research article plasma hulc as a promising novel...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 136106 5 pageshttpdxdoiorg1011552013136106

Research ArticlePlasma HULC as a Promising Novel Biomarker for the Detectionof Hepatocellular Carcinoma

Hui Xie1 Hongwei Ma2 and Danqiu Zhou3

1 Department of Laboratory Medicine Zhengzhou Peoplersquos Hospital 33 Huanghe Road Zhengzhou 450003 China2Henan Red Cross Blood Center Institute of Transfusion Medicine 9 Tongle Road Zhengzhou 450012 China3Department of Laboratory Medicine Jinshan Hospital Shanghai Medical College Fudan University Shanghai 200540 China

Correspondence should be addressed to Danqiu Zhou zhoudqshanghai126com

Received 30 January 2013 Revised 16 April 2013 Accepted 2 May 2013

Academic Editor Vladimir Bajic

Copyright copy 2013 Hui Xie et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Hepatocellular carcinoma (HCC) is a leading cause of cancer death in many Asian and African countries Lack of early diagnosistools is one of the clinical obstacles for effective treatment of HCC Thus enhanced understanding of the molecular changesassociated with HCC is urgently needed to develop novel strategies for the diagnosis and treatment of this dismal disease Whileaberrant expression of long noncoding RNAs (lncRNAs) has been functionally associated with certain cancers the expressionprofiles and biological relevance of lncRNAs in HCC remain unclear Highly upregulated in liver cancer (HULC) lncRNA has beenimplicated in the regulation of hepatoma cell proliferation In this study we demonstrate that HULC expression is significantlyhigher inHCC tumors compared to normal liver tissues Among the tumor tissues higherHULC expression is positively associatedwith Edmondson histological grades or with hepatitis B (HBV) positive status Moreover HULC lncRNA is detected with higherfrequency in the plasma of HCC patients compared to healthy controls Higher HULC detection rates are observed in the plasmaof patients with higher Edmondson grades or with HBV+ status These findings indicate for the first time that the expression ofHULC in plasma can be used as a noninvasive promising novel biomarker for the diagnosis andor prognosis of HCC

1 Introduction

Hepatocellular carcinoma (HCC) is a leading cause of can-cer death in many Asian and African countries [1 2]HCC causes estimated 662000 deaths each year worldwide(ldquoCancerrdquo World Health Organization) and about half ofthem occur in China Although they can be small andslow growing HCC tumors can be successfully treated byaggressive surgery patients often lose the window for surgicalresection due to the lack of effective tools for early diagnosiswhich results in very low 5-year survival rates Therefore toimprove the prognosis of HCC it is important and criticalto develop specific and sensitive diagnostic biomarkers forHCC

Noncoding RNAs (ncRNAs) consist ofmicroRNAs smallinterfering RNAs and various classes of long noncodingRNAs (lncRNAs) NcRNAs play important regulatory roles inthe development and progression of many diseases includingcancers [3 4]

LncRNAs ranging from 200 to over 10000 nucleotidesare abundantly transcribed by themammalian genome [5 6]LncRNAs have been found to be dysregulated in a wide rangeof human diseases and disorders including various typesof cancer For instance lncRNAs PCGEM [7] and DD3 [8]are overexpressed in prostate cancer tissues implicating thatthese lncRNAsmay be involved in prostate tumorigenesis [9]BC200RNAoverexpression has recently been correlatedwiththe progression of breast tumors and proposed as a potentialnovel molecular marker for breast cancer [10] Increasedexpression of the MALAT-1 gene indicates worse prognosisin lung cancer patients [11] Together these observationsprovide evidence and support for the potential roles oflncRNAs in tumor development and progression

miRNAs in human plasma or serum have distinctexpression signatures in different diseases including cancersTherefore serum miRNAs can serve as important diagnosticbiomarkers for certain cancer types [12ndash15] such ascolorectal oral and pancreatic cancers However lncRNA

