microrna-16-5p inhibits osteoclastogenesis in giant cell...

Research ArticleMicroRNA-16-5p Inhibits Osteoclastogenesis inGiant Cell Tumor of Bone

Shang Sang1 Zhichang Zhang1 Shu Qin1 Changwei Li2 and Yang Dong1

1Department of Orthopaedic Surgery Sixth Peoplersquos Hospital Shanghai Jiao Tong University Shanghai China2Shanghai Key Laboratory for Bone and Joint Diseases Shanghai Institute of Orthopaedics and TraumatologyShanghai Ruijin Hospital Shanghai Jiao Tong University School of Medicine Shanghai China

Correspondence should be addressed to Changwei Li changwei393331163com and Yang Dong dongyang6405163com

Received 15 November 2016 Revised 18 March 2017 Accepted 20 April 2017 Published 15 May 2017

Academic Editor Natarajan Muthusamy

Copyright copy 2017 Shang Sang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Giant cell tumor (GCT) of bone is an aggressive skeletal tumor characterized by localized bone resorption MicroRNA-16-5p(miR-16-5p) has been reported to be downregulated in lesions of patients with GCT but little is known about its role in GCTTo explore the underlying function of miR-16-5p in GCT we first detected its expression in patients with GCTThe results showedthat osteoclast formation increased whereas miR-16-5p expression considerably decreased with the severity of bone destructionFurthermore we found that miR-16-5p expression considerably decreased with the progression of receptor activator of nuclearfactor-120581B ligand- (RANKL-) induced osteoclastogenesis Functionally miR-16-5p mimics significantly reduced RANKL-inducedosteoclast formation However treatment with an inhibitor of miR-16-5p significantly promoted osteoclastogenesis These findingsreveal that miR-16-5p inhibits osteoclastogenesis and that it may represent a therapeutic target for giant cell tumor of bone

1 Introduction

Giant cell tumor of bone (GCTB) is a locally aggressiveosteolytic tumor that causes significant bone destruction atthe epiphysis of long bones [1] Both benign and malignantGCTBs have been described [2] Although rarely lethalGCTBs exhibit local recurrence in 27 to 65 of patientsfollowing primary surgical treatments [3] and up to 6 ofGCTBs develop pulmonary metastases [4 5] Histologicallygiant cell tumor (GCT) is a heterogeneous tumor that consistsof three major cell types osteoclast-like multinucleated giantcells spindle-like stromal cells and monocytic round cells[6] These cells have different roles in the promotion ofosteolysis such as cytokine secretion or cellular interaction[7] Since the hallmarks of GCT are its aggressive lyticbehavior and the osteoclast-like tumor giant cells that playa crucial role in the lytic process [8 9] the inhibition ofosteoclastogenesis may represent a therapeutic approach forgiant cell tumor of bone

Recently noncoding microRNAs (miRNAs) haveemerged as important regulatory elements in the devel-opment of tumors These are small (sim20 nt) noncoding

single-stranded RNA molecules that negatively regulatetheir target genes by inducing mRNA degradation orthrough the inhibition of translation [10] miRNAs functionby partially or completely binding to the 31015840-untranslatedregion (31015840-UTR) of their target mRNAs thereby triggeringeither the inhibition of translation or the degradation ofthe mRNA [11] Besides its use as a potential biomarker todetect gastric cancer [12] miR-16-5p has also been shownto be stably expressed in breast cancer [13] In additionwe have previously reported that miR-16-5p is significantlydownregulated in GCT [14] although little is known aboutits role in GCT and osteoclast formation

Here we report that miR-16-5p expression significantlydecreases with the severity of bone destruction and uncoversa crucial role for miR-16-5p in promoting the process ofosteoclastogenesis

2 Results

21 miR-16-5p Expression Is Downregulated in GCT Toinvestigate the underlying function of miR-16-5p in GCT wefirst detected its expression in patientswithGCTBThe results

HindawiBioMed Research InternationalVolume 2017 Article ID 3173547 6 pageshttpsdoiorg10115520173173547

2 BioMed Research International

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(e)

