research article expression of spindle and kinetochore...

Research ArticleExpression of Spindle and Kinetochore-Associated Protein 1 IsAssociated with Poor Prognosis in Papillary Thyroid Carcinoma

Chao Dong Xiao-li Wang and Bin-lin Ma

Department of Head and Neck Surgery Tumor Hospital Affiliated to Xinjiang Medical University Urumqi Xinjiang 830011 China

Correspondence should be addressed to Bin-lin Ma xjmablzohocom

Received 6 February 2015 Accepted 16 April 2015

Academic Editor Kishore Chaudhry

Copyright copy 2015 Chao Dong 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

Aim Spindle and kinetochore-associated protein 1 (SKA1) is one subtype of SKA whose protein can make spindle microtubulesattach steadily to the kinetochore in the middle of mitosis At present there are fewer researches on the relationship between SKA1expression and tumor developmentMethods In this study immunohistochemical analysis was used to determine the expression ofSKA1 in papillary thyroid carcinoma (PTC) and adjacent tissues We used quantitative real-time polymerase chain reaction (qRT-PCR) andWestern blot analysis to further verify the results Results We found that SKA1 expression was significantly higher in PTCtissues than normal adjacent tissues (119875 lt 005) There existed a significant correlation among a higher SKA1 expression includinglymphoid node (119875 = 0005) clinical stage (119875 = 0015) and extrathyroid invasion (119875 = 0004) Survival analysis showed high SKA1expression in PTC patients more likely to relapse after surgery Conclusion High SKA1 expression is predictive of poor prognosisof PTC implying that SKA1 may be a promising new target for targeted therapies for PTC

1 Introduction

Thyroid cancer is the most common malignant tumor ofthe endocrine system and the most common head and necktumor Each year new cases of thyroid cancer account for1ndash5 of all cancer cases [1] Over the past 30 years a rapidincrease in the incidence of thyroid cancer has attractedwidespread attention [2] Thyroid cancer can be classified aspapillary thyroid carcinoma (PTC) follicular thyroid carci-noma (FTC) medullary thyroid carcinoma (MTC) undif-ferentiated thyroid carcinoma (ATC) and others accordingto its pathological characteristics PTC is the most commontype accounting for 79ndash94 of thyroid cancer cases [1]With comprehensive surgery-focused antineoplastic therapythe prognosis of most patients with PTC is good but 20these patients relapse after treatment and require reoperation[3] Consequently analyzing the biological characteristics ofPTC and exploring new targets for therapy have been majorclinical concerns

It is well known that during the cell cycle the proper for-mation and depolymerization of the spindle play an essentialrole in maintaining normal mitosis of eukaryotic cells How-ever chromosomal division abnormalities can cause genetic

instability and ultimately induce PTC development [4] Spin-dle and kinetochore-associated protein 1 (SKA1) is a subtypeof SKA that causes spindle microtubules to attach firmly tothe kinetochore in the middle of mitosis [5] At presentfew research studies have examined the relationship betweenSKA1 expression and tumor development In our study weuse immunohistochemistry to observe SKA1 expression inPTC and we conducted a quantitative analysis of SKA1 usingquantitative real-time PCR and Western blotting with theaim of investigating the association between SKA1 and theprognosis of patients with PTC

2 Materials and Methods

21 Patients and Samples The subjects of this study were 123patients with PTC who had undergone surgical interventionat the Department of Head andNeck Surgery of the AffiliatedTumorHospital of XinjiangMedical University between 2005and 2009 All patients did not receive any therapy prior tosurgery and were diagnosed with primary PTC based on thepathological findings All 123 patients had complete clinicaldata including age sex tumor size lymph node statusclinical stage and extrathyroidal invasion Patients were

Hindawi Publishing CorporationDisease MarkersVolume 2015 Article ID 616541 6 pageshttpdxdoiorg1011552015616541