2 BioMed Research International

expressions in plasma or serum have not been investigatedfor their potential as potential novel biomarkers for HCCdiagnosis or prognosis

Highly upregulated in liver cancer (HULC)was first iden-tified from an HCC-specific gene library as a novel mRNA-like lncRNA which was markedly up-regulated in HCC [16]and is associated with the molecular pathogenesis of HCCAberrant expression of lncRNA HULC has been reported inHCC [17 18] and its potential as a diagnostic biomarker hasbeen proposed [15 19] However the expression of HULC inthe plasma of HCC patients has not been examined In thisstudy we tested the hypothesis that lncRNAHULC is presentin the plasma of HCC patients and can be used clinically as abiomarker to facilitate early diagnosis of HCC We examinedthe expression level of HULC in HCC tumor tissues and livertissues obtained from healthy volunteers and the matchingplasma of these two groups using a real-time quantitativeRT-PCR Our results showed that HULC is markedly up-regulated in the HCC tumor tissues and in plasma of HCCpatients

2 Materials and Methods

21 Tissue and Blood Samples Liver tissue samples from 20healthy volunteers and tumor tissues from 30 HCC patientswere collected from the Zhengzhou Peoplersquos Hospital (Zhen-gzhou China) Healthy volunteers had no history of HCCnor HBV infection and had normal physical examinationsPatients enrolled in the study were provided with writteninformed consent Fresh tissue samples were frozenwithin 30minutes after surgery and stored in liquid nitrogen until useTissue sections from each HCC sample were reviewed andclassified by a pathologist Chronic HBV infection is definedas HBV surface antigen positive for at least 6 months HBVDNA detectable by PCR and HBV infection-compatibleresults in a liver biopsy

Whole blood samples collected in EDTA tubes werecentrifuged at 1200 g for 10min at 4∘C to spin down the bloodcells The supernatants were transferred to microcentrifugetubes and centrifuged at 12000 g for 10min at 4∘C to comp-letely remove cellular components Plasma was then carefullycollected aliquoted and stored at minus80∘Cuntil use Total RNAfrom 1mL plasma was extracted using Trizol (Invitrogen)according to the manufacturerrsquos instructions

22 q-RT-PCR of HULC in HCC Tissues and Blood SamplesReverse transcription reactions were carried out on 1120583gtotal RNA using the PrimeScript RT reagent kit (TaKaRaBIO Shiga Japan) Random hexamer primers were used inthe RT reactions The real-time PCR was then performedusing SYBR Premix DimerEraser kit (TaKaRa Shiga Japan)GAPDH was evaluated as a housekeeping gene for the qPCRreactions using the commercially available primers (TaKaRaShiga Japan) The HULC qPCR primers used are for-ward 51015840-TCATGATGGAATTGGAGCCTT-31015840 reverse 51015840-CTCTTCCTGGCTTGCAGATTG-31015840 All the reactions werecarried out on a Bio-Rad CFX-96 real-time PCR system(Bio-Rad Hercules CA) according to the manufacturesrsquoinstructions To ensure the accuracy of the amplifications we

included a negative control cell line HL-7702 which does notexpress HULC [20] and a positive control cell line Hep3Bwhich expresses high levels of HULC [20] in each run Toprepare the standards with 1 10 50 and 100 of Hep3BcDNA load in a 50 ng120583L background serial dilutions ofHep3B cDNA with HL-7702 cDNA were madeThe standardcurve of HULC expression was plotted using the results ofthe serial dilution standards The amplification efficiency ofeach standard reaction was confirmed to have an 1199032 = 0999The concentrations of all cDNA samples in this study werewithin this standard range Together we confirmed that theHULC expression results are accurateThe expression level ofHULC in each sample was normalized to that of the internalcontrol GAPDH The fold change of HULC expression intumor samples versus healthy controls was calculated by the2

minusΔΔCt method

23 Statistical Analysis Statistical significances betweengroups were determined by two-tailed Studentrsquos 119905-test Theassociation between HULC expression and clinicopatholog-ical characteristics was analyzed using one-way ANOVAwith bonferroni correction All statistical analyses were donewith STATA 100 (StataCorp LP College Station TX) A 119875value of lt005 was considered significant Receiver operatingcharacteristics (ROC) curve was plotted to determine howwell the expression level of HULC discriminated betweentumor samples and healthy control samples

