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Clinical Biochemistry xxx (2014) xxx–xxx

CLB-08710; No. of pages: 3; 4C:

Contents lists available at ScienceDirect

Clinical Biochemistry

j ourna l homepage: www.e lsev ie r .com/ locate /c l inb iochem

Short Communication

Inverse correlation of ribosomal protein S27A and multifunctionalprotein YB-1 in hepatocellular carcinoma

FVinoth Prasanna Gunasekaran, Mathan Ganeshan ⁎Department of Biomedical Science, School of Basic Medical Sciences, Bharathidasan University, Tiruchirappalli 620024, India

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⁎ Corresponding author.E-mail addresses: vinothprasannagm@gmail.com (V.P

mathan@bdu.ac.in (M. Ganeshan).

http://dx.doi.org/10.1016/j.clinbiochem.2014.05.0040009-9120/© 2014 The Canadian Society of Clinical Chem

Please cite this article as: Gunasekaran VP,hepatocellular carcinoma, Clin Biochem (201

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RReceived 6 November 2013Received in revised form 29 April 2014Accepted 3 May 2014Available online xxxx

Keywords:YB-1RPS27AHepatocellular carcinomaLiver cirrhosisTissue microarray

Objective: The detection of possible correlation between ribosomal protein RPS27A andmultifunctional YB-1expression in hepatocellular carcinoma (HCC).

Design andmethods: Tissue microarray slides containing totally 80 cores with 19 tissues of HCC, 1 tissue ofhepatocholangiocarcinoma, 10 tissues of liver cirrhosis and 10 normal liver tissues in duplicates were analyzedfor expression of RPS27A and YB-1 by immunohistochemistry.

Results: Among each of 10 LC and normal liver tissues all (100%) showed RPS27A positive expressionbut only 11 out of 19 HCC tissues (57.89%) showed RPS27A positive expression with significant differ-ence compared (P b 0.05) with both LC and normal tissues. We found positive expression of YB-1 in 17tissues out of 19 HCC tissues (89.47%) but only 4 tissues out of each 10 LC as well as normal liver tissuesshowed positive expression with significant (P b 0.01) difference compared to HCC tissues. A statistical-ly significant inverse weak correlation (rho = −0.293) between YB-1 expression and RPS27A expres-

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Conclusion: The present investigation concludes that the ribosomal protein RPS27A was down-regulated in viral induced HCC patients. RPS27A expression was found to have a weak inverse correlationwith overexpression of multifunctional protein YB-1 in HCC tissues. This study opens up a new windowfor YB-1–RPS27A axis in HCC.

C© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

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RRIntroduction

Hepatocellular carcinoma (HCC) is the sixthmost commonmalignan-cy and third leading neoplastic cause ofmortalitiesworldwide. Hepatitis Bvirus (HBV), hepatitis C virus (HCV), alcoholism and aflatoxicosis are themajor etiological factors for HCC. Infection of HBV and HCV induces livercirrhosis which subsequently progresses to HCC but still the mechanismof HCC progression is poorly understood. The cis/trans activation of vari-ous cellular pathways by the viral core proteins has been observed asthe imperative origin for HCC development and progression. Microarrayanalysis of HCC sample shows up and down-regulation of various cellulargenes involved in the cell proliferation, apoptosis and multidrug resis-tance [1].

A cold shockdomain family protein, Y box binding protein (YB-1) pos-sesses variety of biological activities such as cell proliferation, signalingcascade mechanism, apoptosis, cell growth, embryogenesis and carcino-genesis through its ability to modulate the expression of various cellularproteins at transcriptional, translational and post-translational levels [2].Overexpression of YB-1has been associatedwith themultidrug resistance

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Ganeshan M, Inverse correla4), http://dx.doi.org/10.1016

through its multilevel activation of MDR gene family in many cancerssuch as breast, renal and colorectal cancer [3]. YB-1 is involved in the spa-tial organization of messenger ribonucleoproteins and regulates its ownmRNA translation (autoregulation). Moreover, at higher concentrationsYB-1 inhibits translation of other mRNAs through displacement of eu-karyotic translation initiation factor 4G (eIF4G) and eIF4E by bindingwith the close proximity to the mRNA cap structure [4]. Accumulationstudies have revealed a close association of YB-1 and ribosomal proteinssuch as RPS4X, RPL5, RPS3A, RPS5, RPL18A and RPL23A in differenttypes of cancers [5].

