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miR-6838-5p Affects Cell Growth, Migration, and Invasion by Targeting GPRIN3 via the Wnt/β-Catenin Signaling Pathway in Gastric Cancer

Zhou W.a · Ding X.b · Jin P.a · Li P.b

Author affiliations

aDepartment of Gastroenterology, Hwa Mei Hospital, University of Chinese Academy of Sciences (Ningbo No. 2 Hospital), Ningbo, China
bDepartment of Gastroenterology, Ningbo First Hospital, Ningbo, China

Corresponding Author

Peifei Li

Department of Gastroenterology

Ningbo First Hospital, No. 59 Liuting Street

Ningbo, Zhejiang 315010 (China)

lipeifei@hotmail.com

Keywords: miR-6838-5pGPRIN3Gastric cancerWnt/β-catenin signaling pathway

Related Articles for ""
Pathobiology 2020;87:327–337
https://doi.org/10.1159/000511691

Abstract

Gastric cancer (GC) is a highly prevalent digestive malignant tumor, ranking second in the tumor-related mortality globally. The microRNAs have been confirmed to be connected with GC progression. Accumulative evidence has suggested that miR-6838-5p exerts a suppressive effect on human cancers. Nonetheless, whether miR-6838-5p is involved in the regulation of GC remains to be investigated. During our research, miR-6838-5p was downregulated in GC cells. Upregulated miR-6838-5p repressed GC cell cycle progression, proliferation, migration, and invasion. Furthermore, miR-6838-5p overexpression repressed the nuclear import of β-catenin, thus inactivating Wnt/β-catenin pathway. Moreover, we observed that GPRIN3 was targeted by miR-6838-5p in GC with luciferase reporter and RIP assays. GPRIN3 upregulation reversed the suppression of miR-6838-5p in GC cellular processes. These findings suggest miR-6838-5p restrains the malignant behaviors of GC cells via targeting GPRIN3 to repress Wnt/β-catenin signaling pathway, which may provide novel targets for GC treatment.

© 2020 S. Karger AG, Basel

Introduction

Gastric cancer (GC) is a prevailing digestive malignancy with high mortality [1]. GC is frequently diagnosed at advanced stages with poor prognosis owing to the lack of effective early diagnostic biomarkers. In many cases, conventional treatments for GC fail due to the recurrence and distant metastasis as a result of chemotherapy or radiotherapy resistance [2]. Thus, further understanding of the pathogenesis of GC is needed to promote the diagnosis, treatment, and prognosis of GC.

Being single-stranded small RNAs, microRNAs (miRNAs) negatively modulate gene transcription via interaction with the 3′ untranslated regions (3′-UTRs) of target genes [3]. Accumulative studies have confirmed the roles of miRNAs in tumorigenesis. For instance, miRNA-211-5p represses the proliferative, migratory, and invasive capabilities of breast cancer cells by targeting SETBP1 [4]. miR-205 is associated with breast cancer infiltration and metastasis [5]. miR-137 inhibited by DNA hypermethylation has an anticancer effect in endometrial cancer [6]. Furthermore, miR-6838-5p restrains cell migration in triple-negative breast cancer [7]. Nonetheless, whether miR-6838-5p participates in GC progression still needs to be further explored.

G-protein-regulated inducer of neurite growth 1 and 2 (GPRIN1 and GPRIN2, respectively) function as the downstream effectors of Gαo except for AC with a Gαo-binding region at the C-terminal region [8, 9]. GPRIN3 is also a part of the GPRIN family. It has been proposed that GPRIN3 can regulate dopaminergic behaviors [10]. Nevertheless, the biological roles of GPRIN3 in GC are still unknown.

In this research, we identified that overexpressed miR-6838-5p hampered cell cycle progression, proliferation, migration, and invasion of GC. GPRIN3 was verified to be targeted by miR-6838-5p. Whether miR-6838-5p regulated GC progression by repressing GPRIN3 expression, and the downstream pathway of GPRIN3 were further elucidated in our study. Our findings reveal that miR-6838-5p may be a putative biomarker for GC treatment.

