Yuhua Zhang1, Xiaobo Qin2, Juan Jiang3, Wenjie Zhao1. 1. Reproductive Medicine Centre, Weifang People's Hospital, Weifang, Shandong 261041, P.R. China. 2. Department of Obstetrics and Gynecology, Zhangqiu District Maternal and Child Health Care Hospital, Jinan, Shandong 250200, P.R. China. 3. Department of Nursing, The Third People's Hospital of Qingdao, Qingdao, Shandong 266041, P.R. China.
Ovarian cancer (OC) is a prevalent fatal malignancy in gynecology with chemotherapeutic resistance and high metastatic potential, remaining a serious threat to the lives of females (1). Although advancement has been made in existing therapies for OC patients via the combination of immunotherapy, radiotherapy, chemotherapy and surgery, the survival rates of OC patients have not yet been fully improved (2–4). OC patients frequently present no clear early symptoms owing to the lack of effective approaches for early diagnosis. Moreover, most of OC patients who are diagnosed at advanced-stages have distant metastasis as well as high relapse rate (5–7). In recent years, increasing number of therapeutic targets which are associated with OC progression have been identified, but knowledge concerning OC pathogenesis remains limited. Therefore, identifying specific biomarkers which contribute to OC progression has a great clinical significance for the development of novel therapeutic strategies.Increasing research has shown that miRNAs modulate gene expression via interacting with the target gene 3 untranslated region (3′UTR) and are widely implicated in numerous biological processes, serving an important role in predicting prognosis or tumorigenesis of various tumors (8–10). Emerging evidence has shown that miRNA is frequently abnormally expressed in numerous malignancies, serving as either a tumor suppressor or an oncogene depending on downstream targets involved and the tissue context (11–13). Hence, miRNAs are promising candidate biomarkers in the investigation on initiation, metastasis and development of tumors. For instance, miR-363 and miR-200a in Burkitt's lymphoma (BL) were found to be involved in modulating expression of Yin Yang 1 (YY1), providing further insight into the pathogenesis and treatment strategies of BL (14); miR-145-5p and miR-214-3p may associate with progression of bladder cancer by modulating the epithelial-to-mesenchymal transition (EMT) and NGAL/MMP-9 pathways (15); Yan et al (16) found that miR-495 suppressed colorectal carcinoma cell migration and proliferation via regulating FAM83D; Qi et al (17) reported that miR-21 facilitated gastric cancer growth via the regulation of prostaglandin E2; Cheng et al (18) proposed that miR-183-5p inhibited apoptosis and promoted proliferation in humanbreast carcinoma by modulating PDCD4. miR-126 has been regarded as an antitumor miRNA with altered expression levels in various tumors, including lung cancer (19), hepatocellular carcinoma (20) and colorectal cancer (21). However, miR-126 expression and its specific roles in OC development are still unclear.EMT has been proved to play vital functions in tumor metastases (22). In EMT, cells gain mesenchymal characteristics and lose the epithelial disposition, decreasing the migratory capacities of tumor cells (23). Moreover, the ERK/MAPK signaling pathway plays pivotal roles in multiple key cellular processes including cell proliferation, apoptosis and differentiation. Therefore, it was hypothesized that miR-126 may affect OC cell proliferation, migration and invasion via EMT and ERK/MAPK signaling pathways.Epidermal growth factor-like domain 7 (EGFL7), a secreted protein specifically expressed by endothelial cells during embryogenesis, has emerged as an important factor not only in modulating vascular development but also in tumorigenesis (24,25). Ectopic high-level EGFL7 expression was detected in various tumors including osteosarcoma (26), breast cancer (27) and liver cancer (28). Abnormal EGFL7 expression correlated with the pathologic features including cellular progress, poor prognosis and clinical progression. For example, Shen et al (29) found that EGFL7 promoted pancreatic carcinoma cell invasion and angiogenesis; Wang et al (30) reported that EGFL7 attenuation inhibited humanlaryngocarcinoma cell invasion and growth; Deng et al (31) found that upregulation of EGFL7 expression promoted gastric cancer cell invasion and metastasis. Moreover, studies by Oh et al (32) indicated that EGFL7 expression is a novel predictive factor for the clinical progression of epithelial ovarian cancer (EOC), and may constitute a therapeutic target for antiangiogenesis therapy in patients with EOC. Additionally, previous studies demonstrated that miR-126 is a negative regulator of EGFL7 gene in Systemic sclerosis (33). Therefore, the elevated EGFL7 expression in tumors and its functions in facilitating cancer angiogenesis, invasion and migration make it a candidate target for tumor treatment. As such, we proposed that EGFL7 served as a biomarker in OC progression, which may be regulated by miR-126.In the current study, the expression levels and regulatory functions of miR-126 in OC progression were detected. Briefly, the miR-126 was identified to be downregulated in OC tissues, along with poor prognosis in patients. Moreover, the miR-126 upregulation inhibited OC cell progression via regulation of EGFL7, ERK/MAPK pathway and EMT. Therefore, the present study demonstrated that miR-126 played a critical role in OC tumorigenesis, providing a potential clinical target in OC treatment.
