Research ArticleMicroRNA-520a Suppresses Pathogenesis and Progression of Non-Small-Cell Lung Cancer through Targeting the RRM2/Wnt Axis

Yi Xie ,1 Congyu Xue ,2 Shuai Guo ,1 and Lei Yang 2

1Department of Respiratory Oncology, Shandong Provincial Chest Hospital, Jinan, 250013 Shandong, China2Department of Tuberculosis, Shandong Provincial Chest Hospital, Jinan, 250013 Shandong, China

Correspondence should be addressed to Congyu Xue; congyux010902@163.com

Received 27 April 2020; Revised 14 January 2021; Accepted 25 February 2021; Published 31 March 2021

Academic Editor: Consuelo Amantini

Copyright © 2021 Yi Xie et al. This is an open access article distributed under the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

MicroRNAs (miRNAs) regulate multiple cellular behaviors, and their aberrant expression is frequently associated with diseaseprogression. This research focused on the effects of miR-520a on the development of non-small-cell lung cancer (NSCLC) andthe molecules involved. Tumor and normal tissues from 24 patients with NSCLC were collected. Differentially expressedmiRNAs between tumor tissues and normal tissues were screened using microarrays, and miR-520a was screened to besignificantly poorly expressed in tumor samples. Artificial upregulation of miR-520a reduced proliferation, migration andinvasion, and resistance to death of NSCLC A549 and H460 cells according to the MTT, EdU labeling, transwell, and flowcytometry assays, respectively. miR-520a upregulation suppressed growth and metastasis of xenograft tumors in vivo. Theintegrated bioinformatic analysis and dual luciferase assays suggested that miR-520a targeted ribonucleotide reductase subunit 2(RRM2) mRNA and inactivated the Wnt/β-catenin signaling pathway in NSCLC cells. Upregulation of RRM2 enhanced themalignant behaviors of NSCLCs, but the oncogenic effects of RRM2 were blocked upon miR-520a overexpression. To conclude,this study evidenced that miR-520a inhibits NSCLC progression through suppressing RRM2 and the Wnt signaling pathway.This paper may offer novel insights into NSCLC treatment.

1. Introduction

Lung cancer (LC) is one of the most prevailing and life-threatening cancer types which is estimated to account fornearly a quarter of cancer-related mortality in 2020 [1].According to the pathological type, LC is categorized intotwo major subtypes including non-small-cell lung cancer(NSCLC, accounting for nearly 85% of all cases) and small-cell lung cancer (15% of all cases) [2]. In China, LC has thehighest mortality rate among all cancer types [3]. Despitethe improvements in conventional therapeutic regimensincluding surgery, radiotherapy, and chemotherapy, thetreating outcome of LC patients remains unfavorable [4]. Amajor reason is that a considerable number of LC patientsare initially diagnosed at advanced stages with metastaticproperties, and the overall 5-year survival rate of thesepatients is extremely low at about 5% [1]. Identifying keymolecules involved in growth and metastasis of LC is of greatimportance to develop novel therapeutic options.

Gene-based therapy has been a promising target for dis-ease treatment and aroused wide concerns. Approximately,97% of all human genomes are transcribed to noncoding(nc) RNAs and can regulate the molecular processes atDNA-RNA-protein levels [5]. MicroRNAs (miRNAs) arelargely studied short ncRNAs renowned for their capacityin controlling the gene expression by primarily interactingwith the 3′-UTR of target mRNAs [6]. Owing to the potentgene-modifying functions, miRNAs can regulate diverse fun-damental cellular processes including apoptosis, prolifera-tion, maintenance of cell differentiation, and tumorigenesisin several cancers including LC [7]. A microarray analysisin this study identified miR-520a as an aberrantly poorlyexpressed miRNA in NSCLC tissues. miR-520a has beendemonstrated as a tumor inhibitor in many human malig-nancies [8–11]. Downregulation of miR-520-3p by otherRNA transcripts has been suggested to facilitate the progres-sion of NSCLC [12, 13]. However, the independent roles ofmiR-520a in NSCLC and the downstream molecules

HindawiAnalytical Cellular PathologyVolume 2021, Article ID 9652420, 12 pageshttps://doi.org/10.1155/2021/9652420

involved are not fully explored yet. Here, our integrated bio-informatic analyses suggested ribonucleotide reductase sub-unit 2 (RRM2) mRNA as a potential target transcript ofmiR-520a. RRM2 has shown oncogenic roles in many malig-nancies such as gastric cancer [14] and pancreatic cancer[15]. Herein, we hypothesized that miRNA-520a suppressesprogression of NSCLC through inhibiting RRM2, and bothcell and animal experiments were performed to validate thishypothesis.

2. Materials and Methods

2.1. Clinical Sample Collection. Twenty-four patients withNSCLC diagnosed and treated in Shandong Provincial ChestHospital from January 2015 to January 2016 were recruitedinto the research. Patients with other chronic diseases, witha history of chemo/radio therapy, or with a family historyof malignancy were excluded. The tumor tissues and theadjacent normal tissues (over 5 cm away from lesion sites)were resected during surgery and instantly frozen in liquidnitrogen and preserved at -80°C for further use. A three-year follow-up study was performed. This study was ratifiedand supervised by the Ethics Committee of ShandongProvincial Chest Hospital. Signed informed consent was col-lected from each eligible patient. The clinical characteristicinformation of all participants is presented in Table 1.