Figure 1 miR-16-5p is downregulated in GCTB (a) Plain films of subjects in the GCT group showing different degrees of bone destruction(arrows indicated) (b) HampE staining of GCT slices in patients with different degrees of bone destruction Panel (b) shows the multinucleatedosteoclast-like giant cells (indicated by arrows) in the pathological images of patients for Campanacci Grades I II and III respectively Scalebars represent 10 120583m (c)The average numbers of osteoclast-likemultinucleated cells (d)The average area of the osteoclast-likemultinucleatedcells (e) Quantification ofmiR-16-5pmRNA expression in patients with GCTwith different degrees of bone destruction (fndashh) TRAP stainingof GCT slices in patients with different degrees of bone destruction Scare bar represents 20 120583m lowastlowastlowast119875 lt 0001 and lowastlowast119875 lt 001 P values wereanalyzed using the one-way ANOVA test

showed that osteoclast formation increased whereas miR-16-5p expression decreased significantly with the severity ofbone destruction (Figures 1(a)ndash1(h)) These results predictedthat miR-16-5p might play a role in the pathogenesis of GCT

22 miR-16-5p Inhibited the Process of OsteoclastogenesisHaving observed that miR-16-5p expression was decreasedin GCT we next sought to explore the underlying functionof miR-16-5p in the pathogenesis of GCT Given that GCT ischaracterized by aggressive lytic behavior and by osteoclast-like giant tumor cells that play a crucial role in the lyticprocess we hypothesized that miR-16-5p might play a vitalrole in the process of osteoclastogenesis in GCT To testour hypothesis we first measured miR-16-5p expression in

RANKL-induced osteoclastogenesis of bonemarrow-derivedmacrophages (BMMs) The results showed that miR-16-5pexpression considerably decreased with the progression ofRANKL-induced osteoclastogenesis and the mRNA level ofmiR-16-5p decreased to 38 compared to the levels detectedin the control group after seven days of RANKL stimulation(Figure 2(a)) Furthermore a gain of function experimentrevealed that a miR-16-5p mimic significantly inhibitedRANKL-induced osteoclast formation (Figures 2(b)ndash2(d))whereas a loss of function experiment revealed that themiR-16-5p inhibitor significantly enhanced RANKL-inducedosteoclast formation (Figures 2(e)ndash2(g)) Consistent with theTRAP staining results the expression of osteoclastogenesis-related genes such as tartrate-resistant acidic phosphatase(TRAP) cathepsin K (CK) and matrix metallopeptidase 9

BioMed Research International 3

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ic625

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Mimic control Mimic 625ngml Mimic 125ngml Mim

ic co

ntro

l

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ic625

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ic125

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Inhi

bito

r con

trol

Inhi

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Inhi

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Inhibitor control Inhibitor 625ngml Inhibitor 125ngml

Inhi

bito

r con

trol

Inhi

bito

r625

ngm

l

Inhi

bito

r125

ngm

l

500

400

300

200

100

0

(a)

(c) (d)

(f) (g)

(b)

(e)

Figure 2 miR-16-5p inhibits osteoclast formation (a) Quantification of miR-16-5p mRNA expression during RANKL-induced osteoclasto-genesis (bndashd) TRAP staining showing a decrease in the number of osteoclasts following treatment with the miR-16-5p mimic (endashg) TRAPstaining showing an increase in the number of osteoclasts after treatment with the miR-16-5p inhibitor Mimic represents the agomir for miR-16-5p inhibitor represents the antagomir for miR-16-5p mimic control represents the negative control for miR-16-5p mimic and inhibitorcontrol represents the negative control for miR-16-5p inhibitor Scale bars represent 10 120583m lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 Pvalues were analyzed using the one-way ANOVA test All data are representative of three independent experiments

(MMP9) was detected by real-time PCR and further demon-strated that miR-16-5p functions as a suppressor of RANKL-induced osteoclast formation The upregulated expression ofTRAP CK and MMP9 induced by RANKL was significantlyenhanced by a miR-16-5p inhibitor (Figure 3(a)) whereasthese expression levels were substantially decreased whenusing a miR-16-5p mimic (Figure 3(b)) A well-polarized F-actin ring is required for mature osteoclast formation andefficient bone resorption [15] Therefore we performed F-actin ring staining to estimate the effect of miR-16-5p onosteoclastogenesis The results showed that the miR-16-5pmimic disrupted the structure of the F-actin ring in a dose-dependent manner (Figure 4(a)) whereas the miR-16-5p

inhibitor promoted the clear formation of the F-actin ring(Figure 4(b)) Taken together these results showed that miR-16-5p inhibited the process of osteoclastogenesis in BMMs