2 Disease Markers

selected for this study only if follow-up examinations andclinical data were available The TNM stage was definedaccording to the 7th edition of the TNM classification of theInternational Union Against CancerThe study was approvedby the ethics committee of the Affiliated Tumor Hospital ofXinjiang Medical University Written informed consent wasobtained from each patient for the use of resected samplesfor research

22 Immunohistochemistry Biopsy tissue samples were sec-tioned into 4-120583m slices and embedded with paraffin after fix-ation with 10 formaldehyde Immunohistochemistry withthe SP method (rabbit anti-SKA1 antibody 1 500 SigmaChemical Co St Louis MO USA) was used for testing byapplying PBS instead of primary antibodies as the negativecontrol according to the manufacturerrsquos instructions Theresults were determined by two independent pathologistsusing a double-blind method Five high power fields (200x)were randomly selected and in each field 200 tumor cellswere recorded totaling to 1000 cells According to thestaining intensity the cells were assigned the following pointvalues 0 points for no staining 1 point for pale yellow stain-ing 2 points for brown-yellow staining and 3 points for darkbrown stainingThe proportion of positive cells was assigneda value between 0 and 100 The immunohistochemistryexpression level was determined according to the productof the staining intensity and the proportion of positive cellsWhen the product was below 150 this was considered lowexpression of SKA1 and when the product was above 150this was considered high expression of SKA1

23 RNA Extraction and Quantitative Real-Time PCR AssaysThe tissue samples were removed from liquid nitrogenand RNA was extracted using Trizol reagent and a reversetranscription kit (Invitrogen Carlsbad CA USA) The PCRprimers used were SKA1-F 51015840-TGATGTGCCAGGAAG-GTGAC-31015840 SKA1-R 51015840-CAAAGGATACAGATGAAC-AACAGC-31015840 GAPDH-F 51015840-GTGGACATCCGCAAAGAC-31015840 andGAPDH-R 51015840-AAAGGGTGTAACGCAACTA-31015840 Allprimers were synthesized by Shanghai Yingjun Biotechnol-ogy Company (Shanghai China) The PCR reaction condi-tions were as follows initial denaturation for 2min at 94∘C35 cycles of denaturation for 30 s annealing for 30 s at 94∘Cand extension for 1min at 72∘C followed by 10min at 72∘CPCR reaction product (3 120583L) was used for 2 agarose gelelectrophoresis and the grayscale analysis was performedwith Quantity One software (Bio-Rad Hercules CA USA)The relative expression level of SKA1mRNA was determinedby the ratio of the grayscale value of SKA1mRNA and that ofinternal reference GAPDH mRNA

24 Western Blot Analysis After protein extraction usingtotal protein lysis buffer the protein concentration wasdetermined by the BCA method In brief 80 120583g protein perwell was used for 8 SDS-PAGE and after electrophoreticseparation the protein was wet-transferred to polyvinylidenefluoride 5 nonfat milk powder solution was added andafter a 1 h incubation at room temperature the transferred

protein was incubated with the primary antibodies (1 500dilution SKA1 and GAPDH Sigma Chemical Co St LouisMO USA) at 4∘C overnight Goat anti-rabbit IgG sec-ondary antibody (GenScript Biotechnology Company Nan-jing Jiangsu China) marked with horseradish peroxidaseand diluted at 1 1000 was added and incubated at roomtemperature for 15 hThemembrane was washed three timesin TBST The relative level of SKA1 protein was determinedby the grayscale value of various protein straps and the ratioof the grayscale value of SKA1 to GAPDH protein

25 Statistical Analysis The chi-squared test was used toanalyze the associations between SKA1 expression and theclinicopathological features of PTC SKA1mRNA expressionin PTC was compared with expression in normal adjacenttissues using the Mann-Whitney test (for 2 groups) orthe Kruskal-Wallis test (for more than 2 groups) Kaplan-Meier analysis was used for survival analysis lymph noderecurrence-free survival (LNRFS) and distant recurrence-free survival (DRFS) calculations LNRFS was defined as thetime from the date of surgery to the date of lymph noderelapse and DRFS was defined as the time from the date ofsurgery to the date of distant recurrence [6 7] Cox regressionmodels were used for multiple factor analysis All statisticaltests were two-tailed and statistical significance was assumedfor 119875 lt 005