2.2. Hematoxylin and Eosin (HE) Staining. The collected tis-sues from patients and liver and lung tissues from mice (seebelow) were embedded in paraffin and cut into 5-μm slices.The tissue slices were successively baked at 60°C for onehour, soaked in xylenes I, II, and III (10 minutes for each),dehydrated in an ascending series of alcohol for 5 minutes,and washed in water for 5 minutes. After that, the slices werestained in hematoxylin solution for 8 minutes, differentiatedin 0.5% hydrochloric acid-ethanol mixture for 10 seconds,soaked in ammonia for 40 seconds, and then stained with0.5% eosin for 5 minutes. After that, the slices wererehydrated in a decreasing series of alcohol, sealed with neu-tral balsam (96949-21-2, Solarbio, Science & Technology Co.,Ltd., Beijing, China), and then observed under a microscope(BX53, Olympus Optical Co., Ltd, Tokyo, Japan) with 5 ran-dom fields selected. The nuclei were stained in blueish, whilethe cytoplasm in reddish. To each group, three slices from asame tissue sample were stained.

2.3. Microarray Analysis. Total RNA from 15mg NSCLC andthe adjacent tissues was extracted using a miRNeasy Kit(217061, Qiagen GmbH, Hilden, Germany) and a mirVanamiRNA Isolation Kit (AM1560, Ambion, Austin, Texas,USA), respectively. The RNA purity was examined by NanoDrop (2000/2000C, Thermo Fisher Scientific, Carlsbad, CA,USA). Subsequently, 100 ng RNA was collected and labeledusing a miRNA Complete Labeling and Hyb Kit (5190-0456, Agilent Technologies, Inc. Wakefield, MA, USA).Then, the samples were hybridized with Human V2 miRNA Microarrays (G4470B, Agilent) at 200 rpm at 60°C for20 hours. The images were scanned and analyzed using aDNA Microarray Scanner (G2505-60525, Agilent), while

the data were obtained and analyzed by GeneSpring GX(V11.5, Agilent).

2.4. Bioinformatic Analysis. Data were analyzed using an R-Language Package (version 3.6.3). The microarray resultswere analyzed using a Limma Package (http://www.bioconductor.org/). Differentially expressed miRNAsbetween tumor and normal tissues were screened with ∣log2FC ∣ >2:0 and p < 0:01 as the screening criteria. The heatmap was produced using a pheatmap Package (https://cran.r-project.org/web/packages/pheatmap/index.html). The expres-sion and prognostic value of miR-520a in NSCLC were firstpredicted on The Cancer Genome Atlas (TCGA, https://cancergenome.nih.gov/). Target mRNAs of miR-520a werepredicted on several bioinformatic systems including StarBase(http://starbase.sysu.edu.cn/), TargetScan (http://www.targetscan.org), miRDB (http://www.mirdb.org), and miRbase(http://www.mirbase.org). The predicted results were com-pared using a gplot package (https://cran.r-project.org/web/packages/gplots/), and a Gene Ontology (GO) enrichmentanalysis was performed using a clusterProfiler package(http://www.bioconductor.org/).

2.5. Reverse Transcription-Quantitative Polymerase ChainReaction (RT-qPCR). Total RNA from tissues and cells wasextracted using the TRIzol Reagent (Thermo Fisher Scien-tific). Then, the RNA was reversely transcribed into cDNAusing a high-capacity cDNA Reverse Transcription Kit(4368814, Thermo Fisher Scientific). Subsequently, real-time qPCR was performed using TaqMan microRNA assaykits (4427975, Thermo Fisher Scientific) on a 7900HT fastReal-time PCR system (Applied Biosystems, Foster City,CA). The fold change of the acquired data was measuredusing the 2-ΔΔCt method. The primer sequences are exhibitedin Table 2, in which U6 was used as the internal control formiRNA while GAPDH as the control for RRM2 mRNA.

2.6. Cell Culture and Transfection. Lung large cell carcinomacell lines H273 (CRL-5917) and H460 (HTB-177), lung

Table 1: Detailed characteristics of 24 patients with NSCLC.

Item Group n

GenderMale 14

Female 10

Age≤ 60 11

> 60 13

TNMI and II 5

III and IV 19

Lymph node metastasisNegative 6

Positive 18

Tumor size≤ 3 cm 15

> 3 cm 9

Histology

Adenocarcinoma 10

Squamous cell carcinoma 8

Others 6

Note: TNM: tumor node metastasis.