3 Discussion

GCTB is characterized by numerous osteoclast-like multi-nucleated giant cells that are primarily responsible for theextensive bone resorption by the tumor [16] Although anumber of studies have focused on the causes of GCT theunderlying pathology is not yet fully understood Here wereport that miR-16-5p was significantly downregulated inthe lesions of patients with GCT Furthermore miR-16-5p


Inhibitor control Inhibitor 125ngml

Cathepsin KTRAP

MMP-9

25

20

15

10

5

0

mRN

A ex

pres

sion

(Fol

d ch

ange

)

lowastlowastlowast

lowastlowast

lowast

(a)

Mimic control Mimic 125ngml

Cathepsin K

TRAPMMP-9

15

10

05

00

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A ex

pres

sion

(Fol

d ch

ange

)

lowast

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(b)

Figure 3 TRAP CK and MMP9 expression in RANKL-induced BMM osteoclastogenesis treated with miR-16-5p inhibitor or mimics (a)TRAP CK and MMP9 expression in RANKL-induced BMM osteoclastogenesis treated with miR-16-5p inhibitor (b) TRAP CK andMMP9 expression in RANKL-induced BMMosteoclastogenesis treated with miR-16-5pmimics Mimic represents the agomir for miR-16-5pinhibitor represents the antagomir for miR-16-5p mimic control represents the negative control for miR-16-5p mimic and inhibitor controlrepresents the negative control for miR-16-5p inhibitor lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 P values were analyzed using the one-wayANOVA test


(a)


(b)

Figure 4 Immunostaining of F-actin in RANKL-induced BMMs osteoclastogenesis treated with miR-16-5p inhibitor or mimics (a)Immunostaining of F-actin in RANKL-induced BMMs osteoclastogenesis treated with or without miR-16-5p mimics (b) Immunostainingof F-actin in RANKL-induced BMMs osteoclastogenesis treated with or without miR-16-5p inhibitor Arrows point towards the F-actin ringMimic represents the agomir for miR-16-5p inhibitor represents the antagomir for miR-16-5p mimic control represents the negative controlfor miR-16-5p mimic and inhibitor control represents the negative control for miR-16-5p inhibitor Scale bar represents 10120583mThe originalimage magnification is 40x and the experiments were performed concomitantly

levels were substantially decreased in vitro during the processof RANKL-induced osteoclastogenesis in BMMs Moreoverour gain of function experiment demonstrated that amiR-16-5p mimic significantly reduced RANKL-induced osteoclastformation However treatment with an inhibitor of miR-16-5p clearly promoted osteoclastogenesis Collectively thesefindings reveal thatmiR-16-5p inhibits osteoclastogenesis andmay function as a therapeutic target for giant cell tumor ofbone

As GCTs are known as bone destructive neoplasms theinhibition of bone resorption has been confirmed as aneffective therapeutic strategy to reduce the recurrence ofGCT[17] Recently several effective preventative and nonsurgicalinterventions have been introduced One large recent studyindicates that bisphosphonate has been widely used in theclinical treatment of GCT because of its protective effectagainst osteolysis [16 18] However it has been reported that

the long-term administration of bisphosphonatesmay lead tobone necrosis and atypical fractures in long bones [15] Deno-sumab a monoclonal antibody against RANKL has recentlybeen approved for use in bony metastasis hypercalcemia ofmalignancy recalcitrant to bisphosphonates and certain giantcell tumors This drug is currently in phase II clinical trialsregarding its efficacy in osteolysis [19] Denosumab is mostlyeffective due to its antiresorptive effects as bone alkalinephosphatase did not decrease until a month after injectionwhile bone turnover markers immediately decreased [20]Therefore therapeutic avenues targeting receptor activatorof nuclear factor-120581B (RANK)RANKL and its downstreammolecules might represent good options for the treatment ofGCTB

