Originl Article Downregultion of Notch regulted Ankyrin Repet Protein Exerts Antitumor Activities ginst Growth of Thyroid Cncer Bing Feng Chu 1,2, Yi Yu Qin 3, Sheng Li Zhng 2, Zhi Wei Qun 2, Ming Di Zhng 2, Jin Wei Bi 4 1 Grdute School, Shnghi Second Militry Medicl University, Shnghi 200433, Chin 2 Deprtment of Generl Surgery, Xinhu Hospitl Affilited to Shnghi Jio Tong University School of Medicine, Shnghi 200092, Chin 3 Clinicl College, Yncheng Institute of Helth Sciences, Yncheng, Jingsu 224000, Chin 4 Deprtment of First Generl Surgery, Chnghi Hospitl, Second Militry Medicl University, Shnghi 200433, Chin Bing Feng Chu nd Yi Yu Qin contriuted eqully to this work. Astrct Bckground: The Notch regulted nkyrin repet protein (NRARP) is recently found to promote prolifertion of rest cncer cells. The role of NRARP in crcinogenesis deserves extensive investigtions. This study ttempted to investigte the expression of NRARP in thyroid cncer tissues nd ssess the influence of NRARP on cell prolifertion, poptosis, cell cycle, nd invsion in thyroid cncer. Methods: Thirty four cses with thyroid cncer were collected from the Deprtment of Generl Surgery, Xinhu Hospitl, Shnghi Jio Tong University School of Medicine etween 2011 nd 2012. Immunohistochemistry ws used to detect the level of NRARP in cncer tissues. Lentivirus crrying NRARP shrna (Lenti NRARP shrna) ws pplied to down regulte NRARP expression. Cell viility ws tested fter tretment with Lenti NRARP shrna using 3 (4,5 dimethylthizol 2 yl) 2,5 diphenyltetrzolium romide ssy. Apoptosis nd cell cycle distriution were determined y flow cytometry. Cell invsion ws tested using Trnswell invsion ssy. In ddition, expressions of severl cell cycle ssocited nd poptosis ssocited proteins were exmined using Western lotting fter trnsfection. Student s t test, one wy nlysis of vrince (ANOVA), or Kpln Meier were used to nlyze the differences etween two group or three groups. Results: NRARP ws highly expressed in thyroid cncer tissues. Lenti NRARP shrna showed significntly inhiitory ctivities ginst cell growth t multiplicity of infection of 10 or higher (P < 0.05). Lenti NRARP shrna induced G1 rrest (BHT101: 72.57% ± 5.32%; 8305C: 75.45% ± 5.26%) y promoting p21 expression, induced poptosis y promoting x expression nd suppressing cl 2 expression, nd inhiited cell invsion y suppressing mtrix metlloproteinse 9 expression. Conclusion: Downregultion of NRARP expression exerts significnt ntitumor ctivities ginst cell growth nd invsion of thyroid cncer, tht suggests potentil role of NRARP in thyroid cncer trgeted therpy. Key words: Apoptosis; Cell Cycle Checkpoints; Notch regulted Ankyrin Repet Protein; Thyroid Neoplsms Introduction Thyroid cncer which is the most common mlignncy of the endocrine system hs shown n incresing incidence worldwide in recent yers. According to pthology, thyroid cncer is clssed into ppillry denocrcinom, folliculr crcinom, medullry crcinom, nd nplstic thyroid crcinom (ATC). ATC ccounting for 1% 2% of ll thyroid crcinoms hs high degree of mlignncy nd displys highly invsive ehvior usully presenting with lymph node metstsis or distnt metstsis. In contrst to well differentited cncers (ppillry nd folliculr crcinom), ptients with ATC hve poorer prognosis with 1544 Quick Response Code: Access this rticle online Wesite: www.cmj.org DOI: 10.4103/0366-6999.184465 medin survivl of <1 yer. [1,2] To dte, no effective nd stndrd tretments for ATC were estlished. A numer of studies hve shown tht no survivl enefit from trditionl tretments including resection, rdiotherpy, nd chemotherpy lone or comined were oserved in ptients Address for correspondence: Dr. Jin Wei Bi, Deprtment of Generl Surgery, Chnghi Hospitl, Second Militry Medicl University, No. 168, Chnghi Rod, Shnghi 200433, Chin E Mil: jw3721@126.com This is n open ccess rticle distriuted under the terms of the Cretive Commons Attriution NonCommercil ShreAlike 3.0 License, which llows others to remix, twek, nd uild upon the work non commercilly, s long s the uthor is credited nd the new cretions re licensed under the identicl terms. For reprints contct: reprints@medknow.com 2016 Chinese Medicl Journl Produced y Wolters Kluwer Medknow Received: 19 02 2016 Edited y: Ning-Ning Wng How to cite this rticle: Chu BF, Qin YY, Zhng SL, Qun ZW, Zhng MD, Bi JW. Downregultion of Notch-regulted Ankyrin Repet Protein Exerts Antitumor Activities ginst Growth of Thyroid Cncer. Chin Med J 2016;129:1544-52.