2 Analytical Cellular Pathology

adenocarcinoma cell lines H23 (CRL-5800) and A549 (CCL-185), a normal pulmonary epithelial cell line 16HBE (PCS-300-010), and HEK293T (CRL-3216) cells were purchasedfrom American Type Culture Collection (ATCC, Manassas,VA, USA). The cells were cultivated in RPMI-1640(21870076, Thermo Fisher Scientific) containing 10% fetalbovine serum (FBS, North Andover, MA, USA) at 37°C with5% CO2. The miR-520 mimic and RRM2 overexpressing(OE) vector were synthesized by RiboBio Co., Ltd. (Guang-zhou, Guangdong, China). Cell transfection was performedusing a Lipofectamine 2000 DNA Transfection Reagent(11668027, Thermo Fisher Scientific). In brief, when the cellconfluence reached 80%, the Lipofectamine Reagent wasdiluted in Opti-MEM, while another Opti-MEM was usedto dilute DNA. The diluted DNA was mixed with the dilutedLipofectamine 2000 at a ratio of 1 : 1. After 5 minutes, thecells were filled with DNA-lipid compound and cultured at37°C for 48 hours.

2.7. 3-(4,5-Dimethylthiazol-2-yl)-2,5-DiphenyltetrazoliumBromide (MTT) Assay. Exponentially growing cells wereresuspended to 5 × 104/mL. The cell suspension was sortedinto 96-well plates at 100μL per well till the cells reaching adensity of 5 × 103 cells/well (the marginal pores were filledwith sterile phosphate-buffered saline (PBS) to eliminatethe potential edge effect). Three duplicated wells were setfor each group. The plates were incubated in a 37°C incubatorwith 5% CO2. One plate was taken out every 24 hours formeasurement. In brief, each well was filled with 20μL MTTsolution (M1025, Solarbio), and the cells were incubated foranother 4 hours. Then, the culture medium in wells was dis-carded, and each well was further loaded with 150μLdimethyl sulphoxide to fully dissolve the crystal violets. Then,the optical density at 570nm of each well was determinedusing a microplate reader (Varioskan LUX, Thermo FisherScientific). The obtained data were analyzed to produce anMTT proliferation curve.

2.8. 5-Ethynyl-2′-Deoxyuridine (EdU) Labeling Assay. Expo-nentially growing cells were sorted into 6-well plates. Whenthe cell confluence reached 80%, 10μM EdU solution(Genecopoeia, USA) was loaded into each well. The cellswere incubated for 2 hours, washed in PBS, fixed in 4% para-

formaldehyde for 30 minutes, incubated in glycine solution,and rinsed with PBS containing 0.5% TritonX-100. Subse-quently, the cells were incubated with Andy Fluor™ 555 azide(A004, Genecopoeia, USA) in the dark for 30 minutes,washed in methanol and PBS, and further incubated with4′,6-diamidino-2-phenylindole (DAPI) for 20 minutes. Thelabeling was observed under a fluorescence microscope(XSP-BM13C, Shanghai CSOIF. Co., Ltd., Shanghai, China)at a ×400 magnification with 3 random fields included. TheEdU-positive cells were stained in reddish, and all cells werestained in bluish by DAPI: the cell proliferation rate =number of proliferation cells/total cells × 100%.

2.9. Transwell Assay. The 24-well plates were used for trans-well assays. The Matrigel gel (Corning, Corning, NY, USA)was diluted in serum-free RPMI-1640 at a ratio of 5 : 1 andthen loaded onto the apical chamber (80μL in each apicalchamber) and maintained for 1-2 hours, while each basolat-eral chamber was filled with 500μL 10% FBS-RPMI-1640.The cells were seeded into 24-well plates and incubated for24 hours. The cells that invaded into lower membranes werefixed in 4% paraformaldehyde and stained by crystal violetstaining solution (C0121, Beyotime Biotechnology Co., Ltd.,Shanghai, China) for 10 minutes. The staining was observedunder a microscope (BX53, Olympus Optical Co., Ltd.,Tokyo, Japan) at a ×400 magnification with 5 random fieldsincluded. Cell migration was examined in a similar mannerexcept for pre-coating of Matrigel gel on the apical chambers.The number of invading and migrating cells was counted,and the average value of 3 duplicated wells was calculated.

2.10. Flow Cytometry. Apoptosis of cells was determined byan Annexin V-FITC Apoptosis Detection Kit (15342-54,Nacalai, Tesque, Japan). After transfection, the cells were cul-tivated at 37°C with 5% CO2 for 48 hours, centrifuged at1,000 rpm for 3 minutes, washed in PBS, and then centri-fuged again. Then, 20μL Annexin V Binding Buffer (×10)was added into 200μL PBS. Cells were resuspended in PBSand then incubated with 10μL Annexin V-FITC conjugateand 5μL PI solution at room temperature in the dark for15 minutes. Then, the cells were added with 300μL bindingbuffer, and the excitation wavelength at 488nm was deter-mined using a flow cytometer (6028651, Beckman Coulter,USA).

2.11. Dual Luciferase Reporter Gene Assay. The 3′-UTRsequences of RRM2, SDC1, and YWHAZmRNAs containingputative binding sequences with miR-520a were inserted tothe pGL3 vectors (kl-zl-1031, Ke Lei Biological TechnologyCo., Ltd., Shanghai, China) to construct pGL3-based widetype (WT) vectors. The corresponding mutant type (MT)vectors were constructed as well using the mutant bindingsequences. Well-constructed vectors were cotransfected withmiR-520a mimic or mimic control in HEK293T cells usingthe Lipofectamine 2000 Kit. After 48 h, the cells were col-lected and lysed, and the relative luciferase activity in cellswas evaluated using a Luciferase Reporter Assay Kit (K801-200, Biovision) and a Dual Luciferase Reporter Assay System(E1910, Promega, Madison, WI, USA).

