research article severity of hepatitis c virus (genotype-3...

Research ArticleSeverity of Hepatitis C Virus (Genotype-3) Infection PositivelyCorrelates with Circulating MicroRNA-122 in Patients Sera

Subodh Kumar1 Yogesh Kumar Chawla2 Sujata Ghosh1 and Anuradha Chakraborti1

1 Department of Experimental Medicine and Biotechnology Post Graduate Institute of Medical Education and ResearchChandigarh 160012 India

2Department of Hepatology Post Graduate Institute of Medical Education and Research Chandigarh 160012 India

Correspondence should be addressed to Anuradha Chakraborti superoxidesifycom

Received 3 November 2013 Revised 6 January 2014 Accepted 9 January 2014 Published 18 February 2014

Academic Editor Esperanza Ortega

Copyright copy 2014 Subodh Kumar et alThis 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

Introduction Hepatitis C virus (genotype-3) causes acute and chronic hepatitis infection predomination in India The infectiousphase of the virus requires various host factors for its survival and subsequent viral particle production Small RNAmolecules likemicroRNA-122 (miR-122) are one such factor mostly present in the liver and play a supportive role in viral replicationObjective Inthis study diagnostic potential of miR-122 is evaluated in the sera of chronic hepatitis C patientsMethods miRNAs were isolatedfrom the sera samples of patients as well as controls and miR-122 expression was quantified by real-time PCR Results A significantaugmentation was observed in the level of circulating miR-122 (median level 066 versus 029 119875 = 0001) in patients compared tocontrols with ROC value of 0929 plusmn 0034 (119875 lt 0001) Interestingly miR-122 level also depicted a significant positive correlationwith serum ALT (119903 = 053) AST (119903 = 044) and viral load (119903 = 052) Conclusion The study thus unveiled the role of miR-122 as aplausible diagnostic biomarker during HCV genotype-3 infection in India

1 Introduction

Hepatitis C virus (HCV) is the leading cause of chronichepatitis C (CHC) infection which infects approximately3 of the world population [1] HCV is a positive strandedRNA virus with a genome size of 96 Kb that frequentlyreplicates and survives in the liver tissue HCV is dividedinto six distinct genotypes and multiple subtypes distributedthroughout the world In India approximately 12-13 millionpeople suffer predominantly fromHCV genotype-3 infection(634) followed by genotype-1 (31) and genotype-2 (56)[2 3] In North India more than 76 of CHC cases werespecifically associatedwith genotype-3 [4]The viral infectionis diagnosed by detection of anti-HCV antibodies and HCVRNA in the patientrsquos sera The pathogenic impact of HCV onthe human liver is determined by the routine liver functiontest (LFTs) particularly with alanine transaminase (ALT) andaspartate transaminase (AST) level However an increasedlevel of ALT and AST has been reported during muscle

inflammation and in brain disorders respectively [5] Dufouret al observed a normal ALT level usually in one-thirdof hepatitis C virus infected patients [6] Such uncertainbehavior of these enzymes reduces their prognostic potentialin case of HCV infection

HCV survival and subsequent viral particle produc-tion need various host factors such as CD81 TGF-120573 andCypA proteins as well as microRNAs (miRNAs) specificallymicroRNA-122 (miR-122) [7 8] The expression of thesefactors is altered in host cells during viral infection miRNAsare important regulators of a wide range of physiologicaland pathological processes such as cellular developmentmetabolism viral infection and cancer [9ndash11] miR-122 isa liver specific miRNA whose biological functions are tomaintain liver homeostasis cholesterol metabolism and fetalliver development [12] It also regulates HCV replicationpositively in cultured human hepatoma cells which stablyexpresses HCV replicon by binding to the 51015840UTR of the HCVgenome [8] Furthermore inhibition of miR-122 expression

Hindawi Publishing CorporationDisease MarkersVolume 2014 Article ID 435476 6 pageshttpdxdoiorg1011552014435476

2 Disease Markers

by antisense oligonucleotides reduces the HCV replicationand translation yet the clinical impact of miR-122 as diag-nostic or prognostic molecule is largely unknown [11 13]

Recently few studies have indicated that miR-122 exhibitsdifferent expression profile in the liver and the sera ofhepatitis C patients [11 14] However limited informationis available regarding HCV genotype-3 infection and statusof miR-122 in patients sera as well as its association withviral and the host parameters Hence in the present studyexpression level of miR-122 was assessed in HCV genotype-3infection alongwith other host parameters in order to explorethe association of miR-122 with the disease severity