3 Results

31 Expression of SKA1 in PTC Tissues Immunostainingresults suggested a significantly higher SKA1 expression inPTC tissues compared with adjacent tissues Among 123tumor samples 56 (455) showed high SKA1 expressionwith only 37 of the samples from normal adjacent tissues(see Figure 1) To confirm these observations we investigatedSKA1 mRNA expression in 45 cases of PTC and normaladjacent tissues using qRT-PCR and Western blot analysisThe mRNA levels between PTC tissues and normal adjacenttissues were calculated with the comparative Ct method(2minusΔΔct) The results indicated that SKA1 mRNA and SKA1protein expression was significantly higher in PTC tissuesthan in normal adjacent tissues (119875 lt 005 Figures 2 and 3)

32 Patient Characteristics Table 1 shows the patientsrsquo clini-cal and immunohistochemical data The mean age was 413 plusmn157 years (range 26ndash69 years) for 32 male patients and 442 plusmn145 years (range 24ndash73 years) for 35 female patients Sixty-two patients (504) had lymph node metastasis Eighty-four patients (683) had stage I-II disease based on theTNM staging system and the remaining 39 patients (317)had stage III-IV disease During the median follow-up timewhich was 52 months (range 17ndash102 months) 21 of 123patients (171) suffered lymph node recurrences and 14patients (113) had distant organ recurrences As shown inTable 1 there was a significant association between higherSKA1 expression and lymph node involvement (119875 = 0005)clinical stage (119875 = 0015) and extrathyroidal invasion (119875 =0004) however no association was found between SKA1

Disease Markers 3

(a) (b) (c)

Figure 1 SKA1 expression by immunohistochemistry (SP times 10) Left side is PTC tissue (SP times 40) right side is normal adjacent tissue (SP times40)

C N C N

SKA1

GAPDH

(a)

10

01

001

0001

00001

000001

0000001

n = 45

n = 45

Mann-Whitney test

P = 0004

Normal tissues Cancer tissues

000082

000554

Rela

tive S

KA1

mRN

A ex

pres

sion

RQ (2

minus)

ΔΔ

ct

(b)

Figure 2 SKA1 mRNA was detected by qRT-PCR (N normal tissues C cancer tissues)

C N C N

SKA1

GAPDH

(a)

Cancer tissues Normal tissues

150

100

050

000

Rela

tive S

KA1

prot

ein

expr

essio

n

n = 45 n = 45

P lt 005lowast

(b)

Figure 3 SKA1 protein was detected by Western blot analysis (N normal tissues C cancer tissues)

4 Disease Markers

Table 1 Correlation of high SKA1 expressionwith clinicopathologiccharacteristics of PTC patients

Clinical featuresSKA1 expression

1205942 119875 valueLow (n = 67) High (n = 56)

Agelt45 years 22 15 0531 0466ge45 years 45 41

SexMale 32 23 0552 0457Female 35 33

Hashimotorsquosthyroiditis

Without 64 51 0994 0467With 3 5

Tumor sizelt2 cm 42 26 1008 03799ge2 cm 25 30

MultifocalityWithout 43 34 0156 0712With 24 22

DifferentiationWell 19 17 0059 0808Poor 48 39

Lymphoid nodeWithout 41 20 7922 0005With 26 36

Clinical stageI-II 52 32 5902 0015III-IV 15 24

Extrathyroidinvasion

Without 46 24 8280 0004With 21 32

expression and other clinicopathological features Accordingto the data SKA1 expression in tumors might be useful foridentifying the degree of PTC

33 Expression of SKA1 and Prognosis of PTC Patients Weused the Kaplan-Meier method to analyze the relationshipbetween SKA1 expression levels and the prognosis of patientswith PTC The results showed that high SKA1 expressionwas associated with LNRFS (119875 lt 0001) and DRFS (119875 lt0001) (Table 2 Figures 4 and 5) We performed multivariateanalysis using the Cox regression model The results ofmultivariate analysis for the mentioned parameters showedthat high expression of SKA1 was an independent indicatorof LNRFS and DRFS (Table 3)