Osteoclastogenesis is an intricate multistep process thatbegins with the proliferation and commitment of mononu-cleated precursors and culminates in the formation of large


bone-resorbing polykaryons [21] Some osteolysis-relatedproteins that are produced during this process may betargeted for regulation by miRNAs [14] It has been reportedthat miR-126-5p is significantly downregulated in spindle-like stromal cells ofGCTs and affects osteoclast differentiationand bone resorption by repressing the expression of matrixmetalloproteinase 13 (MMP-13) [6] In addition Huang et alreported that miR-30a can regulate the expression of RUNX2by binding to its 31015840-UTR which regulates osteoclast differen-tiation and promotes osteolysis in GCTB [7] A recent studyalso revealed that miRNA-106b inhibits osteoclastogenesisand osteolysis by targeting RANKL in GCTB [16] Interest-ingly even more miRNAs may modulate the production andfunction of the osteoclast Our study revealed that miR-16-5pinhibits osteoclastogenesis in BMMs and provides additionalproof that miRNAs may function as potential therapeutictargets for the treatment of GCTB However we are awareof the limitations in the experimental design of our studyFirst although our results revealed that miR-16-5p functionsas an inhibitor of osteoclastogenesis the results were acquiredin BMMs from C57 mice and it is less well known whethermiR-16-5p has the same function in human macrophages Inaddition the intratumor heterogeneity of GCTB still needsfurther illustration Lastly the mechanisms by which miR-16-5p regulates the process of osteoclastogenesis remain animportant topic to be addressed by future studies

In conclusion our study provides evidence that miR-16-5p may play a crucial role in GCTB by inhibiting the processof osteoclastogenesis Our findings implicate a potential usefor miR-16-5p as a therapeutic target for the treatment ofGCTB

4 Materials and Methods

41 Clinical Samples Radiological images and the clinicalcharacteristics of 29 Chinese patients (22ndash78 years of age)with GCTB as well as fresh specimens for 17 GCTB tumorswere collected Primary GCT tissues were isolated fromtumor samples derived from tumor resectionsThenontumorinfected cancellous bones from the same patients with GCTwere used as normal controls The tissues were snap-frozenand stored in liquid nitrogen within two hours after surgicalexcision All patients underwent resection for primary GCTin our hospital between 2012 and 2015 All patients withGCTB received extended curettage with no adjuvant therapyThe clinical characteristics of all the patients with GCTBare summarized in Table 1 The progression of GCTB wasevaluated using the Campanacci grading The research wasapproved by the Ethics Committee of the Sixth PeoplersquosHospital of Shanghai and by that of the Jiao Tong Univer-sity (Shanghai China) and written informed consent wasobtained from all participants

42 miRNA Extraction Total RNA was extracted from theGCT tissue (n = 17) the cancellous bone (n = 4) and thein vitro cultured cells using Trizol (Invitrogen Carlsbad CAUSA)

Table 1 Characteristics of the 29 patients with GCT

Patients with GCTBAge (years) 3707 plusmn 1327Sex (malefemale) 1712Disease history (months) 652 plusmn 355Tumor size (cm) 524 plusmn 215Tumor site (spinelimbs) 029Primaryrecurrent tumor 1910Resection (segment resectioncurettage) 1514

43 Cell Lines and Cell Culture For primary cell culturesBMMs were isolated from C57 mice BMM cells were main-tained in MEM (GIBCO) medium supplemented with 10fetal bovine serum (HyClone) and grown in an incubator(37∘C 5 CO2)

44 qRT-PCR for mRNA andmiRNAAnalysis qRT-PCRwasperformed using the iTaq Universal SYBR Green Super-mix (Bio-Rad Laboratories CA USA) on a 7500HT Real-Time PCR System (Life Technologies USA) Real-time PCRprimers used for GAPDH are (forward 51015840-AGGTCGGTG-TGAACGGATTTG-31015840 reverse 51015840-TGTAGACCATGTAGT-TGAGGTCA-31015840) TRAP (forward 51015840-CACTCCCACCCT-GAGATTTGT-31015840 reverse 51015840-CATCGTCTGCACGGTTCT-G-31015840) CK (forward 51015840-GAAGAAGACTCACCAGAAGCAG-31015840 reverse 51015840-TCCAGGTTATGGGCAGAGATT-31015840) MMP-9 (forward 51015840-CTCAGAGATTCTCCGTGTCCTGTA-31015840 re-verse 51015840-GACTGCCAGGAAGACCTTGGTTA-31015840)