with ATC. Therefore, it is impertive to explore novel effective therpeutic strtegy for ATC. Recent dvncement in moleculr iology hs enlightened us on the potentil mechnisms of thyroid cncer tumorigenesis nd development. It hs een documented tht thyroid cncer is complex disese which involves series of gene environment interctions in progressive process ssocited with dysfunction in multiple genes, including norml ctivtion or inctivtion of oncogenes or tumor suppressor genes, such s RET gene rerrngement, C myc gene mplifiction, BRAF, nd p53 muttion inctivtion. [3 7] Notch regulted nkyrin repet protein (NRARP) is negtive feedck regultor in Notch signling pthwy nd regulted y Notch protein. [8] On one hnd, overexpression of Notch protein cn promote NRARP expression; on the other hnd, NRARP hs feedck inhiition on the ctivity of Notch y inducing degrdtion of Notch receptor intrcellulr domin (NICD). [9] In the present study, we found the overexpression of NRARP in thyroid cncer tissues nd thyroid cncer cell lines (BHT101 nd 8305C) nd evluted the effects of downregultion of NRARP expression on cell prolifertion, cell cycle, invsion, nd poptosis. Methods Ptient smples A totl of 34 cses (11 mles nd 23 femles; ge rnge: 39 72 yers) with thyroid crcinom were collected from the Deprtment of Generl Surgery, Xinhu Hospitl, Shnghi Jio Tong University School of Medicine etween 2011 nd 2012. Tumor tissues nd corresponding norml tissues djcent to tumors were otined from ptients undergoing rdicl resection with or without neck lymph node dissection. The smples were stored t 70 C immeditely fter resection. Twenty of 34 cses hd cervicl lymph node metstses. All ptients were followed up for 3 yers fter the opertion, nd no ptient ws lost to follow up. The study ws pproved y the Ethicl Committee of Xinhu Hospitl, Shnghi Jio Tong University School of Medicine nd ll ptients consented to prticipte in the study. Immunohistochemistry Frozen sections were cut t 5 μm thickness nd fixed in cold cetone for 15 min t 4 C nd then rinsed with phosphte uffered sline (PBS) for 5 min. Then, the slides were treted with 3% hydrogen peroxide for 20 min for locking peroxidse in the tissue nd susequently rinsed well with PBS. After locked with got serum, the slides were incuted with monoclonl ntiody ginst NRARP (Snt Cruz, USA) overnight t 4 C. After eing wshed in PBS for 5 min, the slides were incuted for 30 min with the secondry ntiody. After eing wshed three times in PBS for 5 min, the slides were stined with 3,3 diminoenzidine (Merck, Germny). Cell culture nd tretment Humn ATC cell lines BHT 101 [10] nd 8305C [11] were purchsed from Chin Center for Type Culture Collection (Shnghi, Chin). Norml humn thyroid folliculr cell line Nthy ori 3 1 ws purchsed from the Europen Collection of Cell Cultures (Slisury, UK). Nthy ori 3 1 ws grown t 37 C in complete Roswell Prk Memoril Institute 1640 medium (GIBCO, USA), while BHT 101 nd 8305C were mintined t 37 C in Dulecco s modified Egle medium (GIBCO, USA). All medi were supplemented with 10% fetl ovine serum (Sigm, USA) nd 1% penicillin/streptomycin (GIBCO, USA) in 5% CO 2 incutor. Sucultures were mintined t 80% confluence nd pssged y 0.25% trypsin (GIBCO, USA). To produce cells deficient in NRARP, Lentivirus crrying NRARP shrna (Lenti NRARP shrna) (Snt Cruz) nd Lenti CON (Snt Cruz) were trnsfected into cells. In rief, BHT 101 nd 8305C were cultured in 12 well pltes for 24 h, then 5 μg/ml Lenti NRARP shrna or Lenti CON ws dded into the wells. After 24 h with infection, cells were cultured in fresh medium overnight. To otin stle trnsfected cells lines, cells were sucultured with 5 ug/ml puromycin for 2 weeks. Wter solule tetrzolium 1 ssy The viility of cells ws nlyzed using wter solule tetrzolium 1 (WST 1), (Beyotime, Chin) ssy. Briefly, 5 10 3 of BHT 101 nd 8305C cells were seeded in 96 well