Table 2 LNRFS and DRFS of PTC patients with high and low SKA1expressions

119873Survival time (months)

119875 valueM plusmn SE

LNRFSSAK1 low expression 67 8431 plusmn 234

lt0001SAK1 high expression 56 5196 plusmn 338DRFSSAK1 low expression 67 8714 plusmn 206

lt0001SAK1 high expression 56 5625 plusmn 323

High SKA1 expression

Low SKA1 expression

Months

Lym

ph n

ode r

ecur

renc

e-fre

e sur

viva

l rat

e

0 20 40 60 80

10

08

06

04

02

00

Figure 4 SKA1 expression and LNRFS

4 Discussion

An equal distribution of chromosomes depends on theprecise regulation of mitosis during each cell cycle andthe correct formation and depolymerization of the spindleare important for the regulation process [8] Abnormalitiesof mitosis can lead to aneuploidy and genomic instabil-ity ultimately resulting in cell death or tumorigenesis [910] The spindle a temporary organelle directly related tocell division and chromosome movement is composed ofmicrotubules and conjugated proteins and the kinetochorea large protein complex is an important link between mitoticchromosomes and spindle microtubules [11] Based on onesignificant assumption spindle orientation defects can lead tocell number increase by suppressing the asymmetric divisionof stem cells while promoting their symmetric proliferation[12 13] Furthermore tissue architecture disorganization atypical feature of malignant transformation might occur dueto defective spindle orientation [12 14]

The SKA1 gene is located on chromosome 18q211 con-tains 255 amino acids and is approximately 30 kDa in size[15] SKA1 is a necessary component for a stable kineto-chore and microtubule binding and it might = promote

Disease Markers 5

Table 3 Multivariate analysis of the prognosis of PTC

Variable LNRFS119875 value DRFS

119875 valueHR for death (95 CI) HR for death (95 CI)

Lymphoid node 1899 (1054ndash3422) 0033 2089 (1144ndash3817) 0017Clinical stage 2827 (1485ndash5384) 0002 3026 (1573ndash5822) 0001Extrathyroid invasion 2275 (1197ndash4324) 0012 2490 (1305ndash4751) 0006SKA1 high expression 4862 (2071ndash11412) lt0001 4907 (2099ndash11475) lt0001


Low SKA1 expression

Months

Dist

ant r

ecur

renc

e-fre

e sur

viva

l rat

e

0 20 40 60 80

10

08

06

04

02

00

Figure 5 SKA1 expression and DRFS

the combination of kinetochore and tubulin and regulatethe depolymerization of tubulin and the synchronization ofchromosomal migration and movement towards the 2 poles[16] The SKA1 protein might be expressed not only in thespindle but also in the kinetochore [17] Its carboxyl termi-nus contains a conserved microtubule-binding domain withmultiple phosphorylation sites that can be phosphorylatedby Aurora B kinase to further regulate the combination ofSKA1 and microtubules [18] Formation of the SKA complexhelps to ensure the correct positioning of the spindle andkinetochore to promote the transition from middle to latemitosis

Li et al [19] found that SKA1 was overexpressed inhuman prostatic intraepithelial neoplasia by immunohisto-chemistry and quantitative RT-PCR They also carried outin vitro experiments which showed that prostate-specificupregulation of SKA1 in a transgenic mouse model resultedin spontaneous tumorigenesis [19] In addition SKA1 is acritical factor that plays an essential role in the regulationof hepatocellular carcinoma cell proliferation and apoptosisresearch showed that knockdown of SKA1 inhibited hepa-tocellular carcinoma cell proliferation by inducing cell cyclearrest in the G0G1 phase [20] The few studies on thisindicate that abnormal SKA1 expression might be closely