2 Materials and Methods

21 Study Subjects Blood samples were collected fromchronic hepatitis C patients attending the liver clinic ofPGIMER Chandigarh India after receiving their informedconsent Patients positive for anti-HCV antibodies highHCV genotype-3 viral load (gt10000 IUmL) and withoutany antiviral treatment were enrolled in the study (Table 1)HCV viral load and genotype-3 were already estimated bythe clinical laboratories Blood samples were collected fromhealthy individual to be used as control This study wasapproved by the Institute Ethical Committee (Memo no7969-PG-1TRG-0917567)

22 Detection of Anti-HCV Antibody in Patientrsquos SerumInitially 2mL of blood sample was collected from each CHCpatient in plain vials and serum was separated within 2 hrs ofblood collection by centrifugation at 2000 rpm for 10min at4∘C Serum was collected in a fresh tube and stored at minus80∘Ctill further use HCV infection was detected by third gener-ation SD HCV-ELISA 30 kit containing recombinant HCVantigens for core NS3 NS4 and NS5 proteins (StandardDiagnostics Inc Korea) following manufacturerrsquos protocol

23 Liver Function Tests (LFT) of Patient Samples Sampleswere processed for liver function test like ALT AST alkalinephosphatase (ALP) serum bilirubin albumin and globulinusing specific kits (Swemed Diagnostics Bangalore India)according to manufacturerrsquos instructions in fully AutomatedBiochemical Analyzer (Hitachi Tokyo Japan) (Table 1)

24 miRNA Isolation and Detection miRNAs were isolatedfrom the serum samples by using MagMAX viral RNAisolation kit (Ambion USA) as per manufacturerrsquos instruc-tions [15] Briefly 400 120583L of serum sample was mixed withmagnetic beads and separated on the magnetic stand 20 120583Lof elution buffer was added to elute the purified miRNA ina nuclease-free container and further detected on the 12denaturing urea poly acrylamide gel electrophoresis (PAGE)Urea (48 gm) with 40 acrylamide was dissolved in 10x TBEin a final volume up to 10mL with double distilled water Thegel was prepared by mixing above solution with 10 APS and10 120583L of TEMED miRNA samples were denatured in a waterbath and run on urea PAGE at 240 Volt and 40Amp The gel

Table 1 Patients clinical profile

Factors CHC patients group(119873 = 25)

Healthy control group(119873 = 25)

Sex (malefemale) 196 169Age (years) 3808 plusmn 1081 3253 plusmn 963

Anti-HCV antibody(+minus) 250 025

HCV genotype-3(+minus) 250 025

HCV viral load(IUmL) 618 plusmn 1359 (106)

ALT (IUL)1 8656 plusmn 4690

AST (IUL)1 8248 plusmn 4876

Serum bilirubin(mg)2 079 plusmn 069

ALP (IU)3 22748 plusmn 11820

Albumin (gm)4 421 plusmn 102

Globulin (gm)5 355 plusmn 057

[Reference values 1(2ndash40 IUL) 2(02ndash08mg) 3(98ndash306 IU) 4(35ndash5 gm) and 5(20ndash30 gm)]

was stained with ethidium bromide (05120583gmL in 1x TBE)and visualized under UV transilluminator

25 Quantification of miR-122 Expression by Real-Time PCR

251 Polyadenylation of miRNAs Each miRNA moleculewas elongated by polyadenylation reaction at 31015840 end usingmiRNA Ist strand cDNA synthesis kit (Stratagene USA)miRNA (1 120583g) was incubated at 37∘C for 30min in a reactionmixture containing 5x poly A polymerase buffer rATP(10mM) E coli poly A polymerase and RNase-freeH

2OThe

reaction was terminated at 95∘C for 5min and immediatelyproceeded for Ist strand cDNA synthesis

252 cDNA Synthesis of Poly ATailedmiRNA cDNA synthe-sis reaction mixtures were prepared by adding 1x RT bufferdNTPs (100mM) RT adaptor primer (10 120583M) RNase blockpolyadenylated RNA and RNase-free H

2OThe annealing of

adaptor primer to poly A tail creates a universal sequencetag followed by its incorporation at the 51015840 end of cDNAReaction mixture was first incubated at 55∘C for 5min andthen at 25∘C for 15min Thereafter it was transferred to 42∘Cand incubated for 30min to allow reverse transcription Thereaction was terminated at 95∘C for 5min Finally 20120583LRNase-free water was added to each reaction and placed onice for immediate use