Patients and methods
Clinical samples
Fifty-four cases of OC tissues and adjacent tissues (located >3 cm away from the tumor) were collected from OC patients who had undergone surgical resection at Weifang People's Hospital (Weifang, China) between August 2011 and June 2013. Inclusion criteria: i) pathologic biopsy confirmed ovarian cancer; ii) clinical data and follow-up data were complete without loss; iii) did not receive any systemic antitumor treatment before enrollment; iv) have no serious dysfunctions in vital organs (such as heart, liver, kidney and others); v) informed consent. Exclusion criteria: i) combined with other malignant tumors; ii) received surgery, chemotherapy or radiotherapy; iii) less than 18 years old; iv) compliance is poor; v) lost consciousness, unable to communicate in words. All tissue samples were immediately snap-frozen in liquid nitrogen, and stored at −80°C for later use. Written informed consent was obtained from all the patients for the studies. Ethical approval for the study was provided by the Ethics Committee of Weifang People's Hospital.
Cell culture
The normal immortalized human ovarian surface epithelial cell line IOSE29 and OC cells (OVCAR3, SKOV3, and A2780) were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cell lines were maintained in RPMI-1640 medium with 10% FBS (both from Invitrogen; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37°C containing 5% CO2.
Cell transfection
OC cells were seeded at 2×105 per well in 6-well plates for further investigation. miR-126 mimics, inhibitor as well as the negative controls (NC) were synthesized by GenePharma (Shanghai, China). miRNA transfections were carried out by Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) in strict line with the manufacturers' instructions. Further analysis was conducted 48 h after the transfection.
qRT-PCR
Total RNA was isolated from OC cells or tissues with TRIzol reagent (Thermo Fisher Scientifc, Inc.) following the manufacturer's recommendations. Then, PrimeScript RT reagent kit (Takara) was used to transcribe the isolated RNA into cDNA. QRT-PCR was conducted using SYBR Premix Ex Taq (Takara) on ABI 7500 fast real-time PCR system (Applied Biosystems). Relative levels of the RNAs was assessed by the 2−ΔΔCt method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 were endogenous controls for normalization. The sequences of the primers are listed in Table I.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were applied for assessing the influence of miR-126 on OC cell proliferation ability. Briefly, the OC cells transfected with miR-126 mimics or inhibitor were plated into a 96-well plate at a density of 5×103 cells/well. After incubation for specified time (0, 24, 48 and 72 h), MTT solution was added into each well and incubated at 37°C for 4 h. Subsequently, dimethyl sulfoxide (DMSO) was added to dissolve the crystal. The absorbance at 490 nm was examined using a microplate reader (BioTek Instruments, Inc.).
Transwell assays
The invasion or migration capacities of OC cells transfected with miR-126 mimics or inhibitor were examined by Transwell assays. In brief, the transfected cells (5×104) were resuspended in serum-free medium and seeded in the upper chambers of the Transwell chamber inserts (8.0 µm pore size; Corning, Inc.). In addition, the inserts were pre-coated with or without Matrigel (BD Biosciences) for invasion or migration assays. On the other hand, medium supplemented with 10% FBS, which served as a chemoattractant, was added in the lower chambers. Then, cells were incubated at 37°C in a 5% CO2 atmosphere for 48 h. Then, cells left on the upper surface were wiped away with cotton swabs while invaded or migrated cells were fixed by paraformaldehyde and stained with crystal violet. Images of five randomly selected fields of the fixed cells were captured and counted with an inverted microscope (Olympus).