associated with the development of other solid tumorssuch as gastric cancer oral adenosquamous carcinoma andneuronal glioblastoma [5 21 22] In this study we found thatSKA1 expression was associated with the clinicopathologicalfeatures and prognosis of patients with PTC Our results sug-gest that a high expression of SKA1 might be associated withlymph node status clinical stage and extrathyroidal invasionfurther implying an involvement of SKA1 in the incidenceand development of PTC In addition we demonstrated anassociation between high SKA1 expression and the prognosisof patients with PTC using Kaplan-Meier survival curvesOur findings indicate that a high expression of SKA1 indicatesworse LNRFS and DRFS At the same time our multivariateanalysis using Cox proportional hazards regression modelshowed that high expression of SKA1 was related to PTCpatient prognosis Based on these results SKA1 might be animportant prognostic indicator for PTC

To the best of our knowledge this is the first report onthe relationship between SKA1 and prognosis in patients withPTCHigh SKA1 expression is predictive of poor prognosis inPTC implying that SKA1 might be a promising new targetfor targeted therapies for PTC However to determine themechanism by which SKA1 upregulated expression promotesPTC generation and development and whether SKA1 also hasabnormal expression in other solid tumors further study isneeded

Disclaimer

The funders had no role in study design data collection andanalysis decision to publish or preparation of the paper

Conflict of Interests

The authors have declared that no competing interests exist

Acknowledgment

This study was funded by the Natural Science Foundationof the Xinjiang Uyghur Autonomous Region (Grant no2011211A036) and Research and Innovation Foundation ofXinjiang Medical University (Grant no XJC201270)

References

[1] J A Sipos and E L Mazzaferri ldquoThyroid cancer epidemiologyand prognostic variablesrdquo Clinical Oncology vol 22 no 6 pp395ndash404 2010

6 Disease Markers

[2] V Zivaljevic N Slijepcevic S Sipetic et al ldquoRisk factors forwell-differentiated thyroid cancer in menrdquo Tumori vol 99 no4 pp 458ndash462 2013

[3] S Young A Harari S Smooke-Praw P H G Ituarte and MW Yeh ldquoEffect of reoperation on outcomes in papillary thyroidcancerrdquo Surgery vol 154 no 6 pp 1354ndash1362 2013

[4] S Cantara M Pisu D V Frau et al ldquoTelomere abnormali-ties and chromosome fragility in patients affected by familialpapillary thyroid cancerrdquo Journal of Clinical Endocrinology andMetabolism vol 97 no 7 pp E1327ndashE1331 2012

[5] B Zhang K Y Li H Y Chen et al ldquoSpindle and kinetochoreassociated complex subunit 1 regulates the proliferation of oraladenosquamous carcinoma CAL-27 cells in vitrordquo Cancer CellInternational vol 13 no 1 article 83 2013

[6] Y H Park S J Lee E Y Cho et al ldquoClinical relevance of TNMstaging system according to breast cancer subtypesrdquo Annals ofOncology vol 22 no 7 pp 1554ndash1560 2011

[7] NSCLC Meta-analysis Collaborative Group ldquoPreoperativechemotherapy for non-small-cell lung cancer a systematicreview and meta-analysis of individual participant datardquo TheLancet vol 383 no 9928 pp 1561ndash1571 2014

[8] H S Sardar and S P Gilbert ldquoMicrotubule capture by mitotickinesin centromere protein E (CENP-E)rdquo Journal of BiologicalChemistry vol 287 no 30 pp 24894ndash24904 2012

[9] L Kabeche andDA Compton ldquoCyclinA regulates kinetochoremicrotubules to promote faithful chromosome segregationrdquoNature vol 502 no 7469 pp 110ndash113 2013

[10] K Crasta N J Ganem R Dagher et al ldquoDNA breaks andchromosome pulverization from errors in mitosisrdquo Nature vol482 no 7383 pp 53ndash58 2012

[11] HWang I Brust-Mascher and JM Scholey ldquoSliding filamentsand mitotic spindle organizationrdquo Nature Cell Biology vol 16no 8 pp 737ndash739 2014