253 Real-Time PCR for miR-122 The expression of miR-122 was quantified by real-time PCR (qRT-PCR) analysisusing high-specificity miRNAQPCR core reagent kit (Strata-gene USA) containing 10x core PCR buffer 50mM MgCl

2

20mM dNTP mix 20x EvaGreen dye PCR enzyme blendnuclease-free PCR H

2O universal reverse primer (3125 120583M)

(51015840-CTCAACTGGTGTCGTGGA-31015840) and miR-122 specific

Disease Markers 3

forward primer (51015840-TGGAGTGTGACAATGGTGTTTGT-31015840) The highly conserved U6 small nuclear RNA (snRNA)was taken as internal control (forward 51015840-CGCTTCGGC-AGCACATATACTAA-31015840 and reverse 51015840-TATGGAACG-CTTCACGAATTTGC-31015840) [16] Reaction conditions were95∘C for 5min to denature DNA templates followed by40 cycles of 95∘C for 10 sec denaturation 58∘C for 15 secannealing and 72∘C for 25 sec extension The relative levelsof serummiR-122 in patients versus controlswere determinedin terms of their fold change using the formula (2minusΔΔCt) [17]where ΔCt was calculated by subtracting Ct of U6 from Ctof miR-122 and ΔΔCt value was obtained by subtracting ΔCtof miR-122 in controls from the ΔCt of miR-122 in patients[10]TheqRT-PCRwas performed in triplicates and datawereexpressed as the mean plusmn SD from separate experiments

26 Statistical Analysis Data was analyzed using the SPSSsoftware version 170 (USA) The level of miR-122 inCHC patients and controls was calculated using the non-parametric Mann-whitney U test The receiver operatingcharacteristic (ROC) curve was plotted by differentiatingCHCpatients fromhealthy controls [18]Thediagnostic valueof miR-122 for detection of CHC patients was determinedaccording to the area under the ROC curve (AUC) value thesensitivity and the specificity The nonparametric statisticalsignificance for correlation was determined using Spearmanrsquosnonparametric rank testThe correlation coefficients (119903) werecalculated using Spearman correlation 119875 values lt 005 wereconsidered to be significant

3 Results

31 Clinical Profile of Patients The treatment naıve CHCpatients and healthy controls enrolled for the study are sum-marized in Table 1 The average ages of CHC patient groupand control groups were 3808 plusmn 1081 and 3253 plusmn 963 yearsrespectively Patients (119899 = 25) were positive for anti-HCVantibodywith genotype-3 while none of the healthy volunteercarriedHCVViral loadwas high in patients (gt10000 IUmL)with a mean of 618 times 106 (IUmL) Similarly mean values ofALT AST and globulin were higher compared to referencevalue

32 miR-122 Expression in HCV Genotype-3 Infected PatientrsquosSera miRNAs were isolated from the sera samples of CHCpatients (119899 = 25) and analyzed by qRT-PCR Serum U6snRNA and miR-122 expression were detected on the 12denaturing UREA-PAGE (Figures 1(a) and 1(b)) U6 snRNAwas selected as internal references for quantification of serummiRNAs because it is ubiquitously expressed in patientsand even in controls miR-122 expression was quantified bysubtracting the Ct value of U6 from the Ct value of miR-122in patients and controls The relative expression level of miR-122 was determined in both groups and the median level ofmiR-122 was found to be significantly high (066 versus 029119875 = 0001) in patients than controls (Figure 2(a)) confirmingelevation of circulating miR-122 in serum of patients

M

(a)

(b)

1 2 3 4 5 6

75bp

70bp100 bp

Figure 1 12 denaturing UREA-PAGE showing expression (Lanes1ndash6) of (a) U6 snRNA (75 bp) and (b) miR-122 expression (70 bp)detected in different serum samples 119872 = 50 bp DNA ladder(Fermentas USA)

33 miR-122 as DiagnosticMarker To evaluate the diagnosticpotential of circulating miR-122 in CHC patients the areaunder curve (AUC) value from ROC curve was determinedin patients as compared to healthy volunteers The ROCcurve was plotted according to the ΔCt value of the miR-122 in patientrsquos versus controls The calculated AUC valuewas up to 0929 plusmn 0034 (119875 lt 0001) (Figure 2(b)) andthe 95 confidence interval was 0861 to 0996 The cut-offvalue was 299 with a sensitivity of 92 (95 confidenceinterval 7397 to 9902) and specificity was 84 (95confidence interval 6262 to 9526) Further the positivepredictive value (PPV) and negative predictive value (NPV)were 8518 and 9130 respectively Thus ROC curveanalysis confirmedmiR-122 as a valuable molecule capable ofdiscriminating the patients and healthy individuals