Table 2: Primer sequences in RT-qPCR.

Gene Primer sequence (5′-3′)

miR-520aF: GCCACCACCATCAGCCATAC

R: GCACATTACTCTACTCAGAAGGG

RRM2F: CCTGACTGTAAATCGTCCAGTGGTA

R: AGTTTGGAAGCGACTGTAGGT

U6F: CTCGCAGCGCTTCGACA

R: ACTTGCGGCTTCACGAATT

GAPDHF: GATATATGATCAGTCATCTGC

R: TTGGATGATCTGGTTTAGCG

Note: RT-qPCR: reverse transcription quantitative polymerase chain reaction;RRM2: ribonucleotide reductase subunit 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; F: forward; R: reverse.

3Analytical Cellular Pathology

2.12. Western Blot Analysis. Cells were centrifuged andwashed in PBS and resuspended in Tris containing proteaseinhibitor (50mM Tris-HCL, 150mM NaCl). The proteinconcentration was determined using a Pierce™ BCA ProteinAssay Kit (23225, Thermo Fisher Scientific). Then, an appro-priate volume of protein was separated on 8%-12% SDS-PAGE and transferred onto PVDF membranes (LM-937D,LMAI Bio, Shanghai, China). The membranes were blockedand washed in 5% nonfat milk for 1 hour, followed by anincubation with the primary antibodies against B-celllymphoma-2 (Bcl-2, 1 : 1,000, sc-7382, SANTA CRUZ, CA,USA), Bcl-2-associated x, (Bax, 1 : 5,000, sc-7480, SANTACRUZ), caspase-3 (1 : 1,000, ab208161, Abcam, Inc., Cam-bridge, MA, USA), cleaved caspase-3 (1 : 500, ab2302,Abcam), β-catenin (1 : 1,000, ab208161, Abcam), cyclin D1(1 : 500, sc-8396, SANTA CRUZ), and β-actin (1 : 1,000,#3700, Cell Signaling Technology) at 4°C for 16 hours. Afterthat, the membranes were washed and further incubated withsecondary antibodies to IgG (1 : 5,000, ab205719, Abcam)and cleaved caspase (1 : 10,000, ab205718, Abcam) at 20°Cfor 2 hours. The protein bands were developed usingenhanced chemiluminescence western blotting substrate(PE0010, Solarbio), and the images were captured and ana-lyzed using a Western Blot Imaging System (FluorChem M,ProteinSimple).

2.13. Growth of Xenograft Tumors in Nude Mice. A total of 12female nude mice (strain: BALB/c; 3-4 weeks old, 14 ± 2 g)were purchased from the Vital River Laboratory AnimalTechnology Co., Ltd. (Beijing, China). The mice were fed atconstant 25°C with 45% humidity with free access to foodand water. The mice were randomly allocated into 4 groups:miR-520a control group (n = 3, each mouse were injectedwith A549 cells transfected with miR-520a control), miR-520a mimic group (n = 3, each mouse were injected withA549 cells transfected with miR-520a mimic), miR-520a con-trol group (n = 3, each mouse were injected with H460 cellstransfected with miR-520a control), and miR-520a mimicgroup (n = 3, each mouse were injected with H460 cellstransfected with miR-520a mimic). For cell implantation,exponentially growing A549/H460 cells with stable transfec-tion of miR-520a mimic or mimic control were adjusted to1 × 107 cells/mL. Then, each mouse was transplanted with20μL cell suspension through axillary injection, and thenthe growth of tumor in vivo was photographed and recorded.The tumor volume (V) was recorded every 7 days as follows:V = a × b2/2 (where “a” refers to the long diameter while “b”refers to the short diameter), and a growth curve was pro-duced. After 28 days, the mice were euthanized by overdoseof 1% pentobarbital sodium (150mg/kg), and the tumorwas collected and weighed [16]. All procedures were per-formed in line with the guidelines of the Animal Ethics Com-mittee of Shandong Provincial Chest Hospital. Greatattempts were made to reduce the usage and pain of animals.

2.14. Tumor Metastasis in Nude Mice. Another 12 nude micewere numbered by weight and allocated into 4 groups againas performed above (n = 3 in each group). For tumor metas-tasis measurement, A549/H460 cells with stable transfection

of miR-520a mimic or mimic control were injected into micethrough caudal veins. The mice were euthanized in a similarmanner on the 45th day after injection. Then, the lung andliver tissues of mice were collected, embedded in paraffin,and cut into slices for HE staining. The formation of metasta-tic nodules in the tissues was observed under the microscopewith 5 nonoverlapping fields included.

2.15. Statistical Analysis. SPSS 22.0 (IBM Corp. Armonk, NY,USA) was used for data analysis. Measurement data werecollected from three independent experiments andexpressed as mean ± standard deviation (SD). Data betweenevery two groups were analyzed using the t-test, while thoseamong multiple groups were analyzed by one-way or two-way analysis of variance (ANOVA) followed with Tukey’smultiple comparison test. The p value was obtained fromtwo-tailed tests, and p < 0:05 was regarded to show statisti-cal significance.