[12] A Noatynska M Gotta and P Meraldi ldquoMitotic spindle(DIS)orientation and DISease cause or consequencerdquo Journalof Cell Biology vol 199 no 7 pp 1025ndash1035 2012

[13] S J Morrison and J Kimble ldquoAsymmetric and symmetric stem-cell divisions in development and cancerrdquo Nature vol 441 no7097 pp 1068ndash1074 2006

[14] S S McAllister and R A Weinberg ldquoTumor-host interactionsa far-reaching relationshiprdquo Journal of Clinical Oncology vol 28no 26 pp 4022ndash4028 2010

[15] M A Abad B Medina A Santamaria et al ldquoStructural basisfor microtubule recognition by the human kinetochore Skacomplexrdquo Nature Communications vol 5 article 2964 2014

[16] D A Skoufias C Mollinari F B Lacroix and R L MargolisldquoHuman survivin is a kinetochore-associated passenger pro-teinrdquo Journal of Cell Biology vol 151 no 7 pp 1575ndash1582 2000

[17] G J Guimaraes and J G Deluca ldquoConnecting with Ska akey complex at the kinetochorendashmicrotubule interfacerdquo EMBOJournal vol 28 no 10 pp 1375ndash1377 2009

[18] A Boeszoermenyi J C Schmidt I M Cheeseman M ObererG Wagner and H Arthanari ldquoResonance assignments of themicrotubule-binding domain of the C elegans spindle andkinetochore-associated protein 1rdquo Biomolecular NMR Assign-ments vol 8 no 2 pp 275ndash278 2014

[19] J Li J W Xuan V Khatamianfar et al ldquoSKA1 over-expressionpromotes centriole over-duplication centrosome amplificationand prostate tumourigenesisrdquoThe Journal of Pathology vol 234no 2 pp 178ndash189 2014

[20] X Qin B Yuan X Xu H Huang and Y Liu ldquoEffects ofshort interfering RNA-mediated gene silencing of SKA1 onproliferation of hepatocellular carcinoma cellsrdquo ScandinavianJournal of Gastroenterology vol 48 no 11 pp 1324ndash1332 2013

[21] W Sun L Yao B Jiang L Guo and Q Wang ldquoSpindle andkinetochore-associated protein 1 is overexpressed in gastriccancer and modulates cell growthrdquo Molecular and CellularBiochemistry vol 391 no 1-2 pp 167ndash174 2014

[22] X Shi X Chen H Peng et al ldquoLentivirus-mediated silencingof spindle and kinetochore-associated protein 1 inhibits theproliferation and invasion of neuronal glioblastoma cellsrdquoMolecular Medicine Reports vol 11 no 5 pp 3533ndash3538 2015

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


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EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Disease Markers

selected for this study only if follow-up examinations andclinical data were available The TNM stage was definedaccording to the 7th edition of the TNM classification of theInternational Union Against CancerThe study was approvedby the ethics committee of the Affiliated Tumor Hospital ofXinjiang Medical University Written informed consent wasobtained from each patient for the use of resected samplesfor research

22 Immunohistochemistry Biopsy tissue samples were sec-tioned into 4-120583m slices and embedded with paraffin after fix-ation with 10 formaldehyde Immunohistochemistry withthe SP method (rabbit anti-SKA1 antibody 1 500 SigmaChemical Co St Louis MO USA) was used for testing byapplying PBS instead of primary antibodies as the negativecontrol according to the manufacturerrsquos instructions Theresults were determined by two independent pathologistsusing a double-blind method Five high power fields (200x)were randomly selected and in each field 200 tumor cellswere recorded totaling to 1000 cells According to thestaining intensity the cells were assigned the following pointvalues 0 points for no staining 1 point for pale yellow stain-ing 2 points for brown-yellow staining and 3 points for darkbrown stainingThe proportion of positive cells was assigneda value between 0 and 100 The immunohistochemistryexpression level was determined according to the productof the staining intensity and the proportion of positive cellsWhen the product was below 150 this was considered lowexpression of SKA1 and when the product was above 150this was considered high expression of SKA1