Materials and Methods

Clinical Samples

GC tissues and adjacent non-tumor tissues (n = 36) were acquired from GC patients at Ningbo First Hospital (Zhejiang, China). Prior to operation, patients had not received any therapy. The tissue samples were promptly frozen in liquid nitrogen and maintained at –80°C.

Cell Lines and Culture

Cell lines were collected from the American Type Culture Collection (ATCC, USA). GC cell lines (MGC-803, BGC-823, MKN-45 and SGC-7901) and normal human gastric epithelial HFE-145 cell line were applied. Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) containing 10% fetal bovine serum (FBS) was adopted to cultivate cells in humid condition containing 5% CO2 at 37°C.

Cell Transfection

pcDNA3.1-GPRIN3 plasmids were used to upregulate GPRIN3 expression with the empty pcDNA3.1 as negative control (NC), and miR-6838-5p mimics were used to overexpress miR-6838-5p with NC mimics as NC. Cell transfection was performed applying Lipofectamine 2000 (Invitrogen, CA, USA). After transfection, cells were cultivated for 24 h and purified for the next experiments. The pcDNA3.1 vector and miR-6838-5p mimics were bought from GenePharma (Shanghai, China).

RNA Extraction and Real-Time Quantitative PCR

TRIzol reagent (Invitrogen) was employed to extract total RNA. Next, complementary DNA (cDNA; Amersham Pharmacia Biotech, Canada) was reversely transcribed with extracted RNA. Afterwards, cDNA was used for PCR amplification via SYBR Green (Takara, Dalian, China). Thermal Cycler Dice Real-Time PCR System (Takara) was applied for conduction of real-time PCR. We carried out 2–ΔΔCt method for detecting relative RNA expressions, with GAPDH or U6 as internal control. The following primers are utilized for examining:

miR-6838-5p (forward) 5′-GCACTCCTGGATGCCAATCT-3′;

miR-6838-5p (reverse) 5′-CTCTACAGCTATATTGCCAGCCAC-3′;

GPRIN3 (forward) 5′-TCTCACCACAACCAGCTCAG-3′;

GPRIN3 (reverse) 5′-ACTGGCTCTCCCTCACTGAA-3′.

Cell Viability Assay

Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Japan) was added at 0, 24, 48, and 72 h post-transfection. In short, 10 μL of CCK-8 reagent was put in each well, and cells were cultivated at 37°C. A microplate reader (EL340; BioTek Instruments, USA) with a wavelength of 450 nm was used to detect cell viability 4 h later.

Colony Formation Assay

Cells after transfection were planted into 6-well plates. Next, cells were cultured in DMEM containing 10% FBS changed every 3 days. Later, cells were cultured in the humidified atmosphere containing 5% CO2 at 37°C for 2 weeks. Thereafter, methanol was utilized to fixate cells, and crystal violet was used to stain cells.

Cell Cycle Analysis

Transfected cells were reaped and fixated in 70% ethanol at 4°C overnight. 100 µg/mL propidium iodide (Sigma-Aldrich, USA) and 10 µg/mL RNase A were used to treat cells. BD FACSCalibur (BD Biosciences, USA) was employed to detect cell cycle distribution. Subsequently, CellQuest software (BD Biosciences) was utilized for analyzing data.

Transwell Assay

The transfected cells (1 × 105 cells per well) were grown on upper chambers containing serum-free DMEM (Gibco) and coated with Matrigel. Lower chambers were added with DMEM containing 10% FBS. The cells were incubated at 37°C with 5% CO2 for 48 h. Non-invaded cells were cleared by a cotton swab. Invaded cells were fixated, treated with methanol and crystal violet, and then counted from 5 random fields. Later, the migration assays were implemented without Matrigel following the above procedures.

Western Blotting

RIPA buffer with protease was used to lyse the transfected cells. Ten percent SDS-PAGE was utilized for separating proteins. Later, proteins were taken to PVDF membranes. Additionally, the membranes were covered by 5% nonfat milk, and proteins were cultured with primary antibodies overnight at 4°C. Next, the membranes were cultured by secondary antibodies at room temperature for over 2 h. The ECL chemiluminescent Detection System (Thermo Fisher Scientific, USA) was applied for the visualization of protein bands. The following were primary antibodies: cyclin D1 (ab16663), cyclin E1 (ab33911), p-AKT (ab38449), AKT (ab38449), p-ERK1/2 (ab50011), ERK1/2 (ab17942), Wnt (ab142216), β-catenin (ab32572), GAPDH (ab181602), and histone H3 (ab1791). The primary antibodies were provided by Abcam Company in Shanghai, China.