45 Cell Transfection The agomir (mimic) antagomir(inhibitor) and negative controls for miR-16-5p werepurchased from RiboBio (RiboBio Guangzhou China)BMM cells were stimulated with RANK ligand (RANKL)and transfected with the agomir antagomir and thenegative controls for miR-16-5p at different doses (0 625or 125 ngml) For transfection the Lipofectamine 3000Reagent (Invitrogen USA) was used according to themanufacturerrsquos instructions

46 TRAP Staining TRAP staining and F-actin ring for-mation were used to assay osteoclastogenesis For TRAPstaining cells were fixed and stained using the TRAP activ-ity kit (Sigma USA) TRAP-positive multinucleated cellscontaining three or more nuclei were counted as matureosteoclasts

47 Cell Counting We used ImageJ (National Institutes ofHealth USA) to count the number and area of the target cells

48 Statistical Analysis All data are present as mean plusmn SEMWe did analyses of multiple groups by one-way ANOVAwithBonferroni posttest of GraphPad prism version 5 For allstatistical tests we considered P value lt 005 to be statisticallysignificant


Conflicts of Interest

The authors declare no competing financial interest

Authorsrsquo Contributions

Changwei Li Yang Dong and Shang Sang conceived anddesigned the experiments Shang Sang Zhichang Zhang andShu Qin performed the experiments Changwei Li ShangSang and Yang Dong analyzed the data Yang Dong andChangwei Li contributed with reagentsmaterialsanalysistools Changwei Li wrote the paper

Acknowledgments

This work was supported by Shanghai Municipal Science andTechnology Commission support project [16ZR1425500] andShanghai Sailing program [Grant no 16YF1410100]

References

[1] P-F Wu J-Y Tang and K-H Li ldquoRANK pathway in giant celltumor of bone pathogenesis and therapeutic aspectsrdquo TumorBiology vol 36 no 2 pp 495ndash501 2015

[2] K M Skubitz ldquoGiant cell tumor of bone current treatmentoptionsrdquo Current Treatment Options in Oncology vol 15 no 3pp 507ndash518 2014

[3] L van der Heijden P Sander Dijkstra M A J van de Sandeet al ldquoThe clinical approach toward giant cell tumor of bonerdquoOncologist vol 19 no 5 pp 550ndash561 2014

[4] L Xu J Luo R Jin et al ldquoBortezomib inhibits giant cell tumorof bone through induction of cell apoptosis and inhibition ofosteoclast recruitment giant cell formation and bone resorp-tionrdquoMolecular CancerTherapeutics vol 15 no 5 pp 854ndash8652016

[5] S Chawla R Henshaw L Seeger et al ldquoSafety and efficacy ofdenosumab for adults and skeletally mature adolescents withgiant cell tumour of bone interim analysis of an open-labelparallel-group phase 2 studyrdquoThe Lancet Oncology vol 14 no9 pp 901ndash908 2013

[6] Z Wu H Yin T Liu et al ldquoMiR-126-5p regulates osteoclastdifferentiation and bone resorption in giant cell tumor throughinhibition of MMP-13rdquo Biochemical and Biophysical ResearchCommunications vol 443 no 3 pp 944ndash949 2014

[7] Q Huang Z Jiang T Meng et al ldquoMiR-30a inhibits osteolysisby targeting RunX2 in giant cell tumor of bonerdquo Biochemicaland Biophysical Research Communications vol 453 no 1 pp160ndash165 2014

[8] J Kanehisa T IzumoMTakeuchi T Yamanaka T Fujii andHTakeuchi ldquoIn vitro bone resorption by isolated multinucleatedgiant cells from giant cell tumour of bone light and electronmicroscopic studyrdquo Virchows Archiv A Pathological Anatomyand Histopathology vol 419 no 4 pp 327ndash338 1991

[9] J H Lindeman R Hanemaaijer A Mulder et al ldquoCathepsin Kis the principal protease in giant cell tumor of bonerdquo AmericanJournal of Pathology vol 165 no 2 pp 593ndash600 2004

[10] V Ambros ldquoThe functions of animal microRNAsrdquo Nature vol431 no 7006 pp 350ndash355 2004

[11] D P Bartel ldquoMicroRNAs genomics biogenesis mechanismand functionrdquo Cell vol 116 no 2 pp 281ndash297 2004