34 miR-122 Expression Associated with HCV Viral LoadWe investigated the correlation between miR-122 expressionand HCV viral load in the patientrsquos sera Level of miR-122 was calculated in terms of their fold change whichshowed a mean value of 717 plusmn 1125 fold higher expressionin patients as compared to control group Further Spearmanrank correlation coefficient (119903) was determined betweenmiR-122 level and serum HCV viral load (Figure 3(a)) Viral loadof patients 618 plusmn 1359 (106) IUmL was plotted against foldchange in miR-122 which demonstrated a significant positivecorrelation (119903 = 052 95 confidence interval 015 to 076119875 = 0006) Hence the close association of miR-122 withdisease severity in terms of HCV viral load was established

35 miR-122 Expression with Liver Function Tests Spearmanrank correlation (119903) analysis between serum level of miR-122 and liver function parameters was also performed Herevalues of serum ALT (8656 plusmn 4690 IUL) AST (8248plusmn 4876 IUL) ALP (22748 plusmn 11820 IU) serum bilirubin(079 plusmn 069mg) albumin (421 plusmn 102 gm) and globulin(355 plusmn 057 gm) were plotted on 119909-axis and miR-122 foldchange (717 plusmn 1125) values were plotted on 119910-axis The levelof miR-122 was correlated only with serum ALT and ASTactivity with significant positive correlation of (119903 = 05395 confidence interval 015 to 077 119875 = 0009) and (119903 =044 95 confidence interval 0052 to 072 119875 = 0024)

4 Disease Markers

P = 0001

Controls Patients00

05

10

15Re

lativ

e miR

-122

leve

l (lo

g10

)

(a)

00

02

04

06

08

10

00 02 04 06 08 10

Sens

itivi

ty

ROC curve

AUC = 0929

SE = 920

SP = 840

+PV = 8518

minusPV = 9130

miR-1221 minus specificity

Reference line P lt 0001

(b)

Figure 2 (a) Scattered plot diagram showing level of miR-122 expression in CHC patients and controlsThe data are expressed as the mean plusmnSD from separate experiments performed in triplicates Here ΔCt value was normalized by U6 snRNA at log 10 scale which is shown at119910-axisThemedian value of miR-122 level is significantly higher in patients than controls 066 versus 029 (119875 = 0001) (b) Receiver operatingcharacteristic (ROC) curve for miR-122 in 25 chronic hepatitis C patients The diagnostic value of miR-122 was assessed by discriminatingCHC patients from healthy controls based on the ΔCt value SE sensitivity SP specificity +PV positive predictive value and minusPV negativepredictive value

respectively (Figures 3(b) and 3(c)) While other parametersof liver function test did not show any significant correlationAlthough serum HCV viral load showed a positive correla-tion with ALT and AST levels it was insignificant Thereforeassociation of miR-122 with other host parameters furtherconfirmed its decisive role in disease assessment

4 Discussion

HCV infects approximately 18ndash25 of Indian populationaccounting for 15ndash20 of all chronic liver disease and about5ndash10 of hepatocellular carcinoma (HCC) [19 20]The initialdiagnosis of liver diseases is carried out by routine liverfunction tests like serum level of ALT AST ALP bilirubinalbumin globulin and so forth The levels of these enzymesor proteins are altered during hepatic injuries hepatitis Bor C infection liver cirrhosis and HCC [6] Usually inhepatitis C virus infection the levels of both ALT and AST areexamined routinely in the patientrsquos serum however in somecases the level of these enzymes remains unaltered or undernormal value [21] Even though ALT and AST are primarilylocalized in liver their activity also increases heart brainand skeletal muscle disorders [5 22] The activity of ALTand AST is ambiguous as it can be correlated with hepaticand extrahepatic diseases which provoked us to search for analternate biomarker for HCV infection