3. Results

3.1. miR-520a Is Poorly Expressed in NSCLC Tissues andCells. Tissue samples from three NSCLC patients were col-lected for HE staining and miRNA microarray analysis. Thestaining results suggested that the cells in the NSCLC tissuesshowed destructed cell structure and shrinking nuclei, indi-cating severe injury in lungs (Figure 1(a)). Then, the RNAsamples extracted from three pairs of tissues were used formicroarray analysis. Importantly, miR-520a was found tobe significantly downregulated in the NSCLC tumor tissuescompared to the adjacent tissues (Figure 1(b)). To validatethis, tissues from all 24 patients were used for RT-qPCR.Consequently, poor expression of miR-520a was found inthe NSCLC tissues compared to the paired adjacent tissues(Figure 1(c)). The follow-up study results suggested thatpatients with higher levels of miR-520a had better prognosisand longer lifetime (Figure 1(d)). In addition, the miR-520aexpression in NSCLC cell lines H273, H23, A549, and H460and in normal pulmonary epithelial cells 16HBE was deter-mined by RT-qPCR as well. It was found that all NSCLC celllines showed the decreased miR-520a expression relative tothe 16 HBE cells (Figure 1(e)). Among them, the A549 andH460 cells showing the lowest expression of miR-520a weretransfected with miR-520a mimic or mimic control for sub-sequent use (Figure 1(f)). In addition, according to the datafrom the TCGA database, miR-520a was poorly expressedin NSCLC (Figure 1(g)), and the poor expression of miR-520a indicated unfavorable prognosis in patients withNSCLC (Figure 1(h)). These results suggested that miR-520a might have prognostic value in NSCLC.

3.2. Artificial Upregulation of miR-520a Inhibits NSCLCProgression. Subsequently, we found that the overexpressionof miR-520a by miRNA mimic led to a significant decline inthe number of EdU-positive A549 and H460 cells(Figure 2(a)). The MTT assay results suggested that the num-ber of proliferative A549 and H460 cells were reduced follow-ing the miR-520a overexpression (Figure 2(b)). As for cellmigration and invasion, the transwell assay results suggested

4 Analytical Cellular Pathology

that the migration and invasion abilities of both A549 andH460 cell were decreased upon the miR-520a overexpression(Figures 2(c) and 2(d)). In addition, the flow cytometryresults suggested that the overexpression of miR-520increased the number of Annexin V- and PI-positive cells,namely increased apoptosis of A549 and H460 cells(Figure 2(e)). From the molecular perspective, it was foundthat miR-520a mimic decreased the level of Bcl-2 butincreased the levels of Bax and cleaved caspase-3(Figure 2(f)). Collectively, these results suggested that miR-520a inhibits the malignant behaviors of NSCLC cells.

3.3. miR-520a Directly Binds to RRM2 to Mediate the WntSignaling Pathway. To further identify the potential down-stream molecules implicated, we first predicted that the tar-get mRNAs of miR-520a on several bioinformatic systemsincluding StarBase, TargetScan, miRDB and miRBase, and219 mRNAs were found to be intersected (Figure 3(a)). Then,a GO enrichment analysis was performed based on thesegenes, which identified that the Wnt signaling was highlyenriched (Figure 3(b)), and 18 genes were enriched in thissignaling pathway. Then, the levels of Wnt signalingpathway-related factors cyclin D1 and β-catenin in A549

Adjacent-1 Adjacent-2 Adjacent-3

Tumor-1 Tumor-2 Tumor-3

100𝜇m 100𝜇m 100𝜇m

100𝜇m 100𝜇m 100𝜇m

(a)

miR-497miR-338miR-218

miR-130amiR-30d

miR-181amiR-139miR-26b

miR-520a

Adj

acen

t-1

Adj

acen

t-2

Adj

acen

t-3

Tum

or-1

Tum

or-2

Tum

or-3

1110109.358.617.877.136.395.654.914.173.432.691.951.21

miR-101

(b)

Adjacent Tumor0.0

0.5

1.0

1.5

Rela

tive m

iR-5

20 al

evel

⁎

(c)

0 10 20 40300

50

100

Months

Ove

rall

surv

ival

High expression of miR-520a(n = 10)Low expression of miR-520a(n = 14)

P = 0.026

(d)

16HBE H273 H23 A549 H4600.0

0.5

1.0

1.5

Rela

tive m

iR-5

20a l

evel

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⁎

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Rela

tive m

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20a l

evel

miR-520a controlmiR-520a mimic

⁎ ⁎

(f)

TCGA samples

Normal(n=44)

0

1

1.25

0.25

1.5

0.75

0.5

Read

s per

mill

ion

Primary tumor(n=336)

(g)

0 50 100 150

0.0

0.2

0.4

0.6

0.8

1.0hsa−miR−520a

Time (months)

Prob

abili

ty

Number at risk150 34 12 3Low322 65 11 2High

HR = 0.64 (0.48 − 0.85)Logrank P = 0.0019

ExpressionLowHigh

(h)