3 Results

31 HULC lncRNAExpression Is Up-Regulated in HCCTissuesCompared to Normal Liver Tissues To assess the potentialclinical utility of HULC its expression levels in both HCCtissues and healthy control liver specimens were analyzedFirst to determine the reaction efficiencies standard curveswere created using the qPCR results of serial dilutions ofHep3B cDNA which serves as a positive control for HULCexpression The linearity of real-time PCR was confirmedNext the receiver operating characteristics (ROC) analysiswas used to evaluate the suitability of HULC expression todiscriminate between the tumor and control samples Totalarea under the curve (AUC) for HULCwas 086 (Figure 1(a))which suggests thatHULChas adequate sensitivity and speci-ficity to discriminate between tumor and control samplesFurther the expression of HULC in 30 HCC tumor samplesand 20 healthy control liver tissue samples were determinedThe results showed that the expression levels of HULC weremarkedly increased in HCC tissues compared to normal liverspecimens (119875 lt 001) (Figure 1(b))

32 HULC lncRNAExpression Is Associated withHCCGradesTo investigate whether the expression levels of HULClncRNA in the HCC tumor tissues are associated with thedisease severity we analyzed the HULC expression in HCCtissues according to their Edmondson grades The resultsrevealed progressive up-regulation of HULC expression inHCC tissue samples from well-differentiated (Edmondsongrades I-II) to undifferentiated lesions (Edmondson gradesIII-IV) (Figure 2(a))

BioMed Research International 3

0 10 20 30 40 50 60 70 80 90 1000

102030405060708090

100Se

nsiti

vity

1 minus specificity ()(a)

Healthy control HCC0

20

40

60

80

Relat

ive H

ULC

RN

A

(b)

Figure 1 HULC is up-regulated in HCC tissues Receiver-operating characteristic (ROC) curve (a) and box plots (b) of HULC RNAexpression in HCC tissues and healthy control liver tissues (a) The area under the ROC curve was 086 in distinguishing hepatocellularcarcinoma versus healthy control (b) HULC expression was examined in 20 healthy control liver tissues and 30 HCC tissues by qRT-PCRData were analyzed using the 2minusΔΔCt method The upper and lower limits of the boxes and the lines inside the boxes indicate the 75th and25th percentiles and the median respectively The upper and lower horizontal bars denote the 90th and 10th percentiles respectively

Healthy controlHCC Edmondson grade

0

10

20

30

40

50

60

70

Rela

tive H

ULC

RN

A

0

20

40

60

80

Rela

tive H

ULC

mRN

A

Con I-II II-III III-IV

I-II II-III III-IV

(a)

0

10

20

30

40

50

60

70

Healthy controlHCC

0

20

40

60

80

Rela

tive H

ULC

mRN

A

Rela

tive H

ULC

RN

A

HBVminus HBV+Con

HBVminus HBV+

(b)

Figure 2 HULC lncRNA expression is associated with HCC Edmondson grades and HBV status (a) Comparison of the relative levels ofHULC in normal liver tissues and HCC tissues with different Edmondson grades (b) Comparison of the relative levels of HULC in normalliver tissues and HCC tissues from HBVminus and HBV+ patients