micropltes overnight. Cells were divided into three groups: cells with Lenti NRARP shrna (multiplicity of infection [MOI] = 0.01, 0.1, 1, 10, 100, 1000), with Lenti CON (MOI = 0.01, 0.1, 1, 10, 100, 1000), or with PBS. After incution for 48 h t 37 C, the culture medium ws removed nd the cells were rinsed twice with PBS. Then, 10 μl of WST 1 regent ws dded to ech well. The sornce of WST 1 derived formzn ws mesured using microplte reder (Model 550, Bio Rd, Hercules, CA, USA) t 450 nm. Cell survivl rte = (opticl density [A] of experiment group A of ckground)/(a of control group A of ckground) 100%. Mouse xenogrfts To evlute the effects of NRARP on the prolifertion of BHT 101 nd 8305C cells in vivo, mouse models with BHT 101 nd 8305C xenogrfts were used. Nude mice were rised in the specific pthogen free environment. All niml procedures were pproved y the Ethicl Committee of Xinhu Hospitl, Shnghi Jiotong University School of Medicine. Thirty six of BALB/c nude mle mice (4 week old, weight: 25.0 ± 1.5 g) were divided into three groups: the group inoculted with Lenti NRARP shrna trnsfected cells (n = 12), the group inoculted with Lenti CON trnsfected cells (n = 12), nd the group inoculted with culture medium (n = 12). Briefly, BHT 101 nd 8305C cells (1 10 7 cells per mouse) were injected sucutneously into the right flnks of the mice. Tumors were formed fter 2 or 3 weeks. Tumor formtion ws monitored every 5 dys, nd tumor volume sed on cliper mesurements ws clculted y the following formul: tumor volume = 1/2 (length width 2 ). On dy 20 th fter injection, mice were scrificed nd tumors were weighed. 1545
Cell cycle nlysis The effects of NRARP on cell cycle distriution were determined using flow cytometry (FCM) nlysis. BHT 101 nd 8305C cells were seeded in 6 well plte overnight. Lenti NRARP shrna ws dded to the wells of the pltes for 48 h. After wshed twice y PBS, the cells were fixed in 70% precooling ethnol overnight. The cells were then stined with 100 μg/ml RNseA nd 50 μg/ml propidium iodide (PI) (Sigm, USA) in PBS. Smples were run on n fluorescence ctivted cell sorting (FACS) Cliur flow cytometer (Becton Dickinson Bioscience, Frnklin Lkes, NJ, USA) nd DNA content ws nlyzed using FlowJo Softwre 9.1 (Tree Str Inc., Ashlnd, Oreg., USA). Invsion ssys Trnswell ssy (Corning, USA) ws used to detect the invsion of BHT101 nd 8305C cells fter trnsfection with Lenti NRARP shrna. Briefly, 5 10 4 cells in 200 μl of medium were dded to the (Corning, Corning, NY, USA). After 24 h of incution in complete medium, cells tht invded the (Corning, Corning, NY, USA) nd pssed through the filters were stined with 0.1% crystl violet for t lest 15 min. The noninvding cells were then removed from inside with cotton sw. Stined cells were counted under microscope (BX50; Olympus, Tokyo, Jpn). Apoptosis nlysis Annexin V/PI doule stining ssy ws used to nlyze poptosis of BHT101 nd 8305C cells fter trnsfection with Lenti NRARP shrna. Briefly, cells trnsfected Lenti NRARP shrna for 24 h or 48 h were trypsinized, then rinsed with precooled PBS, nd resuspended in nnexin V FITC inding uffer. Annexin V FITC nd PI were dded to the cell suspension. The cell suspension ws gently vortexing nd then incuted for 5 min t 4 C protected from light. After tht smples were nlyzed y the FACS Cliur (Becton Dickinson) within 1 h. Western lotting Western lotting ws used to nlysis protein expression levels. Cells were collected fter trnsfection with Lenti NRARP shrna for 48 h. Totl protein ws extrcted ccording to the mnufcturer s instructions, nd the concentrtion ws determined y the Brdford ssy. Aout 25 μg of protein smples were seprted on sodium dodecyl sulfte polycrylmide gel electrophoresis nd trnsferred to 0.2 μm polyvinylidene difluoride memrne. The memrne ws locked in 5% nonft milk. Memrnes were proed with primry ntiody (1:1000) (Snt Cruz, USA) overnight