Western blot analysis
To analyze the protein expression levels of specific genes, treated cells were lysed using iced lysis buffer including protease and phosphatase inhibitors (Roche Diagnostics). The protein concentration was examined with a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Protein samples were separated by 10% SDS-PAGE and then transferred onto PVDF membranes (Invitrogen; Thermo Fisher Scientific, Inc.). TBST containing 5%-skim milk was utilized to block the membranes for 2 h at room temperature. Subsequently, the membranes were incubated with specific primary antibodies at 4°C overnight, followed by incubation with HRP-conjugated secondary antibodies (1:3,000, ab6721; Abcam). The following specific primary antibodies were used: ERK (1:1,000, ab17942), p-ERK (1:2,000, ab192591), E-cadherin (1:2,000, ab133597), N-cadherin (1:1,000, ab76011), Vimentin (1:1,000, ab137321) and GAPDH (1:1,000, ab9485) (all from Abcam). The protein was visualized with ECL western blot detection reagents (Beyotime Institute of Biotechnology). GAPDH was an internal control.
Dual-luciferase reporter assay
OC cells were seeded into a 24-well plate at a density of 1×105 cells/well and co-transfected with miR-126 mimics or NC and pmir-GLO plasmids (Promega Corporation) containing wild-type (WT) or mutant (MUT) EGFL7 3′UTR by Lipofectamine 2000 following the manufacturers' proposals. Cells were harvested 48 h after transfections and luciferase activities were examined with a Dual-Luciferase Reporter Assay System (Promega Corporation).
Statistical analysis
All experiments were repeated at least 3 times. Statistical analysis was carried out with SPSS software version 17.0 (SPSS, Inc.). Student's t-test and one-way ANOVA followed by Tukey's post hoc test were utilized to analyze two or multiple groups. The Kaplan-Meier curve together with log-rank test was utilized to analyze the overall survival (OS) of OC patients. P<0.05 indicates statistically significant difference.
Results
Declined miR-126 expression in OC tissues indicates poor prognosis of OC patients
To identify potential functions of miR-126 in OC progression, the miR-126 expression in OC tissues was examined. Results revealed that miR-126 expression was dramatically reduced in OC tissues compared to the adjacent tissues (Fig. 1A). Then, the mean miR-126 expression of all patients involved in the present study was used as the cutoff to assign them into two groups: OC patients with low miR-126 level [miR-126 (−)] and OC patients with high miR-126 level [miR-126 (+)]. The clinical relevance of miR-126 in OC was analyzed and results demonstrated that the decline of miR-126 expression was implicated in the worse clinicopathological characteristics of OC patients (Table II). Moreover, the Kaplan-Meier analysis was performed to investigate the functions of miR-126 in the prognosis of OC patients and found that there was a significantly shorter OS in patients who had low miR-126 expression (Fig. 1B). Furthermore, univariate and multivariate Cox analysis showed that miR-126 expression was an independent prognostic indicator for OS in patients with OC (Table III).
Figure 1.
Decreased miR-126 expression indicates poor prognosis of OC patients. (A) Low expressions of miR-126 in OC tissues was confirmed using qRT-PCR. (B) Kaplan-Meier analysis of OC patients with high and low miR-126 expression. ***P<0.001. OC, ovarian cancer.
Table II.
Correlation of miR-126 expression with the clinicopathological characteristics of the ovarian cancer patients.
miR-126[a] expression
Clinicopathological features
Cases (n=54)
High (n=20)
Low (n=34)
P-value
Age (years)
0.4659
>60
27
9
18
≤60
27
11
16
Family history of cancer
0.5126
Yes
27
8
19
No
27
12
15
Tumor size (cm)
0.6653
≥5.0
29
10
19
<5.0
25
10
15
TNM stage
0.0025[b]
I–II
24
15
9
III
30
5
25
Lymph node metastasis
0.0019[b]
Yes
28
4
24
No
26
16
10
Menopause
0.4256
Yes
30
11
19
No
24
9
15
FIGO stage
0.0023[b]
I–II
23
16
7
III–IV
31
4
27
Distant metastasis
0.0034[b]
Yes
30
4
26
No
24
16
8
The mean expression level of miR-126 was used as the cutoff
statistically significant. TNM, tumor-node-metastasis; FIGO, International Federation of Gynecology and Obstetrics.