Chronic HBV infection is a major cause of HCC andthe multifunctional oncoprotein hepatitis B virus X (HBx)plays a crucial role in the development of HCC [21] HBx-mediated up-regulation of HULC promotes the proliferationof hepatoma cells through the reduction of p18 [22] Theevidence prompted us to investigate whether there is anyassociation between HULC expression and HBV infectionstatus in HCC patients We compared the expression ofHULC lncRNA in HBV positive and negative HCC patientsHULC expression and HBV status were positively correlatedin the HCC tissues (Figure 2(b)) HULC expression washigher inHCC tissue samples fromHBV+ patients comparedto those from HBVminus patients (Figure 2(b)) The associationof HULC expression with various clinicopathological param-eters of the HCC patients was further analyzed statistically

(Table 1) The median HULC expression levels were differentin patients with different Edmondson histological grades orwith different HBV status The statistical analyses revealeda striking positive association between HULC expressionlevels and the HCC histological grades (119875 lt 001) (Table 1)Statistical difference in HULC expression was also foundbetween HBV positive and negative groups (119875 lt 001)(Table 1)

33 HULC lncRNA Is Detected in the Plasma of HCC PatientsHULC RNA has been detected in peripheral blood cells ofHCC patients [16]Therefore we investigated the potential ofusing HULC as a novel biomarker for HCC diagnosis HULCexpression levels were examined by qRT-PCR in plasmacollected from healthy volunteers (119899 = 20) and HCC patients


Table 1 Relationship of HULC expression to clinicopathological features of HCC patients

(a)

Normal Tumor (Edmondson grade)119873 = 20 119873 = 30

119899 Median 119875

I-II II-III III-IV119899 Median 119875 119899 Median 119875 119899 Median 119875

20 681 7 2064 lt001 10 2495 lt001 13 4797 lt001

(b)

Normal Tumor (HBV)119873 = 20 119873 = 30

119899 Median 119875

minus +119899 Median 119875 119899 Median 119875

20 681 12 2006 lt001 18 3932 lt001

Table 2 HULC in the plasma of healthy controls andHCC patients

lncRNA Healthy control HCCI-II II-III III-IV

HULC 220 (10) 17 (14) 813 (62) 1010 (100)

Table 3 HULC in the plasma of healthy controls and HCC patientswith different HBV status

lncRNA Healthy control HCCHBV (minus) HBV (+)

HULC 220 (10) 312 (25) 1618 (90)

(119899 = 30) fromwhom the tissue sampleswere collectedHULCwas detected in 63 (1930) of the HCC patients which wasmuch higher than in the healthy control group (10 220)(Tables 2 and 3) Among the HCC patients HULC detectionfrequencies increase with Edmondson grades (Table 2) Thedetection rates are 14 62 and 100 for Edmondsongrades I-II II-III and III-IV respectively (Table 2) HULCwas detected more frequently in the plasma of HBV+ HCCpatients (90) than in HBVminus patients (25) (Table 3)These observations indicate that the presence of HULC isan indication of HCC and also its progression Thereforethe data support the clinical usage of HULC lncRNA as apotential biomarker for HCC diagnosis and prognosis

4 Discussion

LncRNAs belong to a novel class of noncoding RNA mole-cules which are longer than 200 nucleotides [23] A numberof lncRNAs have been reported to control transcriptionalalteration implying that the difference of lncRNA profilingbetween normal and cancer cells may have regulatory rolesin cancer progression instead of being the secondary effect ofcancer transformation [24] LncRNA expressions are stronglyassociated with cancer development and progression [24]Thus differential expression of lncRNAs in cancers maybe used to facilitate cancer diagnosis discover potentialtreatment targets and improve prognosis In this study we

demonstrate that lncRNA HULC is overexpressed in HCCtumor tissues compared to healthy liver tissues Among theHCC patients HULC expression is significantly higher inpatientswith higher Edmondson grades orwith positiveHBVstatusThese results indicate that HULC expression positivelyassociates with the progression of HCC