[12] J Zhang Y Song C Zhang et al ldquoCirculating MiR-16-5pand MiR-19b-3p as two novel potential biomarkers to indicateprogression of gastric cancerrdquoTheranostics vol 5 no 7 pp 733ndash745 2015

[13] G Rinnerthaler H Hackl S P Gampenrieder et al ldquoMiR-16-5p is a stably-expressed housekeeping microRNA in breastcancer tissues from primary tumors and from metastatic sitesrdquoInternational Journal of Molecular Sciences vol 17 no 2 article156 2016

[14] S Qin N-B He H-L Yan and Y Dong ldquoCharacterization ofmicroRNA expression profiles in patients with giant cell tumorrdquoOrthopaedic Surgery vol 8 no 2 pp 212ndash219 2016

[15] H Kang Y Yan P Jia et al ldquoDesferrioxamine reducesultrahigh-molecular-weight polyethylene-induced osteolysisby restraining inflammatory osteoclastogenesis via hemeoxygenase-1rdquo Cell Death amp Disease vol 7 no 10 Article IDe2435 2016

[16] T Wang H Yin J Wang et al ldquoMicroRNA-106b inhibitsosteoclastogenesis and osteolysis by targeting RANKL in giantcell tumor of bonerdquo Oncotarget vol 6 no 22 pp 18980ndash189962015

[17] W Xu X Li W Huang et al ldquoFactors affecting prognosis ofpatients with giant cell tumors of themobile spine retrospectiveanalysis of 102 patients in a single centerrdquo Annals of SurgicalOncology vol 20 no 3 pp 804ndash810 2013

[18] H Yin X Yang W Xu et al ldquoTreatment and outcome ofprimary aggressive giant cell tumor in the spinerdquo EuropeanSpine Journal vol 24 no 8 pp 1747ndash1753 2015

[19] D Thomas R Henshaw K Skubitz et al ldquoDenosumab inpatients with giant-cell tumour of bone an open-label phase2 studyrdquoThe Lancet Oncology vol 11 no 3 pp 275ndash280 2010

[20] A M Kandahari X Yang K A Laroche A S Dighe D Panand Q Cui ldquoA review of UHMWPE wear-induced osteolysisthe role for early detection of the immune responserdquo BoneResearch vol 4 Article ID 16014 2016

[21] M C Horowitz and J A Lorenzo ldquoThe origins of osteoclastsrdquoCurrent Opinion in Rheumatology vol 16 no 4 pp 464ndash4682004

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age a

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휇m

2)

lowastlowastlowast

lowastlowastlowast

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Mir1

6-5

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RNA

expr

essio

n(F

old

chan

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15

10

05

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Gra

de I

Gra

de II

Gra

de II

I

Gra

de I

Gra

de II

Gra

de II

I

(a)

(b)

(c) (d) (h)

(g)(f)

(e)

Figure 1 miR-16-5p is downregulated in GCTB (a) Plain films of subjects in the GCT group showing different degrees of bone destruction(arrows indicated) (b) HampE staining of GCT slices in patients with different degrees of bone destruction Panel (b) shows the multinucleatedosteoclast-like giant cells (indicated by arrows) in the pathological images of patients for Campanacci Grades I II and III respectively Scalebars represent 10 120583m (c)The average numbers of osteoclast-likemultinucleated cells (d)The average area of the osteoclast-likemultinucleatedcells (e) Quantification ofmiR-16-5pmRNA expression in patients with GCTwith different degrees of bone destruction (fndashh) TRAP stainingof GCT slices in patients with different degrees of bone destruction Scare bar represents 20 120583m lowastlowastlowast119875 lt 0001 and lowastlowast119875 lt 001 P values wereanalyzed using the one-way ANOVA test

showed that osteoclast formation increased whereas miR-16-5p expression decreased significantly with the severity ofbone destruction (Figures 1(a)ndash1(h)) These results predictedthat miR-16-5p might play a role in the pathogenesis of GCT

22 miR-16-5p Inhibited the Process of OsteoclastogenesisHaving observed that miR-16-5p expression was decreasedin GCT we next sought to explore the underlying functionof miR-16-5p in the pathogenesis of GCT Given that GCT ischaracterized by aggressive lytic behavior and by osteoclast-like giant tumor cells that play a crucial role in the lyticprocess we hypothesized that miR-16-5p might play a vitalrole in the process of osteoclastogenesis in GCT To testour hypothesis we first measured miR-16-5p expression in