Table III.
Univariate and multivariate Cox proportional hazards analysis of miR-126 expression and overall survival (OS) for patients with ovarian cancer.
Univariate analysis
Multivariate analysis
Variables
HR (95% CI)
P-value
HR (95% CI)
P-value
Age (years)
0.836 (0.421–1.518)
0.369
Family history of cancer
0.733 (0.349–1.483)
0.481
Tumor size (cm)
2.253 (0.970–5.437)
0.181
TNM stage
4.213 (1.355–8.821)
0.001
3.287 (1.043–8.420)
0.005
Lymph node metastasis
3.891 (1.205–6.769)
0.004
2.836 (0.981–6.942)
0.015
Menopause
1.572 (0.846–5.035)
0.262
FIGO stage
3.225 (1.022–5.971)
0.006
3.019 (1.092–7.041)
0.011
Distant metastasis
2.629 (1.021–5.891)
0.022
4.121 (1.700–9.288)
0.002
miR-126
4.436 (1.425–9.021)
<0.001
3.997 (1.310–8.738)
0.003
HR, hazard ratio; 95% CI, 95% confidence interval; TNM, tumor-node-metastasis; FIGO, International Federation of Gynecology and Obstetrics.
miR-126 upregulation in OC cells impaires the proliferation ability
As it is known that miR-126 is significantly downregulated in OC clinical tissues, current study further investigated its functions in OC cells. Similarly, we examined the miR-126 expression in OC cells and qRT-PCR results revealed that miR-126 was dramatically downregulated in all OC cells (Fig. 2A). Subsequently, OVCAR3 and SKOV3 cells were selected for further functional experiments due to their relatively low and high endogenic miR-126 expressions. First, miR-126 mimics or inhibitor was, respectively, transfected into OVCAR3 or SKOV3 cells for overexpressing or inhibiting miR-126 expression. Transfection efficiency was confirmed by qRT-PCR (Fig. 2B and C). Subsequently, MTT assay was conducted to detect the proliferation abilities of OVCAR3 and SKOV3 cells. Through MTT assays, miR-126 overexpression was confirmed to prominently repress cell proliferation while miR-126 inhibition markedly enhanced proliferation abilities in OC cells (Fig. 2D and E).
Figure 2.
miR-126 upregulation significantly inhibits OC cell proliferation. (A) qRT-PCR analysis was conducted to examine miR-126 expression in OC cells. (B and C) miR-126 overexpression or inhibition in OC cells was confirmed by qRT-PCR. (D and E) Effects of miR-126 on OC cell proliferation were assessed by MTT assays. *P<0.05, **P<0.01 and ***P<0.001. OC, ovarian cancer; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
miR-126 overexpression significantly suppressed OC cell invasion and migration
In order to evaluate the potential functions of miR-126 in OC cell invasion and migration, Transwell assays was performed. Results indicated that the invasion and migration abilities of OVCAR3 cells were notably inhibited with the treatment of miR-126 mimics (Fig. 3A and B). On the other hand, the invasion and migration capacities of SKOV3 cells were dramatically promoted by miR-126 inhibitor (Fig. 3C and D). Therefore, the results demonstrated that miR-126 overexpression impaired OC cell invasion and migration abilities.
Figure 3.
miR-126 restoration represses OC cell migration and invasion. (A and B) Invasion and migration abilities of miR-126-overexpressed OVCAR3 cells were observed by Transwell assays. (C and D) Transwell assay was performed to measure the invasion and migration capacities of miR-126-suppressed SKOV3 cells. **P<0.01 and ***P<0.001. OC, ovarian cancer.