Immunofluorescence Assay

The transfected cells were inoculated on cover slips of 6-well plates, fixated by 4% paraformaldehyde and permeabilized in PBS, and covered by 5% bovine serum albumin. Then, cells were cultured using primary antibody (β-catenin) all night at 4°C. Later, cells were washed in PBS and cultured with secondary antibodies at room temperature for 1 h. Thereafter, cells were stained using DAPI. Eventually, images were observed via a fluorescence microscope (Olympus, Beijing, China).

Luciferase Reporter Assay

The synthesized fragments of wild-type (Wt) or mutated (Mut) 3′-UTR of GPRIN3 were subcloned into the pmirGLO (GeneChem, Shanghai, China). Subsequently, Lipofectamine 2000 (Invitrogen) was used for transfection of miR-6838-5p mimics and NC mimics with pmirGLO-GPRIN3-Wt or pmirGLO-GPRIN3-Mut into MKN-45 and SGC-7901 cells. Luciferase activity was evaluated with a Dual-Luciferase Reporter Assay System (Promega Corporation, USA). Relative luciferase activity was measured by comparison with Renilla luciferase activity after 48-h transfection.

RNA Immunoprecipitation Assay

We conducted RNA immunoprecipitation (RIP) assay utilizing the Magna RNA-binding protein immunoprecipitation kit (Millipore, MA). In brief, magnetic beads covered by human Ago2 antibody were put into the blend of RIP buffer and cell lysates. IgG served as NC. Next, RNA was purified using proteinase K. The purified RNAs were examined by real-time quantitative PCR (RT-qPCR).

Immunohistochemistry and in situ Hybridization Assay

The in situ hybridization assay was applied to measure the miR-6838-5p level in GC tissues and normal tissues according to a previous study [11]. The immunohistochemistry assay was utilized to determine the nuclear accumulation of β-catenin according to a previous study [12].

Statistical Analysis

Data analysis was carried out by applying SPSS 21.0 (IBM, USA). All experiments were conducted 4 times. The mean value ± SD was applied to represent the data, and the t test was utilized for comparing data between 2 groups. One-way analysis of variance was applied to compare data among more than 2 groups. p < 0.05 was identified to be statistically significant.

Results

miR-6838-5p Was Downregulated in GC

To probe the function of miR-6838-5p in GC, miR-6838-5p expression in GC tissues and adjacent healthy tissues was detected via RT-qPCR. The result depicted that miR-6838-5p was downregulated in GC tissues in comparison to matched healthy tissues (Fig. 1a). Subsequently, we tested miR-6838-5p expression in HFE-145 cell line and GC cell lines. In contrast to HFE-145, miR-6838-5p was low expressed in GC cell lines (Fig. 1b). As MKN-45 and SGC-7901 cells contained less miR-6838-5p expression than other cell lines, they were chosen for the subsequent assays. Besides, the relationship between miR-6838-5p level and clinical-pathological characteristics in patients with GC was depicted in Table 1. It demonstrated that miR-6838-5p level was tightly linked with tumor size, TNM stage, lymph node metastasis, and histological type in GC (p < 0.05). Meanwhile, the relation between miR-6838-5p expression and age or gender in GC was not statistically significant (p > 0.05; Table 1). Furthermore, we determined the overall survival time of GC patients and found patients with high miR-6838-5p expression had a better survival rate than those with low miR-6838-5p (Fig. 1c). Therefore, we could draw the conclusion: miR-6838-5p might be an effective tumor inhibitor in GC.

Table 1.