It has been widely reported that cancer-specific miRNAsare detectable in blood sputum urine and other biologicalfluids of cancer patients Therefore miRNAs have beeninvestigated as potential biomarkers for cancer diagnosis andprognosis Likewise lncRNAs have demonstrated utility asfluid-based markers of specific cancers Serum and plasmaharbor clinical discriminatory proteomic and transcriptomicbiomarkers which can be easily assessed for clinical use Inthe current study HULC is detected in the plasma of HCCpatients and higher detection rates are found in the plasmaof patients with higher Edmondson grades or positive HBVstatus

Our data suggest that HULC expression in HCC islikely to be associated with the aggressiveness and theprogression of the tumor While the prognostic power ofHULC expression will obviously have to be substantiated bylongitudinal analysis in prospective follow-up studies ourresults represent a significant step towards establishing theutility ofHULCexpression as a prognostic indicator forHCC

In conclusion we have shown that HULC is differentiallyexpressed in the tissues and plasma of the HCC patientscompared with those of healthy controls These findingsindicate for the first time that the expression of HULC inplasma can be used as a novel and a rapid diagnostic andorprognostic biomarker for HCC

Conflict of Interests

There is no conflict of interests from any author

Acknowledgment

This study was supported by the Research Grants fromShanghai Municipal Health Bureau (20114300)


References

[1] A P Venook C Papandreou J Furuse and L L de GuevaraldquoThe incidence and epidemiology of hepatocellular carcinomaa global and regional perspectiverdquo The Oncologist vol 15supplement 4 pp 5ndash13 2010

[2] J Ferlay H R Shin F Bray D Forman C Mathers and DM Parkin ldquoEstimates of worldwide burden of cancer in 2008GLOBOCAN2008rdquo International Journal of Cancer vol 127 no12 pp 2893ndash2917 2010

[3] C D Malone and G J Hannon ldquoSmall RNAs as Guardians ofthe Genomerdquo Cell vol 136 no 4 pp 656ndash668 2009

[4] D Moazed ldquoSmall RNAs in transcriptional gene silencing andgenome defencerdquo Nature vol 457 no 7228 pp 413ndash420 2009

[5] P Bertone V Stolc T E Royce et al ldquoGlobal identification ofhuman transcribed sequences with genome tiling arraysrdquo Sci-ence vol 306 no 5705 pp 2242ndash2246 2004

[6] P Kapranov J Drenkow J Cheng et al ldquoExamples of the com-plex architecture of the human transcriptome revealed byRACEand high-density tiling arraysrdquo Genome Research vol 15 no 7pp 987ndash997 2005

[7] V Srikantan Z Zou G Petrovics et al ldquoPCGEM1 a prostate-specific gene is overexpressed in prostate cancerrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 97 no 22 pp 12216ndash12221 2000

[8] M J G Bussemakers A van Bokhoven G W Verhaegh et alldquoDD3 a new prostate-specific gene highly overexpressed inprostate cancerrdquoCancer Research vol 59 no 23 pp 5975ndash59791999

[9] G Petrovics W Zhang M Makarem et al ldquoElevated expres-sion of PCGEM1 a prostate-specific gene with cell growth-promoting function is associatedwith high-risk prostate cancerpatientsrdquo Oncogene vol 23 no 2 pp 605ndash611 2004

[10] A Iacoangeli Y Lin E J Morley et al ldquoBC200 RNA in invasiveand preinvasive breast cancerrdquoCarcinogenesis vol 25 no 11 pp2125ndash2133 2004

[11] P Ji S Diederichs W Wang et al ldquoMALAT-1 a novel noncod-ing RNA and thymosin 1205734 predict metastasis and survival inearly-stage non-small cell lung cancerrdquo Oncogene vol 22 no39 pp 8031ndash8041 2003

[12] MACortez C Bueso-Ramos J FerdinG Lopez-Berestein AK Sood andG A Calin ldquoMicroRNAs in body fluids-themix ofhormones and biomarkersrdquo Nature Reviews Clinical Oncologyvol 8 no 8 pp 467ndash477 2011