EGFL7 is a direct target of miR-126 in OC cells
To further investigate the molecular mechanism of miR-126 in OC progression, potential targets for miR-126 were predicted by TargetScan online software (TargetScanHuman 7.2, http://www.targetscan.org/) and EGFL7 3′UTR was found to have predicted binding sites of miR-126 (Fig. 4A). Subsequently, luciferase reporter assays were carried out to further confirm the association between EGFL7 and miR-126. OC cells were co-transfected with luciferase reporter vector containing EGFL7 3′UTR-WT or MUT and miR-126 mimics or NC. Our results demonstrated that miR-126 mimic evidently decreased the luciferase activity of OC cells treated with EGFL7 3′UTR-WT whereas it had no prominent suppressive effects on OC cells treated with EGFL7 3′UTR-MUT (Fig. 4B). Furthermore, we evaluated the regulatory effect of miR-126 on EGFL7 expression in OC cells. Data showed that miR-126 overexpression significantly inhibited the EGFL7 expression in OVCAR3 cells (Fig. 4C), while EGFL7 expression was dramatically enhanced by miR-126 inhibition (Fig. 4D), indicating the direct negative regulation of miR-126 in EGFL7 expression.
Figure 4.
EGFL7 is a direct target of miR-126 in OC cells. (A) The binding sites of miR-126 in the EGFL7 3UTR. (B) Relative luciferase activity in OC cells was determined after the co-transfection with WT or MUT EGFL7 3UTR plasmids and miR-126 mimics or NC. (C and D) EGFL7 expression in miR-126-overexpressed OVCAR3 cells or miR-126-suppressed SKOV3 cells. *P<0.05, **P<0.01 and ***P<0.001. OC, ovarian cancer; EGFL7, epidermal growth factor-like domain 7; NC, negative controls; 3UTR, 3 untranslated region; WT, wild-type; MUT, mutant.
miR-126 regulates ERK/MAPK signaling pathway and EMT in OC cells
Next, the expression and the prognostic values of EGFL7 in OC patients were evaluated. First, qRT-PCR was performed to examine the EGFL7 expression in OC tissues and cells, and the results presented significant increase of EGFL7 expression both in OC tissues and cells (Fig. 5A and B). Furthermore, the prognostic value of EGFL7 in OC patients was analyzed by Kaplan-Meier analysis. The results revealed that the OS of OC patients with higher EGFL7 expression [EGFL7 (+)] was worse than that of patients with lower EGFL7 expression [EGFL7 (−)] (Fig. 5C). The underlying mechanisms of the suppressive effects mediated by miR-126 on OC progression were further investigated. Therefore, western blot was carried out to determine the effects of miR-126 in OC cell ERK/MAPK signaling pathway and EMT. It was found that in OVCAR3 cells transfected with miR-126 mimic, p-ERK expression was obviously repressed while there was no significant change in ERK expression; in contrast, miR-126 inhibition in SKOV3 cells markedly promoted p-ERK expression with no evident influence on ERK expression (Fig. 5D). Moreover, miR-126 overexpression was notably increased in E-cadherin expression and declined in N-cadherin and Vimentin expression, respectively, in OVCAR3 cells while the opposite trend occurred when miR-126 was suppressed in SKOV3 cells (Fig. 5D). These data showed that miR-126 may suppress OC progression by targeting EGFL7 and regulating ERK/MAPK signaling pathway and EMT.
Figure 5.
miR-126 restoration modulates ERK/MAPK signaling pathway and EMT in OC cells. (A and B) EGFL7 expression in OC tissues and cells. (C) The OS rate of OC patients with different EGFL7 expressions. (D) Effects of miR-126 on OC cell EMT and ERK/MAPK pathway were determined by western blot analysis. *P<0.05 and **P<0.01. OC, ovarian cancer; OS, overall survival; EGFL7, epidermal growth factor-like domain 7; EMT, epithelial-to-mesenchymal transition.