The relationship between miR-6838-5p expression and clinical-pathological characteristics in GC patients

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Fig. 1.

miR-6838-5p was downregulated in GC tissues and cells lines. a RT-qPCR analysis assessed miR-6838-5p expression in GC tissues and matched healthy tissues. * p < 0.05 vs. normal tissues group. b RT-qPCR revealed the level of miR-6838-5p in GC cells (MGC-803, BGC-823, MKN-45, and SGC-7901) and human gastric epithelial cell line (HFE-145). * p < 0.05 vs. HFE-145 cell line group. c Kaplan-Meier analysis was carried out to assess the effect of miR-6838-5p expression on the overall survival of GC patients.

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miR-6838-5p Overexpression Repressed Cell Proliferation, Migration, and Invasion and Led to Cell Cycle Arrest in GC

Next, the functions of miR-6838-5p in GC cell lines were investigated. To study the influence of miR-6838-5p on the proliferation of GC cells, miR-6838-5p mimics was transfected into MKN-45 and SGC-7901 cells to elevate miR-6838-5p expression (Fig. 2a). CCK-8 and colony formation assays demonstrated that overexpressed miR-6838-5p repressed the proliferative capability of SGC-7901 and MKN-45 cells (Fig. 2b, c). Then, flow cytometry analysis showed miR-6838-5p upregulation resulted in GC cell cycle arrest in the G0/G1 phase (Fig. 2d). Western blotting depicted that upregulation of miR-6838-5p reduced cyclin D1 and cyclin E1 expression levels (Fig. 2e). Furthermore, we found miR-6838-5p restrained GC cell migration and invasion (Fig. 2f, g). In conclusion, high expression of miR-6838-5p repressed GC cell proliferation, migration, and invasion, and resulted in cell cycle arrest at the G0/G1 phase.

Fig. 2.

miR-6838-5p overexpression repressed GC cell proliferation, migration, and invasion, and resulted in cell cycle arrest. a RT-qPCR evaluated the efficiency of miR-6838-5p upregulation in GC cells. * p < 0.05 vs. NC mimics group. b, c CCK-8 and colony formation assays determined cell viability and proliferation. * p < 0.05 vs. NC mimics group. d The flow cytometric analysis determined cell cycle. * p < 0.05 vs. NC mimics group. e Western blotting measured protein levels of cyclin D1 and cyclin E1. f, g Transwell assay determined the migratory and invasive ability of GC cells. * p < 0.05 vs. NC mimics group.

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miR-6838-5p Inactivated Wnt/β-Catenin Signaling Pathway in GC Cells

Many pathways participated in cell proliferation and migration of cancers [13-15]. We detected the key protein levels of the Wnt/β-catenin, PI3K/AKT, and MAPK/ERK pathways. After the treatment of miR-6838-5p mimics, we observed that only the levels of Wnt and β-catenin showed downregulation (Fig. 3a). Besides, since β-catenin nuclear accumulation activated the Wnt/β-catenin signaling pathway [16], we conducted immunofluorescence assay to detect cytoplasmic and nuclear β-catenin protein expression of GC cells. The result suggested high-expressed miR-6838-5p repressed the nuclear import of β-catenin protein in SGC-7901 and MKN-45 cells (Fig. 3b). Figure 3c depicted the downregulation of miR-6838-5p level in tumor tissues relative to normal tissues; β-catenin showed the nuclear accumulation in tumor tissues. All these results implied that miR-6838-5p inactivated Wnt/β-catenin pathway in GC cells.

Fig. 3.

miR-6838-5p inhibited the activation of Wnt/β-catenin pathway in GC cells. a Western blotting was carried out to detect main protein levels of 3 typical pathways in GC cells. b The level of cytoplasmic and nuclear β-catenin in GC cells was measured via immunofluorescence assay. c In situ hybridization (ISH) assay measured miR-6838-5p expression in normal and tumor tissues; immunohistochemistry (IHC) assay depicted the nuclear accumulation of β-catenin.