t 4 C, wshed three times in Tris Buffered Sline Tween 20 (TBST), incuted with nti mouse or nti rit horserdish peroxidse ntiody (1:5000) (Snt Cruz, USA) for 2 h t room temperture nd then wshed three times in TBST. The signl ws visulized y n enhnced chemiluminescence solution (ECL Plus; Pierce, USA) nd ws exposed to Kodk X-OMAT LS film (Estmn Kodk, Rochester, NY, USA). β ctin ws used s loding control. Sttisticl nlysis All sttisticl nlyses were performed y using IBM SPSS Sttistics for Windows, Version 20.0, (IBM Corp, Armonk, 1546 NY, USA) nd dt expressed s men ± stndrd devition (SD). Student s t test, one wy nlysis of vrince (ANOVA), or Kpln Meier were used to nlyze the differences etween two group or three groups. All experiments in this study were performed independently t lest three times. A P < 0.05 ws considered sttisticlly significnt. Results Notch regulted nkyrin repet protein nd Notch re highly expressed in thyroid cncer tissues The immunohistochemistry (IHC) score of NRARP protein ws clculted y multiplying the percentge of positive cells nd the intensity of NRARP protein. The positive rte ws scored s 0 (positive cells: <5%), 1 (5% 25%), 2 (25% 50%), or 3 (>50%). The intensity of stining ws lso scored s 0 (negtive), 1 (wek stining), 2 (moderte stining), or 3 (strong stining). The IHC score were determined s: (score 0 1), + (score 2 3), ++ (score 4 6), nd +++ (score > 6). According to IHC score, the ptients were divided into low expression group ( or +) nd high expression group (++ or +++). As shown in Figure 1 1c, the higher intensity nd more positive cells of NRARP stining were present in thyroid cncer tissues nd involved lymph node tissues thn noncncer tissues. IHC score of NRARP further reveled tht higher level of the protein ws expressed in cncer tissues thn in noncncer tissues (t = 7.86, P = 0.006), nd in metsttic lymph node thn in thyroid cncer tissues (t = 10.22, P = 0.013) [Figure 1d]. The Kpln Meier nlysis showed tht NRARP protein level negtively correlted with survivl of ptients with ATC. Ptients with higher level of NRARP protein hd shorter survivl thn those with lower level ( χ 2 = 6.96, P = 0.031) [Figure 1e]. In vitro study showed tht NRARP nd Notch proteins re highly expressed in BHT101 nd 8305C nd lowly expressed in Nthy ori 3 1 cells, while Lenti NRARP shrna significntly down regulted the level of NRARP protein in BHT101 nd 8305C cells [Figure 1f nd 1g]. Downregultion of Notch regulted nkyrin repet protein expression significntly inhiits the prolifertion of BHT101 nd 8305C cells in vitro nd in vivo WST 1 ssy [Figure 2 nd 2] showed tht fter trnsfection with Lenti NRARP shrna, the cell viility of BHT101 nd 8305C ws significntly decresed (BHT101: F = 79.36, P = 0.025; 8305C: F = 64.59, P = 0.014). Due to the concentrtion dependent mnner of inhiitory effects of Lenti NRARP shrna, cell prolifertion ws not significntly ffected t n MOI of 0.01 1 ut ws significntly suppressed t n MOI of 10 or higher. Mouse xenogrft models showed tht the overll rte of tumor formtion of BHT101 nd 8305C cells trnsfected with Lenti NRARP shrna ws 50% (6/12), while the tumor formtion rte in control groups (including Lenti CON nd Control group) ws 100% (12/12). In ddition, the size nd weight of tumor xenogrfts in Lenti NRARP shrna group were much smller thn those of control groups