Dennis and Chiu discovered a new approach for the clin-ical diagnosis of diseases by detecting the circulating nucleicacids in the peripheral blood [23] The circulating form of

mRNA and miRNA also offered another class of molecularmarker in case of various cancers and pathogenic infections[24ndash26] Wang et al in toxic liver injuries reported a frequentincrease of plasma level of miR-122 than that of ALT [27] Asimilar observation in chronic hepatitis B patients has beenreported by Zhang et al [10] Bihrer et al correlated theserum level of miR-122 ALT and necroinflammatory activityinCHCpatients [14] As liver cells contain high concentrationofmiR-122 it might be speculated that during liver injuries orpathogenic infection miRNAs secretion is a strategy of celladaptation for adverse conditions

In North Indian population genotype-3 HCV infectionis more frequent among all HCV genotypes [4] The clinicalsignificance of miR-122 in HCV genotype-3 infected patientshas not been discussed so farOur study is primarily restrictedto HCV genotype-3 virus infection with viral load beyond10000 IUmL Here an elevation in the level of serum miR-122 and a significant positive association between miR-122level andHCVRNAwere observedAlthough there is no suchbiological explanation regarding miR-122 elevation in thepatientsrsquo sera it may be speculated that upon viral infectionmiRNAs might be passively released from the damaged ordying cells While other possible mechanismmight be linkedto the virus itself that can also elicit the secretion of miRNAsfrom host cells to influence the cellular physiology for theirown production [28] These assumptions may be correlatedwith our observations that miR-122 level elevated as theseverity of infection increases Our results are also supportedby the previous study of Morita et al where hepatic miR-122

Disease Markers 5

0 20 40 60 800

10

20

30

40

50

HCV viral load (IUmL)

miR

-122

fold

chan

ge

times106

(r = 052 P = 0006)

(a)

0 50 100 150 200 2500

10

20

30

40

50

Serum ALT level (IUL)

miR

-122

fold

chan

ge

(r = 053 P = 0009)

(b)

50 100 150 200 2500

20

40

60

Serum AST level (IUL)

miR

-122

fold

chan

ge

minus20

(r = 044 P = 0024)

(c)

Figure 3 Scattered plot diagrams showing the significant positive correlation of serummiR-122 level with (a) HCV viral load (b) serumALTlevel and (c) serum AST level

expression was weakly but positively correlated with serumHCV load [11] Similarly we also ascertained the associationof miR-122 level with ALT and additionally with AST activityHowever in patients infected with other HCV genotypes (1and 2) recently it has been reported that serum miR-122level was correlated only with hepatic histology activity index(HAI) score and ALT level but not with HCV viral load andAST activity [29]

The study depicted the correlation of serummiR-122 levelwith HCV viral load ALT and AST activity in patientrsquos seraBesides viral RNA also showed a positive correlation withALT andAST activity it was not significant (data not shown)While HCV viral RNA is significantly associated with miR-122 and likewise miR-122 showed a positive correlation withALT and AST activity These observations further enhancethe diagnostic importance ofmiR-122 Apparently miR-122 isquite stable andmore sensitive in the serumwhereas ALT andAST enzymes are comparatively less stable [30 31]Moreoverquantification of viral RNA is quite expensive and timeconsuming procedure Hence these findings authenticatemiR-122 as a promising biomolecule to investigate HCVinfection and monitor liver dysfunctions

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors are grateful to the Indian Council of MedicalResearch (ICMR) New Delhi for providing Junior andSenior Research Fellowship to Mr Subodh Kumar

References

[1] V Pazienza S Clement P Pugnale et al ldquoGene expressionprofile ofHuh-7 cells expressing hepatitis C virus genotype 1b or3a core proteinsrdquo Liver International vol 29 no 5 pp 661ndash6692009

[2] P K Mishra A Bhargava S Khan N Pathak R P Pundeand S Varshney ldquoPrevalence of hepatitis C virus genotypes andimpact of T helper cytokines in achieving sustained virologicalresponse during combination therapy a study from CentralIndiardquo Indian Journal of Medical Microbiology vol 28 no 4 pp358ndash362 2010

6 Disease Markers

[3] A Chakravarti G Dogra V Verma and A P SrivastavaldquoDistribution pattern of HCV genotypes amp its association withviral loadrdquo Indian Journal ofMedical Research vol 133 no 3 pp326ndash331 2011

[4] B R Das B Kundu R Khandapkar and S Sahni ldquoGeograph-ical distribution of hepatitis C virus genotypes in Indiardquo IndianJournal of Pathology ampMicrobiology vol 45 no 3 pp 323ndash3282002