RANKL-induced osteoclastogenesis of bonemarrow-derivedmacrophages (BMMs) The results showed that miR-16-5pexpression considerably decreased with the progression ofRANKL-induced osteoclastogenesis and the mRNA level ofmiR-16-5p decreased to 38 compared to the levels detectedin the control group after seven days of RANKL stimulation(Figure 2(a)) Furthermore a gain of function experimentrevealed that a miR-16-5p mimic significantly inhibitedRANKL-induced osteoclast formation (Figures 2(b)ndash2(d))whereas a loss of function experiment revealed that themiR-16-5p inhibitor significantly enhanced RANKL-inducedosteoclast formation (Figures 2(e)ndash2(g)) Consistent with theTRAP staining results the expression of osteoclastogenesis-related genes such as tartrate-resistant acidic phosphatase(TRAP) cathepsin K (CK) and matrix metallopeptidase 9


Mir1

6-5

p m

RNA

expr

essio

n(F

old

chan

ge)

15

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Day

0

Day

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5

Day

7

lowastlowast




lowastlowastlowast

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age n

umbe

r

100

150

50

0

Aver

age n

umbe

r 100

150

50

0

100

150

200

50

0

Aver

age a

rea (

휇m

2)

Aver

age a

rea (

휇m

2)

Mim

ic co

ntro

l

Mim

ic625

ngm

l

Mim

ic125

ngm

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ic co

ntro

l

Mim

ic625

ngm

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Mim

ic125

ngm

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Inhi

bito

r con

trol

Inhi

bito

r625

ngm

l

Inhi

bito

r125

ngm

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Inhi

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trol

Inhi

bito

r625

ngm

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Inhi

bito

r125

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500

400

300

200

100

0

(a)

(c) (d)

(f) (g)

(b)

(e)




3 Discussion




Cathepsin KTRAP

MMP-9

25

20

15

10

5

0

mRN

A ex

pres

sion

(Fol

d ch

ange

)

lowastlowastlowast

lowastlowast

lowast

(a)


Cathepsin K

TRAPMMP-9

15

10

05

00

mRN

A ex

pres

sion

(Fol

d ch

ange

)

lowast

lowastlowast

lowastlowast

(b)



(a)


(b)

























Acknowledgments


References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Mir1

6-5

p m

RNA

expr

essio

n(F

old

chan

ge)

15

10

05

00

Day

0

Day

1

Day

3

Day

5

Day

7

lowastlowast




lowastlowastlowast

lowastlowast

Aver

age n

umbe

r

100

150

50

0

Aver

age n

umbe

r 100

150

50

0

100

150

200

50

0

Aver

age a

rea (

휇m

2)

Aver

age a

rea (

휇m

2)

Mim

ic co

ntro

l

Mim

ic625

ngm

l

Mim

ic125

ngm

l


ic co

ntro

l

Mim

ic625

ngm

l

Mim

ic125

ngm

l

Inhi

bito

r con

trol

Inhi

bito

r625

ngm

l

Inhi

bito

r125

ngm

l


Inhi

bito

r con

trol

Inhi

bito

r625

ngm

l

Inhi

bito

r125

ngm

l

500

400

300

200

100

0

(a)

(c) (d)

(f) (g)

(b)

(e)




3 Discussion




Cathepsin KTRAP

MMP-9

25

20

15

10

5

0

mRN

A ex

pres

sion

(Fol

d ch

ange

)

lowastlowastlowast

lowastlowast

lowast

(a)


Cathepsin K

TRAPMMP-9

15

10

05

00

mRN

A ex

pres

sion

(Fol

d ch

ange

)

lowast

lowastlowast

lowastlowast

(b)



(a)


(b)

























Acknowledgments


References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Cathepsin KTRAP

MMP-9

25

20

15

10

5

0

mRN

A ex

pres

sion

(Fol

d ch

ange

)

lowastlowastlowast

lowastlowast

lowast

(a)


Cathepsin K

TRAPMMP-9

15

10

05

00

mRN

A ex

pres

sion

(Fol

d ch

ange

)

lowast

lowastlowast

lowastlowast

(b)



(a)


(b)

























Acknowledgments


References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































Acknowledgments


References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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