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GPRIN3 Was the Downstream Target of miR-6838-5p in GC Cells

Furthermore, the downstream target of miR-6838-5p was searched. Based on prediction from starBase [17] (screened by 4 cancer types in Pan-Cancer), 6 potential targets of miR-6838-5p (GPRIN3, SINHCAF, ZNF449, EFNB2, CHD9, and WDR47) were revealed. RT-qPCR demonstrated that GPRIN3 expression showed the most significant downregulation in response to miR-6838-5p overexpression in SGC-7901 and MKN-45 cells (Fig. 4a). Additionally, GPRIN3 expression was significantly upregulated in GC cells than in control cells (Fig. 4b). The binding sequences between GPRIN3 and miR-6838-5p predicted from starBase were shown in Figure 4c. To further validate the connection between GPRIN3 and miR-6838-5p, luciferase reporter and RIP assays were carried out. The results depicted enhanced miR-6838-5p weakened the luciferase activity of pmirGLO-GPRIN3-Wt reporters, but no distinct change was detected in pmirGLO-GPRIN3-Mut reporters in GC cells (Fig. 4d). GPRIN3 and miR-6838-5p tended to enrich in Ago2 groups instead of IgG groups, revealing that GPRIN3 was targeted by miR-6838-5p (Fig. 4e). Afterwards, RT-qPCR depicted that overexpressed miR-6838-5p decreased the level of GPRIN3 (Fig. 4f). To sum up, GPRIN3 was the downstream target of miR-6838-5p.

Fig. 4.

GPRIN3 was targeted by miR-6838-5p in GC cells. a Six candidate targets for miR-6838-5p were obtained from starBase; expression of the 6 candidates was subjected to RT-qPCR analysis after miR-6838-5p overexpression in GC cells. * p < 0.05 vs. NC mimics group. b RT-qPCR detected the expression of GPRIN3 in GC cells lines. * p < 0.05 vs. HFE-145 cell line group. c StarBase predicted the underlying binding sequence between miR-6838-5p and GPRIN3. d Luciferase reporter assay validated the association of miR-6838-5p with GPRIN3 in GC cells. * p < 0.05 vs. NC mimics group. e RIP assay revealed the relative enrichment of miR-6838-5p and GPRIN3 precipitated by anti-Ago2 or anti-IgG in GC cells. * p < 0.05 vs. IgG group. f RT-qPCR evaluated GPRIN3 expression after miR-6838-5p overexpression. * p < 0.05 vs. NC mimics.

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miR-6838-5p Promoted GC Progression by Regulating GPRIN3

To verify whether miR-6838-5p promoted GC cell malignant behaviors by targeting GPRIN3, we conducted the rescue assays in MKN-45 cells. GPRIN3 was effectively overexpressed via transfection of pcDNA3.1/GPRIN3 into GC cells before the rescue assays (Fig. 5a). GPRIN3 upregulation counteracted the inhibition arising from miR-6838-5p overexpression in cell viability and proliferation (Fig. 5b, c). As shown in Figure 5d, e, upregulation of GPRIN3 countervailed the suppression from miR-6838-5p in cell cycle progression. Additionally, GPRIN3 overexpression rescued the suppressing influence induced by miR-6838-5p overexpression on the migratory and invasive abilities of GC cells (Fig. 5f, g). Thereafter, the influence of GPRIN3 on miR-6838-5p-mediated β-catenin nuclear transport was assessed. The result indicated that miR-6838-5p-induced inhibition on β-catenin protein expression, and its nuclear import was counteracted by upregulated GPRIN3 (Fig. 5h). In summary, miR-6838-5p facilitated GC cell malignant behaviors via downregulation of GPRIN3 to inhibit Wnt/β-catenin signaling.

Fig. 5.

miR-6838-5p promoted GC cell malignancy by regulating GPRIN3. a RT-qPCR analysis determined the efficiency of GPRIN3 upregulation in MKN-45 cells. * p < 0.05 vs. pcDNA3.1 group. b, c CCK-8 and colony formation assays determined the influence of GPRIN3 on miR-6838-5p mediated cell viability and proliferation. * p < 0.05 vs. NC mimics group and miR-6838-5pmimics+pcDNA3.1/GPRIN3 group. d Flow cytometry analysis detected cell cycle in MKN-45 cells by indicated transfections. * p < 0.05 vs. NC mimics group and miR-6838-5pmimics+pcDNA3.1/GPRIN3 group. e Western blotting measured protein levels of cyclin D1 and cyclin E1. f, g Transwell assay measured the migratory and invasive abilities of MKN-45 cells by indicated transfections. * p < 0.05 vs. NC mimics group and miR-6838-5pmimics+pcDNA3.1/GPRIN3 group. h β-Catenin protein and its nuclear import was assessed via Western blotting.