[5] R A Nathwani S Pais T B Reynolds and N KaplowitzldquoSerum alanine aminotransferase in skeletal muscle diseasesrdquoHepatology vol 41 no 2 pp 380ndash382 2005

[6] D R Dufour J A Lott F S Nolte D R Gretch R S Koffand L B Seeff ldquoDiagnosis and monitoring of hepatic injuryI Performance characteristics of laboratory testsrdquo ClinicalChemistry vol 46 no 12 pp 2027ndash2049 2000

[7] Q Li A L Brass A Ng et al ldquoA genome-wide genetic screenfor host factors required for hepatitis C virus propagationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 38 pp 16410ndash16415 2009

[8] C L Jopling M Yi A M Lancaster S M Lemon and PSarnow ldquoMolecular biology modulation of hepatitis C virusRNA abundance by a liver-specific microRNArdquo Science vol309 no 5740 pp 1577ndash1581 2005

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

[10] Y Zhang Y Jia R Zheng et al ldquoPlasma microRNA-122 asa biomarker for viral- alcohol- and chemical-related hepaticdiseasesrdquoClinical Chemistry vol 56 no 12 pp 1830ndash1838 2010

[11] K Morita A Taketomi K Shirabe et al ldquoClinical significanceand potential of hepatic microRNA-122 expression in hepatitisCrdquo Liver International vol 31 no 4 pp 474ndash484 2011

[12] R Doddapaneni Y K Chawla A Das J K Kalra SGhosh and A Chakraborti ldquoOverexpression of microRNA-122enhances in vitro hepatic differentiation of fetal liver-derivedstemprogenitor cellsrdquo Journal of Cellular Biochemistry vol 114no 7 pp 1575ndash1583 2013

[13] J Chang J-T Guo D Jiang H Guo J M Taylor and TM Block ldquoLiver-specific microRNA miR-122 enhances thereplication of hepatitis C virus in nonhepatic cellsrdquo Journal ofVirology vol 82 no 16 pp 8215ndash8223 2008

[14] V Bihrer M F Rust B Kronenberger et al ldquoSerum miR-122 as a biomarker of necroinflammation in patients withchronic hepatitis C virus infectionrdquo The American Journal ofGastroenterology vol 106 no 9 pp 1663ndash1669 2011

[15] W Zhu W Qin U Atasoy and E R Sauter ldquoCirculatingmicroRNAs in breast cancer and healthy subjectsrdquo BMCResearch Notes vol 2 no 89 pp 1ndash5 2009

[16] H Xu J-H He Z-D Xiao et al ldquoLiver-enriched transcriptionfactors regulate microRNA-122 that targets CUTL1 during liverdevelopmentrdquo Hepatology vol 52 no 4 pp 1431ndash1442 2010

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[18] M H Zweig and G Campbell ldquoReceiver-operating charac-teristic (ROC) plots a fundamental evaluation tool in clinicalmedicinerdquo Clinical Chemistry vol 39 no 4 pp 561ndash577 1993

[19] S S Hissar A Goyal M Kumar et al ldquoHepatitis C virusgenotype 3 predominates in North and Central India andis associated with significant histopathologic liver diseaserdquoJournal of Medical Virology vol 78 no 4 pp 452ndash458 2006

[20] S P D Ponamgi M Chandra Y N Kumar et al ldquoGenotypeanalysis and assessment of antigenic sensitivity for recombinantHCV proteins by indigenous SIBA for detection of hepatitisC virus infection a comparison with 3rd EIA and RT-PCRrdquoIndian Journal of Biotechnology vol 8 no 1 pp 33ndash39 2009

[21] M G Ghany D B Strader D L Thomas and L B SeeffldquoDiagnosis management and treatment of hepatitis C anupdaterdquo Hepatology vol 49 no 4 pp 1335ndash1374 2009

[22] J Ozer M Ratner M Shaw W Bailey and S Schomaker ldquoThecurrent state of serumbiomarkers of hepatotoxicityrdquoToxicologyvol 245 no 3 pp 194ndash205 2008

[23] L Y Dennis and R W Chiu ldquoPrenatal diagnosis progressthrough plasma nucleic acidsrdquo Nature Reviews Genetics vol 8no 1 pp 71ndash77 2007

[24] S C C Wong S F E Lo M T Cheung et al ldquoQuantificationof plasma 120573-catenin mRNA in colorectal cancer and adenomapatientsrdquo Clinical Cancer Research vol 10 no 5 pp 1613ndash16172004