c d e f Figure 1: Immunohistochemicl stining of NRARP in norml thyroid tissues, thyroid cncer tissues, nd metsttic lymph nodes. Immunohistochemicl stining, originl mgnifiction 200 of NRARP in norml thyroid tissues (), in thyroid cncer tissues (), nd in metsttic lymph nodes (c). (d) Immunohistochemistry score of NRARP. (e) The Kpln Meier nlysis of overll survivl of thyroid cncer ptients ccording to the NRARP level in thyroid cncer. (f) Western lotting nlysis of NRARP expression in BHT101, 8305C, nd Nthy ori 3 1 cells. (g) Western lotting nlysis of Notch expression in BHT101, 8305C, nd Nthy ori 3 1 cell. *P < 0.05. NRARP: Notch regulted nkyrin repet protein. (size: BHT101, F = 31.55, P = 0.000; 8305C: F = 42.67, P = 0.000. Weight: BHT101: F = 26.13, P = 0.000; 8305C: F = 30.57, P = 0.000) [Figure 2c nd 2d]. Downregultion of Notch regulted nkyrin repet protein expression induces G1 phse rrest in BHT101 nd 8305C cells As shown in Figure 3, G1 popultion of BHT101 (72.575 ± 5.32%) nd 8305C (75.455 ± 5.26%) cells ws significntly incresed fter trnsfection with Lenti NRARP shrna compred to the two control groups (BHT101: F = 109.26, P = 0.000; 8305C: F = 209.31, P = 0.000). In ddition, Lenti NRARP shrna induced p21 protein expression, while it inhiited the expression of cyclin D1 protein [Figure 3]. Downregultion of Notch regulted nkyrin repet protein expression induces poptosis in BHT101 nd 8305C cells As shown in Figure 4 4c, the popultion of poptotic cells ws significntly incresed fter trnsfection with Lenti NRARP shrna for 24 h or 48 h in BHT 101 cell line (24 h: F = 11.42, P = 0.009; 48 h: F = 25.51, P = 0.000), nd in 8305C cell line (24 h: F = 8.97, P = 0.016; 48 h: F = 17.44, P = 0.000). In ddition, poptosis ssocited proteins including x nd cl 2 were lso detected in cell lystes y immunolotting. Lenti NRARP shrna promoted the expression of x nd the ctivtion of cspse 3, while it inhiited the expression of cl 2 protein [Figure 4d]. Downregultion of Notch regulted nkyrin repet protein expression ttenutes BHT101 nd 8305C cell invsion Invsion ssys showed tht the numer of migrtory cells ws significntly decresed fter tretment with Lenti NRARP shrna (BHT 101: F = 23.12, P = 0.000; 8305C: F = 29.75, P = 0.000) [Figure 5 nd 5]. Western lotting nlysis showed tht Lenti NRARP shrna significntly decresed expression of mtrix metlloproteinse 9 (MMP 9) protein [Figure 5c]. 1547 g
c d Figure 2: Knockdown of NRARP significntly inhiits the prolifertion of BHT101 nd 8305C cells in vitro nd in vivo. () Knockdown of NRARP significntly inhiits the prolifertion of BHT101 cells in vitro. () Knockdown of NRARP significntly inhiits the prolifertion of 8305C cells in vitro. (c) The volume of tumor treted with or without knockdown of NRARP. (d) The weight of tumor treted with or without knockdown of NRARP, *, indictes the difference ws sttisticlly significnt compred to Lenti control nd control, respectively. NRARP: Notch regulted nkyrin repet protein. Figure 3: Knockdown of NRARP induces G1 phse rrest in BHT101 nd 8305C cells. () The effects of knockdown of NRARP on cell cycle of BHT101 nd 8305C cells. () Western lotting nlysis of p21 nd cyclin D1 expression in NRARP knockdown BHT101 nd 8305C cells. *, indicte the difference ws sttisticlly significnt compred to Lenti-control nd control, respectively. NRARP: Notch-regulted nkyrin repet protein. Discussion Thyroid crcinom is one of the most ggressive mlignncies of hed nd neck tumors nd is responsile for the mjority of deth from ll endocrine mlignncies. [12] The nnul incidence of thyroid cncer for mle is pproximtely 3/100,000, while it is out 9/100,000 for femle, which rnks first in the incresing incidence of cncers for femle cncers. [13] ATC, n undifferentited thyroid cncer with ggressive phenotype nd poor prognosis, is responsile for more thn hlf deths of ll thyroid cncer. Clinicl presenttion is frequently chrcterized s rpidly growing neck mss with ssocited compressive symptoms nd lymph node metstsis. [14] Until now, few pulished ppers hve shown significnt survivl enefit from the current therpies. In such circumstnce, new nd effective therpeutic strtegies sed on moleculr underpinnings of crcinogenesis nd growth of ATC received our ttention. Notch signling is known to ply importnt roles in the regultion of diverse developmentl processes including cell prolifertion, differentition nd poptosis, nd deregultion nd this signling cscde hs een linked to mny cncers. A numer of studies hve reveled tht Notch gene my hve dul effects on tumorigenesis vrying with cncer types. In rest cncer or prostte cncer, Notch seems to ct s n oncogene, [15] 1548