Figure 1: miR-520a is poorly expressed in NSCLC tissues and cells. (a) NSCLC tissues and the adjacent normal tissues observed by HEstaining. (b) Differentially expressed miRNAs between cancer and the adjacent normal tissues screened out by miRNA microarrayanalysis. (c) The miR-520a expression in NSCLC and the adjacent normal tissues determined by RT-qPCR (∗p < 0:05, two-way ANOVA).(d) Overall survival rate of patients with different levels of miR-520a according to a 3-year followed-up study. (e) The miR-520aexpression in NSCLC cell lines and in normal pulmonary epithelial cell line 16HBE determined by RT-qPCR (∗p < 0:05, one-wayANOVA). (f) The miR-520a expression in A549 and H460 cells after miR-520a mimic/control transfection determined by RT-qPCR(∗p < 0:05, two-way ANOVA). (g) The expression of miR-520a in NSCLC predicted on the TCGA database. (h) Relevance of miR-520a tothe survival of patients predicted on the TCGA database.

5Analytical Cellular Pathology

and H460 cell lines were measured. The western blot analysisresults showed that the cyclin D1 and β-catenin levels in bothcells were declined following the miR-520a overexpression(Figure 3(c)), indicating that miR-520a inactivated the Wntsignaling. Subsequently, we tested the expression of mRNAsenriching on the Wnt signaling in tissues using RT-qPCR.It was found that the expression of SDC1, YWHAZ, andRRM2 was significantly increased in tumor tissues as com-pared to the paired normal tissues (Figure 3(d)). Then, theluciferase assays validated that only RRM2 presented a bind-ing relationship with miR-520a (Figure 3(e)). The correlation

analysis suggested that the expression of miR-520a was neg-atively correlated with expression of RRM2 mRNA inNSCLC tumor tissues (Figure 3(f)). In concert with the abovefindings, the RRM2 expression was found higher in NSCLCcell lines than that in 16HBE cells (Figure 3(g)). Also, miR-520a mimic was found to decrease the RRM2 expression inA549 and H460 cells (Figure 3(h)). In addition, the overex-pression of RRM2 in cells increased the levels of cyclin D1and β-catenin in A549 and H460 cells (Figure 3(i)), indicat-ing that miR-520a inactivates the Wnt signaling pathwaypossibly through downregulating the RRM2 expression.

A549 H4600

10

20

30

40

EdU

-pos

itive

cells

(%)

miR-520a control miR-520a mimic

A549

H460

100𝜇m 100𝜇m

100𝜇m 100𝜇m

⁎⁎

(a)

0 12 24 480.0

0.5

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Hour (h)0 12 24 48

Hour (h)

Cell

viab

ility

(OD

)

Cell

viab

ility

(OD

)

miR-520a controlmiR-520a mimic

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miR-520a control miR-520a mimic

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Num

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100𝜇m 100𝜇m

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60

80

100

Num

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H460

100𝜇m 100𝜇m

100𝜇m 100𝜇m

⁎⁎

(d)

A549 H460

miR-520a control miR-520a mimic

miR-520a control miR-520a mimic

0

10

20

30

40

Apo

ptos

is ce

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tio (%

)

A549

H460miR-520a controlmiR-520a mimic

⁎⁎

101010Annexin-V-FITC1010101010

Annexin-V-FITC1010

10

10

10PI

10

10

10

10

10PI

10

10

10

10

10PI

10

10

10

10

10PI

10

10

101010Annexin-V-FITC1010 101010

Annexin-V-FITC1010

(e)

0

1

2

3

4 A549

Prot

ein

expr

essio

n

Ccaspase-3

Bcl-2

BaxCleaved-

caspase-3

Ccas

pase

-3

Bcl-2 Ba

x

Clea

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ase-

3

Ccas

pase

-3

Bcl-2 Ba

x

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casp

ase-

3

𝛽-Actin

31 kDa

26 kDa

24 kDa

17 kDa

45 kDa

0

1

2

3

4

5 H460

Prot

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expr

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n

A549 H460

miR

-520

a mim

ic

miR

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miR-520a controlmiR-520a mimic

⁎⁎

⁎

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⁎

(f)

Figure 2: Artificial upregulation of miR-520a inhibits NSCLC progression. (a) Viability of A549 and H460 cells determined by the EdUlabeling assay (∗p < 0:05, two-way ANOVA). (b) Proliferation of A549 and H460 cells after miR-520a transfection determined by theMTT assay (∗p < 0:05, two-way ANOVA). (c, d) Changes in number of migrating (c) and invading cells (d) detected using the transwellassays (∗p < 0:05, two-way ANOVA). (e) The apoptosis rate of A549 and H460 cells determined by flow cytometry (∗p < 0:05, two-wayANOVA). (f) Protein levels of apoptosis-related factors Bcl-2, Bax, caspase-3, and cleaved caspase-3 determined by western blot analysis(∗p < 0:05, two-way ANOVA).