Discussion
OC is a highly lethal malignancy which lacks effective early diagnostic approaches (34). Most of OC patients are frequently diagnosed at late stages when the tumor has largely metastasized. Moreover, current radical surgery cannot excise the metastatic tumor tissues completely (35). Therefore, looking for specific biomarkers that contribute to the progression of OC is necessary for the development of novel treatment. The present study was designed to investigate the role of miR-126 in OC progression and to undertake a preliminary study of the clinical significance of miR-126 in OC treatments. The results showed that miR-126 expression was decreased in OC tissues and cells, indicating its potential role in the progression of OC. The role of miR-126 in OC was then explored in OC cells, revealing the suppressive role of miR-126 in OC cell proliferation, invasion and migration. EGFL7 was predicted to be a target of miR-126 and directly negatively regulated by miR-126. Additionally, EGFL7 upregulation was associated with poor OS for OC patients. Furthermore, it was discovered that miR-126 exerts its function on OC cell ERK/MAPK pathway and EMT. Taken together, this study revealed that miR-126 may be served as a cancer suppressor and a potential therapy target in OC.Accumulating studies have revealed that miRNAs are promising molecular biomarkers for tumor diagnosis and prognosis, and also therapeutic targets for malignancies (36–38), including OC (39). For example, Lv et al (40) demonstrated that miR-34a reduced OC cell chemoresistance and proliferation via targeting HDAC1; Zheng et al (41) proposed that miR-101 repressed OC cell invasion and proliferation by down-regulating SOCS-2 expressions; Li et al (42) claimed that miR-221 overexpression promoted OC cell proliferation via regulating apoptotic protease activating factor-1, indicating a poor prognosis. Our study focused on a deeper investigation of clinical significance and the molecular mechanisms of miR-126 in OC therapies.Numerous studies have demonstrated that miR-126 might serve as a regulator and prognostic biomarker for patients suffering from various tumors (43,44). For instance, Feng et al (45) found that downregulated serum miR-126 was related to poor prognosis and aggressive progression of gastric carcinoma; Li et al (46) indicated that miR-126 repressed glioma cell proliferation and invasion via modulating ERK pathway through KRAS; Wen et al (47) proposed that miR-126 suppressed cell growth by regulating LRP6 in papillary thyroid carcinoma. In the current study, results revealed that miR-126 was markedly down-regulated in OC tissues and cells. Moreover, we found that the low miR-126 expression in OC tissues was related to the aggressive progression of OC patients. In addition, miR-126 overexpression suppressed OC cell proliferation, migration and invasion capability, which was inconsistent with a previous report that miR-126 affected OC cell differentiation and invasion (48).To explore the underlying mechanism of miR-126 in OC, the identification of regulatory targets is crucial. Our western blot results indicated that miR-126 overexpression led to upregulation of E-cadherin and p-ERK and the decreased expression of N-cadherin and Vimentin, which may cause tumor progression. Thus we proposed miR-126 may suppress OC tumor metastasis and growth. By bioinformatics analysis, EGFL7 was identified as a candidate downstream target of miR-126. Therefore, we explored the role of EGFL7 in OC to further understand the mechanisms underlying OC progression.EGFL7 which is an emerging therapeutic biomarker in tumorigenesis has been proven to be up-regulated in various tumors, playing pivotal functions (49). However, how EGFL7 contributes to the progression of OC remains to be further elucidated. Luciferase reporter assay identified EGFL7 as a functional target of miR-126 in OC cells, indicating that EGFL7 was implicated in the suppressive functions of miR-126 in OC cell progress. In addition, it was verified that upregulated EGFL7 in OC tissues were related to the poor prognosis of OC patients. These results suggested that EGFL7 may become a novel potential target for OC treatments.This study showed the suppression impacts of miR-126 on OC progression and its possible mechanisms. However, there may exist other target genes or lncRNAs and possible mechanisms of miR-126 in OC. The relationship between EGFL7 and ERK/MAPK signaling pathway and EMT, and whether miR-126 regulates EMT through miR-126/EGFL7/ERK/MAPK axis still need further study.In summary, the results of this study revealed that miR-126 expression was decreased in OC. Additionally, the ectopic overexpression of miR-126 suppressed OC cell proliferation, invasion and migration, and miR-126 may suppress OC tumor metastasis and growth by regulating ERK/MAPK signaling pathway and EMT. Furthermore, EGFL7 which contributes to the OC progression was identified as a potential target of miR-126 and negatively regulated by it, indicating that miR-126 may serve as an OC tumor suppressor by targeting EGFL7. These findings may help us better understand the molecular mechanism of miR-126 in OC carcinogenesis and may have potentially diagnostic and therapeutic value.
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