23 RNA Extraction and Quantitative Real-Time PCR AssaysThe tissue samples were removed from liquid nitrogenand RNA was extracted using Trizol reagent and a reversetranscription kit (Invitrogen Carlsbad CA USA) The PCRprimers used were SKA1-F 51015840-TGATGTGCCAGGAAG-GTGAC-31015840 SKA1-R 51015840-CAAAGGATACAGATGAAC-AACAGC-31015840 GAPDH-F 51015840-GTGGACATCCGCAAAGAC-31015840 andGAPDH-R 51015840-AAAGGGTGTAACGCAACTA-31015840 Allprimers were synthesized by Shanghai Yingjun Biotechnol-ogy Company (Shanghai China) The PCR reaction condi-tions were as follows initial denaturation for 2min at 94∘C35 cycles of denaturation for 30 s annealing for 30 s at 94∘Cand extension for 1min at 72∘C followed by 10min at 72∘CPCR reaction product (3 120583L) was used for 2 agarose gelelectrophoresis and the grayscale analysis was performedwith Quantity One software (Bio-Rad Hercules CA USA)The relative expression level of SKA1mRNA was determinedby the ratio of the grayscale value of SKA1mRNA and that ofinternal reference GAPDH mRNA

24 Western Blot Analysis After protein extraction usingtotal protein lysis buffer the protein concentration wasdetermined by the BCA method In brief 80 120583g protein perwell was used for 8 SDS-PAGE and after electrophoreticseparation the protein was wet-transferred to polyvinylidenefluoride 5 nonfat milk powder solution was added andafter a 1 h incubation at room temperature the transferred

protein was incubated with the primary antibodies (1 500dilution SKA1 and GAPDH Sigma Chemical Co St LouisMO USA) at 4∘C overnight Goat anti-rabbit IgG sec-ondary antibody (GenScript Biotechnology Company Nan-jing Jiangsu China) marked with horseradish peroxidaseand diluted at 1 1000 was added and incubated at roomtemperature for 15 hThemembrane was washed three timesin TBST The relative level of SKA1 protein was determinedby the grayscale value of various protein straps and the ratioof the grayscale value of SKA1 to GAPDH protein

25 Statistical Analysis The chi-squared test was used toanalyze the associations between SKA1 expression and theclinicopathological features of PTC SKA1mRNA expressionin PTC was compared with expression in normal adjacenttissues using the Mann-Whitney test (for 2 groups) orthe Kruskal-Wallis test (for more than 2 groups) Kaplan-Meier analysis was used for survival analysis lymph noderecurrence-free survival (LNRFS) and distant recurrence-free survival (DRFS) calculations LNRFS was defined as thetime from the date of surgery to the date of lymph noderelapse and DRFS was defined as the time from the date ofsurgery to the date of distant recurrence [6 7] Cox regressionmodels were used for multiple factor analysis All statisticaltests were two-tailed and statistical significance was assumedfor 119875 lt 005

3 Results

31 Expression of SKA1 in PTC Tissues Immunostainingresults suggested a significantly higher SKA1 expression inPTC tissues compared with adjacent tissues Among 123tumor samples 56 (455) showed high SKA1 expressionwith only 37 of the samples from normal adjacent tissues(see Figure 1) To confirm these observations we investigatedSKA1 mRNA expression in 45 cases of PTC and normaladjacent tissues using qRT-PCR and Western blot analysisThe mRNA levels between PTC tissues and normal adjacenttissues were calculated with the comparative Ct method(2minusΔΔct) The results indicated that SKA1 mRNA and SKA1protein expression was significantly higher in PTC tissuesthan in normal adjacent tissues (119875 lt 005 Figures 2 and 3)