6 Analytical Cellular Pathology

15.4%

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R-HSA-512988: Interleukin-3, Interleukin-5 and GM-CSF signalingGO:0009792: embryo development ending in birth or egg hatchinghsa04340: Hedgehog signaling pathwayGO:0034063: stress granule assemblyGO:0051261: protein depolymerizationhsa04520: Adheren junctionGO:0046777: protein autophosphorylationGO:1903320: regulation of protein modification by small protein conjugation or removalM145: PID P53 Downstream pathwayGO:0007264: small GTPase mediated signal transductionhsa04810: Regulation of actin cytoskeletonGO:0002573: myeloid leukocyte differentiationGO:0021766: hippocampus developmentGO:1904019: epithelial cell apoptotic processhsa05220: Chronic myeloid leukemiaGO:1900153: positive regulation of nuclear-transcribed mRNA catabolic process, deadenylation-dependent decayhsa05210: Colorectal cancerGO:0016055: Wnt signaling pathwayGO:0030099: myeloid cell differentiationR-HSA-4086398: Ca2+ pathway

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7Analytical Cellular Pathology

3.4. miR-520a Inhibits the Oncogenic Effects of RRM2 onNSCLC Progression. To confirm the involvement of RRM2in the miR-520a-mediated events, A549 and H460 cellstransfected with RRM2 OE vector were further transfectedwith miR-520a mimic (Figure 4(a)). It was found that the cellviability increased by RRM2 was reversed after further upreg-ulation of miR-520a (Figure 4(b)). In addition, cotransfectionof miR-520a and RRM2 OE vector decreased the number ofmigrating and invading cells, and it increased the numberof apoptotic cells compared to transfection of RRM2 OEvector alone (Figures 4(c)–4(e)). These results, collectively,validated that miR-520a directly binds to RRM2 to inhibitthe malignant behaviors of NSCLC cells.

3.5. miR-520a Inhibits Tumor Growth and Metastasis InVivo. A549 and H460 cells with stable miR-520a mimic/con-trol transfection were implanted into nude mice throughaxillary injection (for growth measurement) or tail vein injec-tion (for metastasis measurement). The tumor volume inmice was measured every 7 days. It was found that the over-expression of miR-520a significantly reduced the tumorgrowth rate in mice (Figure 5(a)). Twenty-eight days laterwhen the mice were euthanized, it was found that miR-520a mimic also reduced the tumor weight in nude mice(Figure 5(b)). In addition, the HE staining results showedthat the number of metastatic nodules in lung (Figure 5(c))and liver (Figure 5(d)) tissues was decreased by miR-520amimic.

4. Discussion

NSCLC remains the most prevailing cancer and conse-quently the biggest cause of cancer-related morbidity world-wide, leaving understanding the biology of NSCLC andidentifying novel molecules involved in pathogenesis of greaturgency [17]. miRNA-targeted therapies, including miRNAreplacement and miRNA reduction where oligonucleotides,virus-based constructs, or small molecule compounds are

administrated to restore the expression of suppressive miR-NAs or inhibit expression of oncogenic miRNAs, havearoused increasing concerns in LC treatment [18]. The pres-ent study suggested that miR-520a played potent tumor sup-pressing roles in NSCLC in both cell models and animalmodels through the downregulation of RRM2 and inactiva-tion of the Wnt signaling.

Aberrant expression of miRNA is often linked to theonset and development of diseases including cancers [19].Here, a miRNA microarray analysis and RT-qPCR resultssuggested that miR-520a was downregulated in NSCLCtumor samples. A higher level of miR-520a was associatedwith a better prognosis in NSCLC patients and artificialupregulation of miR-520a inhibited proliferation, migration,invasion, and resistance to death of NSCLC cell lines. There isincreasing evidence that emphasized the core functions ofmiRNAs, either oncogenic or antioncogenic, in NSCLC path-ogenesis. For instance, miR-218 was reported as a strongtumor suppressor in NSCLC which inhibited the malignantbehaviors of NSCLC cells and tumor growth by targetinginterleukin-6 receptor and JAK3 [20]. miR-421 was foundas an oncogene and promoted proliferation and invasionpotentials in NSCLC cells by targeting PCDC4 [21]. As formiR-520a, it has been reported as a tumor suppressor inmany neoplastic diseases. Upregulation of miR-520a-3p bylidocaine, for example, has been found to block proliferationand trigger apoptosis of colorectal cancer cells [22]. Similartumor-suppressing functions of miR-520a have been evi-denced in osteosarcoma [23] and breast cancer [24]. This isalso true for NSCLC. Recent studies have noted that down-regulation of miR-520a by other RNA transcripts increasedproliferation, migration, and invasion of NSCLC cells [12,13, 25]. Likewise, a previous study by Lv X et al. suggestedthat miR-520a-3p suppressed growth and aggressiveness ofNSCLC cells through mediating the PI3K/AKT/mTOR sig-naling pathway [26]. Here, our study validated the anticancerroles of miR-520a in NSCLC cells. In addition, animal exper-iments were further performed in our study, by which we