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Discussion

GC is a prevalent malignancy with significant mortality rate [18]. Accumulating evidence has showed that miRNAs exert crucial influence on the progression of GC [19-21]. miR-6838-5p serves as an anti-cancer gene in triple-negative breast cancer [7]. The downregulation of miR-6838-5p in GC cell lines was identified in this study. Furthermore, upregulation of miR-6838-5p repressed the proliferative, migratory, and invasive capabilities of GC cells, and resulted in cell cycle arrest at the G0/G1 stage.

As a highly preserved pathway [22, 23], Wnt/β-catenin signaling modulates multiple cellular processes [24-26]. Mounting evidence revealed that nuclear accumulation of β-catenin promotes the activation of the downstream Wnt-responsive genes, and triggers Wnt/β-catenin pathway [27-29]. We found miR-501-5p upregulation drives Wnt/β-catenin pathway and elevates stem cell-like phenotypes in GC [30]. LINC01606 promotes GC cell migration through activating Wnt/β-catenin pathway [31]. TGM1 facilitates stemness of GC cells through Wnt/β-catenin signaling [32]. In our investigation, miR-6838-5p repressed Wnt/β-catenin signaling in GC cells.

miRNAs have been shown to regulate mRNA expressions via binding complementary sites in the 3′-UTR of target mRNAs [33, 34]. miR-6838-5p was verified to be targeted by GPRIN3 in GC cells, and its role has not been explored in cancers. In this paper, we discovered that GPRIN3 can interact with miR-6838-5p in GC cells. GPRIN3 was upregulated in GC cell lines and was negatively modulated by miR-6838-5p in GC. GRIN3 is also reported to be upregulated in striatum and interact with β-arrestin 2 [35]. Besides, rescue assays in our study demonstrated that GPRIN3 overexpression counteracted the inhibition from miR-6838-5p upregulation in the proliferative, migratory, and invasive capabilities of GC cells.

In summary, our findings suggested that miR-6838-5p suppressed cell growth, migration and invasion through the regulation of GPRIN3 and Wnt/β-catenin signaling pathway in GC cells, which might provide practical meaning for the therapeutic strategies of GC.

Acknowledgement

We thank all participants for their assistance.

Statement of Ethics

Written informed consent was signed by all participants. The research gained approval of the Ethics Committee of Ningbo First Hospital (Zhejiang, China).

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

The study was funded by Zhejiang Provincial Natural Science Foundation of China (LQ18H160015).


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  35. Mototani Y, Okamura T, Goto M, Shimizu Y, Yanobu-Takanashi R, Ito A, et al. Role of G protein-regulated inducer of neurite outgrowth 3 (GRIN3) in β-arrestin 2-Akt signaling and dopaminergic behaviors. Pflugers Arch. 2018 Jun;470(6):937–47.
    External Resources

Author Contacts

Peifei Li

Department of Gastroenterology

Ningbo First Hospital, No. 59 Liuting Street

Ningbo, Zhejiang 315010 (China)

lipeifei@hotmail.com

Article / Publication Details

First-Page Preview
Abstract of Research Article

Received: March 02, 2020
Accepted: September 16, 2020
Published online: November 30, 2020
Issue release date: December 2020

Number of Print Pages: 11
Number of Figures: 5
Number of Tables: 1

ISSN: 1015-2008 (Print)
eISSN: 1423-0291 (Online)

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  35. Mototani Y, Okamura T, Goto M, Shimizu Y, Yanobu-Takanashi R, Ito A, et al. Role of G protein-regulated inducer of neurite outgrowth 3 (GRIN3) in β-arrestin 2-Akt signaling and dopaminergic behaviors. Pflugers Arch. 2018 Jun;470(6):937–47.
    External Resources
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2020, Vol.87, No. 6

December 2020

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