[Downloded free from http://www.cmj.org on Wednesdy, Septemer 28, 2016, IP: 89.68.4.152] c d Figure 4: Knockdown of NRARP induces poptosis in BHT101 nd 8305C cells. () The poptotic cell deth ws ssessed y mesuring the fluorescence intensity of cells stining with nnexin V/FITC nd PI. Legend for cytogrm: the lower left qudrnt includes the vile cells, which re negtive for nnexin V/FITC inding (nnexin V ) nd exclude PI (PI ); the lower right qudrnt include erly poptosis cells, which re positive for nnexin V/FITC inding (nnexin V+) ut PI ; the upper right qudrnt represent the lte poptotic cells, which re nnexin V+ nd show PI uptke (PI+); the upper left qudrnt represents necrotic cells, which re nnexin V /PI+. () FACS nlysis confirmed tht the erly nd lte poptotic cell deth in NRARP knockdown BHT101 cells ws significntly higher thn tht in control group. (c) FACS nlysis confirmed tht the erly nd lte poptotic cell deth in NRARP knockdown 8305C cells ws significntly higher thn tht in control group. (d) Western lotting nlysis the expression of x, cl 2, nd cspse 3. *, indictes the difference ws sttisticlly significnt compred to Lenti control nd control, respectively. PI: Propidium iodide; NRARP: Notch regulted nkyrin repet protein; FACS: Fluorescence ctivted cell sorting. 1549
c Figure 5: Knockdown of NRARP ttenutes BHT101 nd 8305C cell invsion. () Migrtory cells were stined with crystl violet (originl mgnifiction 100). () Less Lenti NRARP shrna trnsfected cells invding to the ottom chmer, suggested decresed invsiveness. (c) Western lotting showed the reduced expression of MMP 9 in NRARP knockdown BHT101 nd 8305C cells. *, indictes the difference ws sttisticlly significnt compred to Lenti control nd control, respectively. NRARP: NOTCH regulted nkyrin repet protein; Lenti NRARP shrna: Lentivirus crrying NRARP shrna; MMP 9: Mtrix metlloproteinse 9. while in ldder cncer it possesses chrcteristics s tumor suppressor gene. [16] Moreover, it my ct s oth oncogene nd tumor suppressor gene in prostte cncer simultneously. [17] An importnt role for Notch signling in thyroid cncer hs een well documented nd the memers of Notch signling re normlly expressed in thyroid cncer. [18,19] NRARP encodes smll evolutionrily conserved protein contining two nkyrin repets which is component of negtive feedck system to ttenute Notch pthwy medited signling. Notch my 1550 promote the expression of NRARP, nd NRARP suppresses the ctivtion of Notch signling y promoting degrdtion of Notch intrcellulr domin (NICD). To our knowledge, the present study originlly reported the expression nd regultion of NRARP nd its ssocition with cncer cell functions in vitro nd in vivo in thyroid cncer. In our study, NRARP protein ws highly expressed in ATC tissues nd ATC cell lines compred to norml thyroid tissue nd folliculr cells. The expression of Notch incresed in
prllel with NRARP in ATC tissues nd cell lines, which indicted tht the overexpression of NRARP my result from the normlly high expression of Notch in ATC cells. Overexpression of Notch ws lso found in other types of thyroid cncer. The feedck of NRARP on Notch signling seemed through inhiiting the ctivtion of downstrem pthwy of Notch ut it hd little effect on the expression of Notch. The prdoxicl effects of overexpressed Notch on cncer cells my depend on the cellulr context. In the cse of ATC, it is more likely tht high concentrtion of Notch protein would ply n ntitumor role. [20] We found tht downregultion of NRARP expression could inhiit the growth of ATC cells oth in vitro nd in vivo. Moreover, downregultion of NRARP induced