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Figure 3: miR-520a directly binds to RRM2 to mediate the Wnt signaling pathway. (a) A Venn diagram for the intersected target mRNAs ofmiR-520a predicted on StarBase, TargetScan, miRDB, and miRbase. (b) The enriched signaling pathway by the putative mRNAs identified byGO enrichment analysis. (c) Protein levels of cyclin D1 and β-catenin in A549 and H460 cells after miR-520a mimic transfection determinedby western blot analysis (∗p < 0:05, two-way ANOVA). (d) The expression of Wnt-enriched mRNAs in NSCLC tumor tissues determined byRT-qPCR (∗p < 0:05, two-way ANOVA). (e) Binding relationships between miR-520a and SDC1, YWHAZ, or RRM2 validated through dual-luciferase reporter gene assays (∗p < 0:05, two-way ANOVA). (f) Correlation analysis of the RRM2 andmiR-520a expression in tumor tissues.(g) The RRM2 expression in NSCLC cell lines H273, H23, A549, and H460 and in normal pulmonary epithelial cell line 16HBE determined byRT-qPCR (∗p < 0:05, one-way ANOVA). (h) The RRM2 expression in A549 and H460 cells after miR-520a mimic transfection detected byRT-qPCR (∗p < 0:05, two-way ANOVA). (i) Protein levels of cyclin D1 and β-catenin in A549 and H460 cells after the RRM2 overexpressiondetermined by western blot analysis (∗p < 0:05, two-way ANOVA).

8 Analytical Cellular Pathology

confirmed that upregulation of miR-520a suppressed thegrowth and metastasis of xenograft tumors formed byNSCLC cells in vivo.

Subsequently, 219 candidate target mRNAs of miR-520awere predicted using an integrated bioinformatic analysis.Then, a GO enrichment analysis confirmed that the Wnt sig-naling pathway was enriched by these mRNAs. The Wnt sig-naling is an evolutionary highly conserved signaling pathwaythat is important for the development and homeostasis, andits aberrant activation is a critical factor in the onset, mainte-nance, and development of many cancers [27–29]. There isno exception for NSCLC, and Wnt inactivation has beenreviewed as a promising therapeutic option for NSCLC treat-ment [30, 31]. Intriguingly, miR-520a has been demonstrated

as one of the core miRNAs mediating the sensitivity ofesophageal squamous cell carcinoma cells to neoadjuvantchemoradiotherapy through the Wnt signaling pathway[32]. Here, in this paper, we found that miR-520a decreasedthe levels of cyclin D1 and β-catenin in NSCLC cells, indicat-ing the implication of Wnt defect in the miR-520a-mediatedevents. Furthermore, our study found that SDC1, YWHAZ,and RRM2 were enriched on this signaling pathway, whileonly RRM2 was confirmed to have a binding relationshipwith miR-520a according to the luciferase assays. RRM2 isa cell cycle dependent factor that was suggested to play onco-genic roles in many cancers such as adrenocortical cancer[33], glioma [34], and neuroblastoma [35]. Interestingly,RRM2 has also been involved in several ceRNA networks,

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9Analytical Cellular Pathology

and its upregulation has been found to trigger cell prolifera-tion, drug resistance, and tumor growth in NSCLC [36, 37].Importantly, artificial upregulation of RRM2 in cells pro-moted cell viability, migration, invasion, and resistance toapoptosis. Accordingly, further experiments found that over-expression of miR-520a blocked the above events, indicatingthat RRM2 is a downstream target of miR-520a during itsregulation in NSCLC progression. Interestingly, RRM2silencing has been suggested to inactivate the Wnt/β-cateninsignaling pathway by enhancing phosphorylation of glucosesynthase kinase 3β [38]. Collectively, miR-520a possibly sup-presses the RRM2/Wnt/β-catenin axis to block progressionof NSCLC.

5. Conclusions

To sum up, the current study evidenced that miR-520acould inhibit malignant behaviors of cells and tumorgrowth in NSCLC by directly binding to RRM2 and thesubsequent Wnt signaling defect. These findings can pro-vide novel insights into the gene-based therapy for NSCLCtreatment that miR-520a may serve as a potential tool while

RRM2 may serve as a potential therapeutic target in theclinical practice. However, the sample size in this studywas not that huge since a larger sample size is being ana-lyzed but the three-year follow-up study of those patientshas not been completed yet. We further analyzed the eval-uation of the power of this research using the Minitab soft-ware. According to the data form the TCGA Database, theD value of the miR-520 expression between cancer tissuesand normal tissues was 0.18, the SD was 0.25, and thepower value we defined was 0.8 (a large effect size). In thissetting, a sample size of 18 can indicate the significance ofthe statistical results. We would like to include data froma larger sample size in our future researches. We also hopemore researches in this field will be conducted to developmore understandings to improve the therapeutic efficacyin NSCLC treatment.

Data Availability

The data used to support the findings of this study areincluded within the article.

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Figure 5: miR-520a inhibits tumor formation and metastasis in nude mice. (a) Role of miR-520a mimic in tumor volume changes (∗p < 0:05,two-way ANOVA). (b) Role of miR-520a mimic in tumor weight change (∗p < 0:05, two-way ANOVA). (c, d) Formation of metastaticnodules in lung (c) and liver (d) nodules in rats (∗p < 0:05, two-way ANOVA).

10 Analytical Cellular Pathology

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Yi Xie and Congyu Xue contributed equally to this work.

Acknowledgments

The authors would like to thank the Health Science andTechnology Development Plan of Shandong Province(2018WS239).

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