Currently ribosomal proteins are gathering a great significance as di-agnostic markers and their extra-ribosomal functions have close rela-tion with oncogenesis. Ribosomal proteins are involved in DNA repair,cell cycle arrest, apoptosis and proliferation, and moderately expressedin various cancers [6]. However, enhanced expression of ribosomal pro-teins such as RPL23A, RPL12, RPL27, RPS8, RPL36A and RPL30 has beenreported in HCC [7]. Among these, RPS27A is a ubiquitin C-terminalextension protein, member of 40s subunit and involved in p53activation through MDM2 in response to ribosomal stress [8]. RPS27Ahas been reported as an early growth response gene having overexpres-sion in colorectal cancer but not in gastric cancer [9]. Interestingly,transcriptomic profile of X15-myc transgenic mice for HCC has revealedthat the two multi-potential genes RPS27A and YB-1 are expressed in

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high frequencies [10]. In this context the present study was designed toassess the expression of multifunctional protein YB-1 and RPS27A andtheir correlation in HCC.

Methodology

Tissue microarray and immunohistostaining

Two replicate tissue microarrays were used for this retrospec-tive analysis of YB-1 and RPS27A expression. Tissue microarrayslides containing totally 80 cores with 19 tissues of HCC, 1 tissueof hepatocholangiocarcinoma, 10 tissues of each liver cirrhosis(LC) and normal liver in duplicates were purchased from USBIOMAX, catalogue no. LV8013 (tissues were collected under highethical standard with HIPPA approval). Out of 19 HCC tissues 9were HCV positive and 10 were HBV positive. All the 10 liver cir-rhosis specimens were HBV positive. For immunohistochemistry,anti-YB-1 antibody was purchased from Abcam (catalogue no.ab12148) and RPS27A specific antibody was purchased fromNovus Biologicals (catalogue no. H00006233-D01P). Immunohis-tochemical staining was carried out using polymer labeling tech-nique. Antigen retrieval was carried out in a pressure cooker with10 mM Citra solution for 15 min and endogenous peroxidase wasblocked by using 3% hydrogen peroxide in methanol at room tem-perature for 10 min. Slide were then washed with PBS briefly andincubated with primary antibody YB-1 (dilution 1:5000) andRPS27A (dilution 1:70) for 60 min. After washing with PBS the sec-tions were incubated with the polymer for 30 min followed bywashing with PBS. Diaminobenzidine (DAB) was used as the chro-mogen in hydrogen peroxide for 10 min after which the sectionswere counterstained with hemotoxylin and mounted.

Microscopic analyses and scoring of sections

The stained sections were analyzed by using Nikon eclipse 50imicro-scope with attached camera. Positive staining was indicated by browncolor. Photomicrographs were taken for all the sections and scoring wasdone based on the intensity of the staining. The stained cells were catego-rized into four groups i.e. 0 (not stained), 1+ (≤30% intensity; weaklystained), 2+ (30% to 60% intensity; moderately stained) and 3+ (60%to 100% intensity; strongly stained). The sections containing more than

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Fig. 1. Representative images of the immunohistochemically stained tissues from hepatocellulaof RPS27a (A–C), and YB-1 (D–F). Compound arrows, solid arrows, small dashed arrows, and l

Please cite this article as: Gunasekaran VP, Ganeshan M, Inverse correlahepatocellular carcinoma, Clin Biochem (2014), http://dx.doi.org/10.1016

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50% of cells with a score of 2+ and/or 3+ were considered as positive.All the analyses and scorings were done by a blind investigator whowas unaware of the pathological status of TMA slide.

Statistical analysis

Statistical analysis of categorical variables was performed by usingFisher's exact test. Two tailed Student's t-test was used to compare thedifferences between groups. All statistical analyses were made usingGraphPad PRISM Software; GraphPad Software, Inc., CA, USA. Pearson'scorrelation coefficient was used to find the correlation. Results weregiven asmean± SE and values with P b 0.05 were considered as statis-tically significant.

