transwell assay showing the effects of miR-22 dysregulation on cell migration. E:

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[image:6.612.86.522.75.622.2]

Roles of miR-22 in human OA chondrocytes

Figure 3. SP1 as a direct target of miR-22 and miR-22 and possibly regulating cell apoptosis and migration via

targeting SP1. A: The predicted information of TargetScan 7.1. B: Dual-luciferase reporter assay showing SP1 as the

direct target of miR-22. C: The expression levels of SP1 in si-SP1 and control groups. D: Flow cytometry showing the cell apoptosis in different transfected groups. E: Transwell assay showing the cell migration in different transfected groups. Compared with control group: *, P<0.05, **, P<0.01. Compared with miR-22 inhibitor group: #, P<0.05, ##, P<0.01.

(P>0.05) was observed. Moreover, as

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Suppression of miR-22 promoted proliferation, inhibited apoptosis, and enhanced migration of chondrocytes

The effect of miR-22 dysregulation on cell via-bility was evaluated using the MTT assay. The results showed that cell viability of miR-22 mimic group was significantly reduced as com -pared to the scramble or the control groups, while the cell viability of miR-22 inhibitor group was markedly increased (P<0.05, Figure 2A). Furthermore, the effect of miR-22 dysregula-tion on cell apoptosis was assessed by flow cytometry. As shown in the Figure 2B, the per-centage of apoptotic cells in the miR-22 inhibi-tor group significantly declined in comparison to the scramble or the control groups, while it markedly increased in the miR-22 mimic group (P<0.05). Such findings indicate that an over-expression of miR-22 significantly induces the apoptosis of chondrocytes. Moreover, we also analyzed the expression levels of apoptosis-related proteins, including the Bax, Bcl-2, and caspase-3 (Figure 2C). In contrast to the scram-ble or control groups, Bax and caspase-3 pro -tein levels showed a remarkable down-regula -tion; while the Bcl-2 protein levels demonstrated an obvious up-regulation in the miR-22 inhibitor group (P<0.05). Entirely opposite expression levels of the apoptosis-related proteins were observed in the miR-22 mimic group (P<0.05). To support further these findings, the effect of miR-22 dysregulation on cell migration was also studied using the Transwell assay (Figure 2D). The number of migrated cells in miR-22 inhibitor group was considerably higher as com-pared to the scramble or the control groups, while it was markedly decreased in the miR-22 mimic group (P<0.05). Moreover, in compari-son to the scramble or the control group, MMP1, MMP3 and COL2A1 were found to be significantly up-regulated in the miR-22 inhibi -tor group; whereas these showed expected down-regulation in the miR-22 mimic group (P<0.05, Figure 2E).

SP1 was a direct target of miR-22, and miR-22 may regulate cell apoptosis and migration via SP1

Based on the information of the TargetScan 7.1, SP1 seems to be a potential target of miR-22 (Figure 3A). Further, we conducted the

dual-luciferase reporter assay to validate if SP1 is a direct target of miR-22 (Figure 3B). Our results showed that the over-expression of miR-22 post transfection with miR-22 mimic signifi -cantly reduced the WT-SP1-3’-UTR reporter luciferase activity (P<0.05), but not the MUT-SP1-3’-UTR reporter luciferase activity. We then used the si-SP1 to knock down the expression of SP1 gene. The analysis exhibited significant -ly reduced expression levels of SP1 in the si-SP1 group than the control group (P<0.05,

Figure 3C), indicating successful down-regula-tion of the SP1 gene expression. Addidown-regula-tionally, the percentage of the apoptotic cells that decreased after transfection with miR-22 inhib-itor showed considerable elevation when the cells were simultaneously transfected with miR-22 inhibitor and si-SP1 (P<0.05, Figure 3D). The effect of miR-22 on cell migration was confirmed by a decrease in the otherwise increased number of migrated cells after co-transfection with miR-22 inhibitor and si-SP1 into the cells (P<0.05, Figure 3E). Such data indicate that miR-22 may regulate cell apopto-sis and migration by targeting SP1.

miR-22 may be involved in OA development

through regulation of ERK/NF-κB pathway

To further explore the regulatory mechanism of miR-22 in the OA development, the relationship between miR-22 and ERK/NF-κB signal path -way was investigated. The results showed that p-ERK and p-p65 significantly increased and the expression levels of IκBa markedly decreased in the miR-22 inhibitor group in con-trast to the scramble or the control groups, while entirely different expression profiles were observed in the miR-22 mimic group (P<0.05). In addition, an ERK inhibitor, PD98059 and an inhibitor of NF-κB, PDTC were incubated with the cells. As shown in the Figure 4B, the increased expression of p-ERK after transfec-tion with the miR-22 inhibitor was significantly reversed after simultaneous transfection of the cells with miR-22 inhibitor and PD98059 (P<0.05). Moreover, the increased expression levels of p-p65 and decreased expression lev-els of IκBa after the transfection with miR-22 inhibitor alone were also greatly reversed after the cells were co-transfected with miR-22 inhib-itor and PDTC (P<0.05, Figure 4C).

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Roles of miR-22 in human OA chondrocytes

apoptosis of chondrocytes, increased the ex- pression of Bcl-2 and decreased the Bax and caspase-3 expression levels. These observa-tions showed significant contrasting patterns

upon co-transfection of cells with miR-22 inhib-itor and PD98059 or PDTC (P<0.05, Figure 5A-D). In addition, the suppression of miR-22 after the transfection of miR-22 inhibitor signifi -Figure 4. The expression levels of p-ERK, p-p65, and IκBa in different groups. PD98059 is an ERK inhibitor and PDTC is an inhibitor of NF-κB. Compared with control group: *, P<0.05, **, P<0.01. Compared with miR-22 inhibitor

[image:8.612.88.522.72.614.2]
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Roles of miR-22 in human OA chondrocytes

cantly increased the number of migrated chon-drocytes and elevated the expression levels of MMP-1, MMP-3, and COL2A1, while were sig-nificantly reversed after co-transfection of cells with miR-22 inhibitor and PD98059 or PDTC (P<0.05, Figure 5E-H).