[25] C L Bartels andG J Tsongalis ldquoMicroRNAs novel biomarkersfor human cancerrdquo Clinical Chemistry vol 55 no 4 pp 623ndash631 2009

[26] HMHeneghanNMiller A J Lowery K J Sweeney J Newelland M J Kerin ldquoCirculating microRNAs as novel minimallyinvasive biomarkers for breast cancerrdquo Annals of Surgery vol251 no 3 pp 499ndash505 2010

[27] K Wang S Zhang B Marzolf et al ldquoCirculating microRNAspotential biomarkers for drug-induced liver injuryrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 106 no 11 pp 4402ndash4407 2009

[28] O Waidmann V Bihrer T Pleli et al ldquoSerum microRNA-122levels in different groups of patients with chronic hepatitis Bvirus infectionrdquo Journal of Viral Hepatitis vol 19 no 2 pp e58ndashe65 2012

[29] TH Su C-H Liu C-J Liu C-L Chen T-T Ting T-C Tsenget al ldquoSerum microRNA-122 level correlates with virologicresponse to pegylated interferon therapy in chronic hepatitis CrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 19 pp 7844ndash7849 2013

[30] B L Boyanton Jr and K E Blick ldquoStability studies of twenty-four analytes in human plasma and serumrdquo Clinical Chemistryvol 48 no 12 pp 2242ndash2247 2002

[31] J S McDonald D Milosevic H V Reddi S K Grebe and AA Schimnich ldquoAnalysis of circulatingmicroRNA preanalyticaland analytical challengesrdquo Clinical Chemistry vol 57 no 6 pp833ndash840 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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Behavioural Neurology

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

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Disease Markers

by antisense oligonucleotides reduces the HCV replicationand translation yet the clinical impact of miR-122 as diag-nostic or prognostic molecule is largely unknown [11 13]

Recently few studies have indicated that miR-122 exhibitsdifferent expression profile in the liver and the sera ofhepatitis C patients [11 14] However limited informationis available regarding HCV genotype-3 infection and statusof miR-122 in patients sera as well as its association withviral and the host parameters Hence in the present studyexpression level of miR-122 was assessed in HCV genotype-3infection alongwith other host parameters in order to explorethe association of miR-122 with the disease severity

2 Materials and Methods

21 Study Subjects Blood samples were collected fromchronic hepatitis C patients attending the liver clinic ofPGIMER Chandigarh India after receiving their informedconsent Patients positive for anti-HCV antibodies highHCV genotype-3 viral load (gt10000 IUmL) and withoutany antiviral treatment were enrolled in the study (Table 1)HCV viral load and genotype-3 were already estimated bythe clinical laboratories Blood samples were collected fromhealthy individual to be used as control This study wasapproved by the Institute Ethical Committee (Memo no7969-PG-1TRG-0917567)

22 Detection of Anti-HCV Antibody in Patientrsquos SerumInitially 2mL of blood sample was collected from each CHCpatient in plain vials and serum was separated within 2 hrs ofblood collection by centrifugation at 2000 rpm for 10min at4∘C Serum was collected in a fresh tube and stored at minus80∘Ctill further use HCV infection was detected by third gener-ation SD HCV-ELISA 30 kit containing recombinant HCVantigens for core NS3 NS4 and NS5 proteins (StandardDiagnostics Inc Korea) following manufacturerrsquos protocol

23 Liver Function Tests (LFT) of Patient Samples Sampleswere processed for liver function test like ALT AST alkalinephosphatase (ALP) serum bilirubin albumin and globulinusing specific kits (Swemed Diagnostics Bangalore India)according to manufacturerrsquos instructions in fully AutomatedBiochemical Analyzer (Hitachi Tokyo Japan) (Table 1)

24 miRNA Isolation and Detection miRNAs were isolatedfrom the serum samples by using MagMAX viral RNAisolation kit (Ambion USA) as per manufacturerrsquos instruc-tions [15] Briefly 400 120583L of serum sample was mixed withmagnetic beads and separated on the magnetic stand 20 120583Lof elution buffer was added to elute the purified miRNA ina nuclease-free container and further detected on the 12denaturing urea poly acrylamide gel electrophoresis (PAGE)Urea (48 gm) with 40 acrylamide was dissolved in 10x TBEin a final volume up to 10mL with double distilled water Thegel was prepared by mixing above solution with 10 APS and10 120583L of TEMED miRNA samples were denatured in a waterbath and run on urea PAGE at 240 Volt and 40Amp The gel