Results

Among each of 10 LC and normal liver tissues all (100%) showedRPS27A positive expression but only 11 out of 19 HCC tissues (57.89%)showed RPS27A positive expression with significant difference com-pared (P b 0.05) with both LC and normal tissues (SupplementaryTable 1). We found positive expression of YB-1 in 17 tissues out of 19HCC tissues (89.47%) but only 4 tissues out of each 10 LC, aswell as, nor-mal liver tissues showed positive expression with significant (P b 0.01)difference compared to HCC tissues (Fig. 1). A statistically significant in-verse weak correlation (rho= −0.293) between YB-1 and RPS27A ex-pression was found (Fig. 2).

Discussion

To the best of our knowledge, this is the first clinical report of ribo-somal protein RPS27A expression and its correlation with YB-1 proteinin HCC tissues. Apart from the structural organization of ribosomes,RPS27A plays an important role in pathogenesis of various cancersand its overexpression has been reported in early stages of the colorec-tal cancer [9]. Likewise, Grace et al. reported a comparative higher he-patic expression of RPS27A in X15-myc transgenic mice (a model forHCC) as compared to normal mice. Moreover, silencing of RPS27Ashowed a remarkable reduction in cell proliferation by cell cycle arrestat G0/G1 phase in HBx transfected HCC cell line [12]. On the contrary,Sun et al. reported that knockdown of RPS27A in cell lines attenuatescell cycle arrest by inhibiting p53 activation via MDM2 [8].

r carcinoma (A, D), liver cirrhosis (B, E) and normal human liver (C, F) showing expressionarge dashed arrows represent the scores 0, 1+, 2+ and 3+ respectively.

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Fig. 2. YB-1 and RPS27A correlation in hepatocellular carcinoma.

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However, in the present study RPS27A was down-regulated in HCCtissues as compared to LC and normal liver tissues. The contentiousexpression of RPS27A in previous studies of Grace et al. in X15-myctransgenic mice might be driven by the sustained overexpression ofHBX and c-myc, unlike the clinical samples. The down-regulation ofRPS27A in present study is concurrent with the decreased p53 activa-tion and cell cycle arrest in HCC [13]. Moreover, extensive molecularinvestigation might be required to explore the exact role of RPS27A inHCC progression. The down-regulation of RPS27A in HCC may be dueto the transcriptional and/or translational regulation of various cellularor viral proteins which are moderately expressed in HCC. By analysisof ~1 kbupstream region of human RPS27A genewe found that the pro-moter contains several Y box sequence (data not shown), which is thetarget for multifunctional YB-1 protein. Hence, we further focused onthe correlation between RPS27A and YB-1 in HCC tissues. Overexpres-sion of YB-1 has been suggested as a marker for multi-drug resistancein various cancers such as prostate, lung and breast cancer [3]. Similarly,in the present study we observed overexpression of YB-1 in HCC as com-pared to normal and LC patients.Moreover, therewas aweak inverse cor-relation in YB-1 and RPS27A expression in HCC tissues.

YB-1 has a role in down-regulation of several proteins at tran-scriptional level by binding with the Y Box region of the promoterand/or with non-specific region [2]. Apart from the transcriptionalregulation, YB-1 controls translation of various mRNA due to itsrelative concentration with the mRNA. A low YB-1/mRNA ratiostimulates whereas a high YB-1/mRNA ratio suppresses proteinsynthesis [4].

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Conclusion

From this study it is speculated that YB-1 down-regulates RPS27A at atranscription level and/or the overexpression of YB-1 relative to RPS27AmRNAmay attenuate the translational expression of RPS27A. The presentinvestigation concludes that the ribosomal protein RPS27A was down-regulated in virus induced HCC patients. Further, an inverse correlationof RPS27A with the up-regulation of multifunctional protein, YB-1 inHCC opens up a new window for future researches in YB-1–RPS27A axisin HCC.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.clinbiochem.2014.05.004.

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Conflict of interest

None.

Please cite this article as: Gunasekaran VP, Ganeshan M, Inverse correlahepatocellular carcinoma, Clin Biochem (2014), http://dx.doi.org/10.1016

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ROUncited references

[11,14]

Acknowledgement

The authors are grateful to the Department of Biotechnology(DBT), India for financial support to the project (Grant No. BT/PR6112/GBD/27/371/2012) and Dr. P. Selvamani, Assistant Profes-sor, Department of Pharmaceutical Technology, BIT Campus, AnnaUniversity, Tiruchirappalli for providing necessary microscopefacility.

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