[13] Z Huang D Huang S Ni Z Peng W Sheng and X DuldquoPlasma microRNAs are promising novel biomarkers for earlydetection of colorectal cancerrdquo International Journal of Cancervol 127 no 1 pp 118ndash126 2010

[14] R Liu X Chen Y Du et al ldquoSerummicroRNA expression pro-file as a biomarker in the diagnosis and prognosis of pancreaticcancerrdquo Clinical Chemistry vol 58 pp 610ndash618 2012

[15] C J Liu S Y Kao H F Tu M M Tsai K W Chang and S CLin ldquoIncrease of microRNA miR-31 level in plasma could be apotential marker of oral cancerrdquoOral Diseases vol 16 no 4 pp360ndash364 2010

[16] K Panzitt M M O Tschernatsch C Guelly et al ldquoCharacter-ization of HULC a novel gene with striking up-regulation inhepatocellular carcinoma as noncoding RNArdquo Gastroenterol-ogy vol 132 no 1 pp 330ndash342 2007

[17] T S Wong X B Liu B Y H Wong R W M Ng A P WYuen and W I Wei ldquoMature miR-184 as potential oncogenic

microRNA of squamous cell carcinoma of tonguerdquo ClinicalCancer Research vol 14 no 9 pp 2588ndash2592 2008

[18] N J Park H Zhou D Elashoff et al ldquoSalivary microRNAdiscovery characterization and clinical utility for oral cancerdetectionrdquo Clinical Cancer Research vol 15 no 17 pp 5473ndash5477 2009

[19] M Gorenchtein C F Poh R Saini andC Garnis ldquoMicroRNAsin an oral cancer contextmdashfrom basic biology to clinical utilityrdquoJournal of Dental Research vol 91 pp 440ndash446 2012

[20] J Wang X Liu H Wu et al ldquoCREB up-regulates longnon-coding RNA HULC expression through interaction withmicroRNA-372 in liver cancerrdquo Nucleic Acids Research vol 38no 16 pp 5366ndash5383 2010

[21] X Zhang H Zhang and L Ye ldquoEffects of hepatitis B virusX protein on the development of liver cancerrdquo Journal ofLaboratory and Clinical Medicine vol 147 no 2 pp 58ndash662006

[22] Y Du G Kong X You et al ldquoElevation of highly up-regulated in liver cancer (HULC) by hepatitis B virus X proteinpromotes hepatoma cell proliferation via down-regulating p18rdquoThe Journal of Biological Chemistry vol 287 pp 26302ndash263112012

[23] L Lipovich R Johnson and C Y Lin ldquoMacroRNA underdogsin a microRNAworld evolutionary regulatory and biomedicalsignificance of mammalian long non-protein-coding RNArdquoBiochimica et Biophysica ActamdashGene Regulatory Mechanismsvol 1799 no 9 pp 597ndash615 2010

[24] M Huarte and J L Rinn ldquoLarge non-coding RNAs missinglinks in cancerrdquo Human Molecular Genetics vol 19 no 2 ppR152ndashR161 2010

Submit your manuscripts athttpwwwhindawicom

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Oxidative Medicine and Cellular Longevity


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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


expressions in plasma or serum have not been investigatedfor their potential as potential novel biomarkers for HCCdiagnosis or prognosis

Highly upregulated in liver cancer (HULC)was first iden-tified from an HCC-specific gene library as a novel mRNA-like lncRNA which was markedly up-regulated in HCC [16]and is associated with the molecular pathogenesis of HCCAberrant expression of lncRNA HULC has been reported inHCC [17 18] and its potential as a diagnostic biomarker hasbeen proposed [15 19] However the expression of HULC inthe plasma of HCC patients has not been examined In thisstudy we tested the hypothesis that lncRNAHULC is presentin the plasma of HCC patients and can be used clinically as abiomarker to facilitate early diagnosis of HCC We examinedthe expression level of HULC in HCC tumor tissues and livertissues obtained from healthy volunteers and the matchingplasma of these two groups using a real-time quantitativeRT-PCR Our results showed that HULC is markedly up-regulated in the HCC tumor tissues and in plasma of HCCpatients