G1 rrest, inhiited invsion, nd promoted poptosis. How NRARP gene prticiptes in the regultion of growth, poptosis, nd invsion of ATC cells is not cler, ut we ssumed tht overexpression of NRARP could lock Notch signling nd inhiited the ctivtion of Notch trget genes y degrding NICD. When NRARP ws down regulted y Lenti NRARP shrna, the inhiitory feedck on the downstrem pthwy of Notch ws reduced, Notch signling ws over ctivted nd the ntitumor ction of Notch ws restored. Our study suggested tht the expression of x nd the ctivtion of cspse 3 were reinforced nd the expression of cl 2 protein ws ttenuted in Lenti NRARP shrna treted ATC cells indicting possile pthwy involved in NRARP regulted poptosis of ATC cells. In ddition, it hs een demonstrted tht ctivtion of Notch suppressed the expression of WNT trget genes in humn colorectl cncer cells through epigenetic modifiction. [21] We found the expression of MMP 9, downstrem trget of WNT signling, ws significntly suppressed nd invsion ws lso inhiited fter tretment with Lenti NRARP shrna. It ws very likely tht downregultion of NRARP promoted ctivtion of Notch, which susequently inhiits WNT signling nd cell invsion. Apprently, NRARP/Notch regulted prolifertion, poptosis, differentition, nd invsion of cncer cells remin to e explored in more detils. It ws interesting to find significntly higher expression of NRARP in metsttic lymph node tissue thn in ATC tissue. The highly ggressive nture of ATC ws closely relted to extremely high expressed NRARP. On the other hnd, incresed NRARP my lso correlte to less differentited cncer, nd therefore, the expression of NRARP in poorly differentited ATC would e compred with other types of thyroid cncer in the future study. Nevertheless, our reserch dt long with the clinicl finding showing tht NRARP protein level could e negtively correlted with ptient prognosis which ws similr to the rest cncer dt [22] suggesting tht NRARP my serve s prognostic mrker nd s potentil trget for thyroid cncer trgeted therpy. Our investigtion suggested tht Notch could e up regulted in thyroid cncer to over ctivte Notch signling pthwy which my inhiit ATC cell prolifertion, induce G1 rrest, inhiit cell invsion, nd promote poptosis. However, NRARP s feedck regultor of Notch pthwy would e ctivted y overexpressed Notch, exerting negtive effect on the ctivtion of the trget genes of Notch, nd ttenuting ntitumor effects ccordingly. Downregultion of NRARP expression could reverse the inhiitory feedck of NRARP on Notch signling. Therefore, NRARP gene my ct s n oncogene in the tumorigenesis nd development of thyroid cncer. NRARP gene my serve s potentil trget for thyroid cncer therpy. Though series of functionl studies were crried out to demonstrted the role of NRARP in prolifertion, poptosis, nd invsion of thyroid cncer, the exct moleculr mechnisms y which NRARP regulted prolifertion, poptosis, nd invsion hve not een completely elucidted in this study. In ddition, the optiml wy to down regulte the expression of NRARP needs to e investigted in future studies. Finncil support nd sponsorship This study ws supported y technicl fund of Shnghi Jio Tong University School of Medicine (No. 13xj22003), nd the Key Progrm of Shnghi Municipl Commission of Helth nd Fmily Plnning (No. 20124015). Conflicts of interest There re no conflicts of interest. References 1. Punovic IR, Sipetic SB, Zoric GV, Diklic AD, Svic DV, Mrinkovic J, et l. Survivl nd prognostic fctors of nplstic thyroid crcinom. Act Chir Belg 2015;115:62 7. 2. Hsu KT, Yu XM, Audhy AW, Jume JC, Lloyd RV, Miymoto S, et l. Novel pproches in nplstic thyroid cncer therpy. Oncologist 2014;19:1148 55. doi: 10.1634/theoncologist.2014 0182. 3. Sores P, Trovisco V, Roch AS, Lim J, Cstro P, Preto A, et l. BRAF muttions nd RET/PTC rerrngements re lterntive events in the etiopthogenesis of PTC. Oncogene 2003;22:4578 80. doi: 10.1038/sj.onc.1206706. 