32 Patient Characteristics Table 1 shows the patientsrsquo clini-cal and immunohistochemical data The mean age was 413 plusmn157 years (range 26ndash69 years) for 32 male patients and 442 plusmn145 years (range 24ndash73 years) for 35 female patients Sixty-two patients (504) had lymph node metastasis Eighty-four patients (683) had stage I-II disease based on theTNM staging system and the remaining 39 patients (317)had stage III-IV disease During the median follow-up timewhich was 52 months (range 17ndash102 months) 21 of 123patients (171) suffered lymph node recurrences and 14patients (113) had distant organ recurrences As shown inTable 1 there was a significant association between higherSKA1 expression and lymph node involvement (119875 = 0005)clinical stage (119875 = 0015) and extrathyroidal invasion (119875 =0004) however no association was found between SKA1

Disease Markers 3

(a) (b) (c)


C N C N

SKA1

GAPDH

(a)

10

01

001

0001

00001

000001

0000001

n = 45

n = 45

Mann-Whitney test

P = 0004


000082

000554

Rela

tive S

KA1

mRN

A ex

pres

sion

RQ (2

minus)

ΔΔ

ct

(b)


C N C N

SKA1

GAPDH

(a)


150

100

050

000

Rela

tive S

KA1

prot

ein

expr

essio

n

n = 45 n = 45

P lt 005lowast

(b)


4 Disease Markers



1205942 119875 valueLow (n = 67) High (n = 56)


SexMale 32 23 0552 0457Female 35 33


Without 64 51 0994 0467With 3 5







Without 46 24 8280 0004With 21 32










Low SKA1 expression

Months

Lym

ph n

ode r

ecur

renc

e-fre

e sur

viva

l rat

e

0 20 40 60 80

10

08

06

04

02

00


4 Discussion



Disease Markers 5






Low SKA1 expression

Months

Dist

ant r

ecur

renc

e-fre

e sur

viva

l rat

e

0 20 40 60 80

10

08

06

04

02

00






Disclaimer




Acknowledgment


References


6 Disease Markers



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 3

(a) (b) (c)


C N C N

SKA1

GAPDH

(a)

10

01

001

0001

00001

000001

0000001

n = 45

n = 45

Mann-Whitney test

P = 0004


000082

000554

Rela

tive S

KA1

mRN

A ex

pres

sion

RQ (2

minus)

ΔΔ

ct

(b)


C N C N

SKA1

GAPDH

(a)


150

100

050

000

Rela

tive S

KA1

prot

ein

expr

essio

n

n = 45 n = 45

P lt 005lowast

(b)


4 Disease Markers



1205942 119875 valueLow (n = 67) High (n = 56)


SexMale 32 23 0552 0457Female 35 33


Without 64 51 0994 0467With 3 5







Without 46 24 8280 0004With 21 32










Low SKA1 expression

Months

Lym

ph n

ode r

ecur

renc

e-fre

e sur

viva

l rat

e

0 20 40 60 80

10

08

06

04

02

00


4 Discussion



Disease Markers 5






Low SKA1 expression

Months

Dist

ant r

ecur

renc

e-fre

e sur

viva

l rat

e

0 20 40 60 80

10

08

06

04

02

00






Disclaimer




Acknowledgment


References


6 Disease Markers



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















4 Disease Markers



1205942 119875 valueLow (n = 67) High (n = 56)


SexMale 32 23 0552 0457Female 35 33


Without 64 51 0994 0467With 3 5







Without 46 24 8280 0004With 21 32










Low SKA1 expression

Months

Lym

ph n

ode r

ecur

renc

e-fre

e sur

viva

l rat

e

0 20 40 60 80

10

08

06

04

02

00


4 Discussion



Disease Markers 5






Low SKA1 expression

Months

Dist

ant r

ecur

renc

e-fre

e sur

viva

l rat

e

0 20 40 60 80

10

08

06

04

02

00






Disclaimer




Acknowledgment


References


6 Disease Markers



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 5






Low SKA1 expression

Months

Dist

ant r

ecur

renc

e-fre

e sur

viva

l rat

e

0 20 40 60 80

10

08

06

04

02

00






Disclaimer




Acknowledgment


References


6 Disease Markers



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















6 Disease Markers



























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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