2 Materials and Methods

21 Tissue and Blood Samples Liver tissue samples from 20healthy volunteers and tumor tissues from 30 HCC patientswere collected from the Zhengzhou Peoplersquos Hospital (Zhen-gzhou China) Healthy volunteers had no history of HCCnor HBV infection and had normal physical examinationsPatients enrolled in the study were provided with writteninformed consent Fresh tissue samples were frozenwithin 30minutes after surgery and stored in liquid nitrogen until useTissue sections from each HCC sample were reviewed andclassified by a pathologist Chronic HBV infection is definedas HBV surface antigen positive for at least 6 months HBVDNA detectable by PCR and HBV infection-compatibleresults in a liver biopsy

Whole blood samples collected in EDTA tubes werecentrifuged at 1200 g for 10min at 4∘C to spin down the bloodcells The supernatants were transferred to microcentrifugetubes and centrifuged at 12000 g for 10min at 4∘C to comp-letely remove cellular components Plasma was then carefullycollected aliquoted and stored at minus80∘Cuntil use Total RNAfrom 1mL plasma was extracted using Trizol (Invitrogen)according to the manufacturerrsquos instructions

22 q-RT-PCR of HULC in HCC Tissues and Blood SamplesReverse transcription reactions were carried out on 1120583gtotal RNA using the PrimeScript RT reagent kit (TaKaRaBIO Shiga Japan) Random hexamer primers were used inthe RT reactions The real-time PCR was then performedusing SYBR Premix DimerEraser kit (TaKaRa Shiga Japan)GAPDH was evaluated as a housekeeping gene for the qPCRreactions using the commercially available primers (TaKaRaShiga Japan) The HULC qPCR primers used are for-ward 51015840-TCATGATGGAATTGGAGCCTT-31015840 reverse 51015840-CTCTTCCTGGCTTGCAGATTG-31015840 All the reactions werecarried out on a Bio-Rad CFX-96 real-time PCR system(Bio-Rad Hercules CA) according to the manufacturesrsquoinstructions To ensure the accuracy of the amplifications we

included a negative control cell line HL-7702 which does notexpress HULC [20] and a positive control cell line Hep3Bwhich expresses high levels of HULC [20] in each run Toprepare the standards with 1 10 50 and 100 of Hep3BcDNA load in a 50 ng120583L background serial dilutions ofHep3B cDNA with HL-7702 cDNA were madeThe standardcurve of HULC expression was plotted using the results ofthe serial dilution standards The amplification efficiency ofeach standard reaction was confirmed to have an 1199032 = 0999The concentrations of all cDNA samples in this study werewithin this standard range Together we confirmed that theHULC expression results are accurateThe expression level ofHULC in each sample was normalized to that of the internalcontrol GAPDH The fold change of HULC expression intumor samples versus healthy controls was calculated by the2

minusΔΔCt method

23 Statistical Analysis Statistical significances betweengroups were determined by two-tailed Studentrsquos 119905-test Theassociation between HULC expression and clinicopatholog-ical characteristics was analyzed using one-way ANOVAwith bonferroni correction All statistical analyses were donewith STATA 100 (StataCorp LP College Station TX) A 119875value of lt005 was considered significant Receiver operatingcharacteristics (ROC) curve was plotted to determine howwell the expression level of HULC discriminated betweentumor samples and healthy control samples