4. Zhu X, Zho L, Prk JW, Willinghm MC, Cheng SY. Synergistic signling of KRAS nd thyroid hormone receptor ß mutnts promotes undifferentited thyroid cncer through MYC up regultion. Neoplsi 2014;16:757 69. doi: 10.1016/j. neo.2014.08.003. 5. McFdden DG, Vernon A, Sntigo PM, Mrtinez McFline R, Bhutkr A, Crowley DM, et l. p53 constrins progression to nplstic thyroid crcinom in Brf mutnt mouse model of ppillry thyroid cncer. Proc Ntl Acd Sci U S A 2014;111:E1600 9. doi: 10.1073/pns.1404357111. 6. Jing FJ, Ling J, Ling ZY, Meng C, Long W, Li XY, et l. BRAF(V600E) muttion is not positive predictor for distnt metstsis in spordic ppillry thyroid crcinom. Chin Med J 2013;126:3013 8. 7. Morni F, Phdngm S, Follo C, Titone R, Thongrkrd V, Gletto A, et l. PTEN deficiency nd mutnt p53 confer glucose ddiction to thyroid cncer cells: Impct of glucose depletion on cell prolifertion, cell survivl, utophgy nd cell migrtion. Genes Cncer 2014;5:226 39. 8. Kres LT, Deftos ML, Bevn MJ, Gridley T. The NRARP gene encodes n nkyrin repet protein tht is trnscriptionlly regulted y the notch signling pthwy. Dev Biol 2001;238:110 9. doi: 10.1006/dio.2001.0408. 9. Yun TJ, Bevn MJ. Notch regulted nkyrin repet protein inhiits Notch1 signling: Multiple Notch1 signling pthwys involved in T cell development. J Immunol 2003;170:5834 41. doi: 10.4049/ jimmunol.170.12.5834. 1551
10. Pályi I, Péter I, Duner D, Vincze B, Lõrincz I. Estlishment, chrcteriztion nd drug sensitivity of new nplstic thyroid crcinom cell line (BHT 101). Virchows Arch B Cell Pthol Incl Mol Pthol 1993;63:263 9. 11. Ito T, Seym T, Hyshi Y, Hyshi T, Dohi K, Mizuno T, et l. Estlishment of 2 humn thyroid crcinom cell lines (8305c, 8505c) ering p53 gene muttions. Int J Oncol 1994;4:583 6. doi: 10.3892/ijo.4.3.583. 12. Shermn SI. Thyroid crcinom. Lncet 2003;361:501 11. 13. Nix P, Nicolides A, Cotesworth AP. Thyroid cncer review 2: Mngement of differentited thyroid cncers. Int J Clin Prct 2005;59:1459 63. doi: 10.1111/j.1368 5031.2005.00672.x. 14. Elisei R, Ugolini C, Viol D, Lupi C, Bigini A, Ginnini R, et l. BRAF (V600E) muttion nd outcome of ptients with ppillry thyroid crcinom: A 15 yer medin follow up study. J Clin Endocrinol Met 2008;93:3943 9. doi: 10.1210/jc.2008 0607. 15. Guo S, Liu M, Gonzlez Perez RR. Role of Notch nd its oncogenic signling crosstlk in rest cncer. Biochim Biophys Act 2011;1815:197 213. doi: 10.1016/j.cn.2010.12.002. 16. Rmpis T, Vgenopoulou P, Avgeris M, Polyzos A, Strvodimos K, Vlvnis C, et l. A new tumor suppressor role for the Notch pthwy in ldder cncer. Nt Med 2014;20:1199 205. doi: 10.1038/ nm.3678. 17. Leong KG, Go WQ. The Notch pthwy in prostte development nd cncer. Differentition 2008;76:699 716. doi: 10.1111/j.1432 04 36.2008.00288.x. 18. Geers C, Colin IM, Gérrd AC. Delt like 4/Notch pthwy is differentilly regulted in enign nd mlignnt thyroid tissues. Thyroid 2011;21:1323 30. doi: 10.1089/thy.2010.0444. 19. Zhng J, Wng Y, Li D, Jing S. Notch nd TGF ß/Smd3 pthwys re involved in the interction etween cncer cells nd cncer ssocited firolsts in ppillry thyroid crcinom. Tumour Biol 2014;35:379 85. doi: 10.1007/s13277 013 1053 z. 20. Yu XM, Jskul Sztul R, Ahmed K, Hrrison AD, Kunnimliyn M, Chen H. Resvertrol induces differentition mrkers expression in nplstic thyroid crcinom vi ctivtion of Notch1 signling nd suppresses cell growth. Mol Cncer Ther 2013;12:1276 87. doi: 10.1158/1535 7163.MCT 12 0841. 21. Kim HA, Koo BK, Cho JH, Kim YY, Seong J, Chng HJ, et l. Notch1 countercts WNT/ß ctenin signling through chromtin modifiction in colorectl cncer. J Clin Invest 2012;122:3248 59. doi: 10.1172/JCI61216. 22. Imok T, Okutni T, Dino K, Iizuk D, Nishimur M, Shimd Y. Overexpression of NOTCH regulted nkyrin repet protein is ssocited with rest cncer cell prolifertion. Anticncer Res 2014;34:2165 71. 1552