Discussion

In the present study, we found that miR-22 was significantly down-regulated in the chondro -cytes that were isolated from the articular car-tilage of OA patients, while SP1 was markedly up-regulated. The suppression of miR-22 sig-nificantly promoted proliferation, inhibited apoptosis, and enhanced the migration of chondrocytes. In addition, SP1 was found to be a direct target of miR-22. The knockdown of SP1 significantly reversed the effects of miR-22 inhibition on the apoptosis and migration of chondrocytes after co-transfection of cells with miR-22 inhibitor and si-SP1. Besides, the sup -pression of miR-22 resulted in significant increase in the expression of p-ERK and p-p65 and a decrease in the expression of IκBa. It was found that PD98059 or PDTC could significantly reverse the effects of miR-22 inhibition on apoptosis and migration of chondrocytes after transfection with miR-22 inhibitor and D98059 or PDTC at the same time. Our findings eluci -date the key mechanism of miR-22 in OA development.

In a previous study, SP1 was reported to play a key role in the maintenance of chondrocyte pro -liferation [21]. Chondrocyte viability is consid-ered essential for the sustenance of the integ-rity of articular cartilage [22]. In addition, SP1 is shown to be involved in the interleukin-1 beta (IL-1β)-mediated down-regulation or insulin-like growth factor-I-mediated up-regulation of the gene expression of human type II collagen in articular chondrocytes [23, 24]. Type II collagen is reported to be involved in the consecutive stages of human OA development [25]. The

allelic imbalance of SNP rs143383 may be mediated by SP1 transcriptional repressors, which is associated with OA susceptibility [26]. Besides, an increase of empty lacunae in the cartilage is linked to the apoptosis of cells in the OA patients [27, 28]. In our study, miR-22 was significantly down-regulated in the chon -drocytes that were isolated from OA patients. The suppression of miR-22 significantly pro -moted the proliferation, and the migration of chondrocytes, while inhibiting the cell apopto-sis. Furthermore, SP1 was found to be the direct target of miR-22, as previously reported by Guo [14]. The knockdown of SP1 significantly reversed the effects of miR-22 inhibition on the apoptosis and the migration of chondrocytes after co-transfection with miR-22 inhibitor and si-SP1. The current data imply that the sup-pression of miR-22 may inhibit apoptosis of chondrocytes and may promote their migration via SP1 during the development of OA.

Furthermore, ERK activation is considered to be a key mechanism in the determination of the synthesis or degradation of the matrix during the OA development. Mutual inhibition of ERK has been reported to regulate MMP-13 expres-sion in human OA chondrocytes [29]. Moreover, the silencing of ERK2 significantly inhibits the cartilage degeneration of post-traumatic OA [30]. In addition, NF-κB pathway seems to be involved in the inflammatory response by chon -drocytes, and thereby, contributes to the pro-gressive extracellular matrix damage and the cartilage destruction [31]. Inappropriate regula-tion of NF-κB is implicated in the pathogenesis of several diseases including the OA [32]. In the present work, the suppression of miR-22 result -ed in a significantly high expression of p-ERK and p-p65 and low expression of IκBa. Moreover, PD98059 or PDTC were observed to greatly reverse the effects of miR-22 inhibition on the expression levels of the ERK/NF-κB pathway-related proteins, and also the apopto-sis and migration of chondrocytes after simul-Figure 5. miR-22 regulating cell apoptosis and migration through regulating ERK/NF-κB pathway. A: Flow cytometry showing the effects of miR-22 inhibition and PD98059 on cell apoptosis. B: The expression levels of apoptosis-relat

-ed proteins, including Bax, Bcl-2, and caspase-3 in different groups. C: Flow cytometry showing the effects of miR-22 inhibition and PDTC on cell apoptosis. D: The expression levels of apoptosis-related proteins, including Bax, Bcl-2,

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taneous transfection with miR-22 inhibitor and D98059 or PDTC. Collectively, it is speculated that miR-22 may regulate the apoptosis and migration of chondrocytes by involving in ERK/ NF-κB pathway during the development of OA in humans.

Thus, the authors would like to conclude that the suppression of miR-22 may inhibit the apoptosis and may promote the migration of OA chondrocytes by targeting SP1 and activating the ERK/NF-κB pathway. Our study presents imperative implications for the understanding of OA progression and its therapy.

Disclosure of conflict of interest

None.

Address correspondence to: Qichun Zhao, Depart- ment of Orthopedics, Anhui Provincial Hospital, Anhui Medical University, NO. 17 Lujiang Road, Hefei 230001, Anhui, China. E-mail: qichunzhao06@126. com

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Figure

Table 1. The primers used for the amplification of targets
Figure 1. The inverse expression of miR-22 and SP1 in human OA chondrocytes. A: The expression of miR-22 in sion of SP1 in OA chondrocytes
Figure 2. The suppression of miR-22 promoted proliferation, inhibited apoptosis, and enhanced migration of chondrocytes
Figure 3. SP1 as a direct target of miR-22 and miR-22 and possibly regulating cell apoptosis and migration via targeting SP1
+2

References

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