3 Results

31 HULC lncRNAExpression Is Up-Regulated in HCCTissuesCompared to Normal Liver Tissues To assess the potentialclinical utility of HULC its expression levels in both HCCtissues and healthy control liver specimens were analyzedFirst to determine the reaction efficiencies standard curveswere created using the qPCR results of serial dilutions ofHep3B cDNA which serves as a positive control for HULCexpression The linearity of real-time PCR was confirmedNext the receiver operating characteristics (ROC) analysiswas used to evaluate the suitability of HULC expression todiscriminate between the tumor and control samples Totalarea under the curve (AUC) for HULCwas 086 (Figure 1(a))which suggests thatHULChas adequate sensitivity and speci-ficity to discriminate between tumor and control samplesFurther the expression of HULC in 30 HCC tumor samplesand 20 healthy control liver tissue samples were determinedThe results showed that the expression levels of HULC weremarkedly increased in HCC tissues compared to normal liverspecimens (119875 lt 001) (Figure 1(b))

32 HULC lncRNAExpression Is Associated withHCCGradesTo investigate whether the expression levels of HULClncRNA in the HCC tumor tissues are associated with thedisease severity we analyzed the HULC expression in HCCtissues according to their Edmondson grades The resultsrevealed progressive up-regulation of HULC expression inHCC tissue samples from well-differentiated (Edmondsongrades I-II) to undifferentiated lesions (Edmondson gradesIII-IV) (Figure 2(a))


0 10 20 30 40 50 60 70 80 90 1000

102030405060708090

100Se

nsiti

vity



20

40

60

80

Relat

ive H

ULC

RN

A

(b)



0

10

20

30

40

50

60

70

Rela

tive H

ULC

RN

A

0

20

40

60

80

Rela

tive H

ULC

mRN

A


I-II II-III III-IV

(a)

0

10

20

30

40

50

60

70

Healthy controlHCC

0

20

40

60

80

Rela

tive H

ULC

mRN

A

Rela

tive H

ULC

RN

A

HBVminus HBV+Con

HBVminus HBV+

(b)







(a)


119899 Median 119875


20 681 7 2064 lt001 10 2495 lt001 13 4797 lt001

(b)

Normal Tumor (HBV)119873 = 20 119873 = 30

119899 Median 119875


20 681 12 2006 lt001 18 3932 lt001



HULC 220 (10) 17 (14) 813 (62) 1010 (100)



HULC 220 (10) 312 (25) 1618 (90)


4 Discussion








Acknowledgment



References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 10 20 30 40 50 60 70 80 90 1000

102030405060708090

100Se

nsiti

vity



20

40

60

80

Relat

ive H

ULC

RN

A

(b)



0

10

20

30

40

50

60

70

Rela

tive H

ULC

RN

A

0

20

40

60

80

Rela

tive H

ULC

mRN

A


I-II II-III III-IV

(a)

0

10

20

30

40

50

60

70

Healthy controlHCC

0

20

40

60

80

Rela

tive H

ULC

mRN

A

Rela

tive H

ULC

RN

A

HBVminus HBV+Con

HBVminus HBV+

(b)







(a)


119899 Median 119875


20 681 7 2064 lt001 10 2495 lt001 13 4797 lt001

(b)

Normal Tumor (HBV)119873 = 20 119873 = 30

119899 Median 119875


20 681 12 2006 lt001 18 3932 lt001



HULC 220 (10) 17 (14) 813 (62) 1010 (100)



HULC 220 (10) 312 (25) 1618 (90)


4 Discussion








Acknowledgment



References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)


119899 Median 119875


20 681 7 2064 lt001 10 2495 lt001 13 4797 lt001

(b)

Normal Tumor (HBV)119873 = 20 119873 = 30

119899 Median 119875


20 681 12 2006 lt001 18 3932 lt001



HULC 220 (10) 17 (14) 813 (62) 1010 (100)



HULC 220 (10) 312 (25) 1618 (90)


4 Discussion








Acknowledgment



References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article plasma hulc as a promising novel...

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