Table 1 Patients clinical profile

Factors CHC patients group(119873 = 25)

Healthy control group(119873 = 25)

Sex (malefemale) 196 169Age (years) 3808 plusmn 1081 3253 plusmn 963

Anti-HCV antibody(+minus) 250 025

HCV genotype-3(+minus) 250 025

HCV viral load(IUmL) 618 plusmn 1359 (106)

ALT (IUL)1 8656 plusmn 4690

AST (IUL)1 8248 plusmn 4876

Serum bilirubin(mg)2 079 plusmn 069

ALP (IU)3 22748 plusmn 11820

Albumin (gm)4 421 plusmn 102

Globulin (gm)5 355 plusmn 057

[Reference values 1(2ndash40 IUL) 2(02ndash08mg) 3(98ndash306 IU) 4(35ndash5 gm) and 5(20ndash30 gm)]

was stained with ethidium bromide (05120583gmL in 1x TBE)and visualized under UV transilluminator

25 Quantification of miR-122 Expression by Real-Time PCR

251 Polyadenylation of miRNAs Each miRNA moleculewas elongated by polyadenylation reaction at 31015840 end usingmiRNA Ist strand cDNA synthesis kit (Stratagene USA)miRNA (1 120583g) was incubated at 37∘C for 30min in a reactionmixture containing 5x poly A polymerase buffer rATP(10mM) E coli poly A polymerase and RNase-freeH

2OThe

reaction was terminated at 95∘C for 5min and immediatelyproceeded for Ist strand cDNA synthesis

252 cDNA Synthesis of Poly ATailedmiRNA cDNA synthe-sis reaction mixtures were prepared by adding 1x RT bufferdNTPs (100mM) RT adaptor primer (10 120583M) RNase blockpolyadenylated RNA and RNase-free H

2OThe annealing of

adaptor primer to poly A tail creates a universal sequencetag followed by its incorporation at the 51015840 end of cDNAReaction mixture was first incubated at 55∘C for 5min andthen at 25∘C for 15min Thereafter it was transferred to 42∘Cand incubated for 30min to allow reverse transcription Thereaction was terminated at 95∘C for 5min Finally 20120583LRNase-free water was added to each reaction and placed onice for immediate use

253 Real-Time PCR for miR-122 The expression of miR-122 was quantified by real-time PCR (qRT-PCR) analysisusing high-specificity miRNAQPCR core reagent kit (Strata-gene USA) containing 10x core PCR buffer 50mM MgCl

2

20mM dNTP mix 20x EvaGreen dye PCR enzyme blendnuclease-free PCR H

2O universal reverse primer (3125 120583M)

(51015840-CTCAACTGGTGTCGTGGA-31015840) and miR-122 specific

Disease Markers 3



3 Results



M

(a)

(b)

1 2 3 4 5 6

75bp

70bp100 bp





4 Disease Markers

P = 0001

Controls Patients00

05

10

15Re

lativ

e miR

-122

leve

l (lo

g10

)

(a)

00

02

04

06

08

10

00 02 04 06 08 10

Sens

itivi

ty

ROC curve

AUC = 0929

SE = 920

SP = 840

+PV = 8518

minusPV = 9130



(b)



4 Discussion





Disease Markers 5

0 20 40 60 800

10

20

30

40

50


miR

-122

fold

chan

ge

times106

(r = 052 P = 0006)

(a)

0 50 100 150 200 2500

10

20

30

40

50


miR

-122

fold

chan

ge

(r = 053 P = 0009)

(b)

50 100 150 200 2500

20

40

60


miR

-122

fold

chan

ge

minus20

(r = 044 P = 0024)

(c)






Acknowledgment


References



6 Disease Markers



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 3



3 Results



M

(a)

(b)

1 2 3 4 5 6

75bp

70bp100 bp





4 Disease Markers

P = 0001

Controls Patients00

05

10

15Re

lativ

e miR

-122

leve

l (lo

g10

)

(a)

00

02

04

06

08

10

00 02 04 06 08 10

Sens

itivi

ty

ROC curve

AUC = 0929

SE = 920

SP = 840

+PV = 8518

minusPV = 9130



(b)



4 Discussion





Disease Markers 5

0 20 40 60 800

10

20

30

40

50


miR

-122

fold

chan

ge

times106

(r = 052 P = 0006)

(a)

0 50 100 150 200 2500

10

20

30

40

50


miR

-122

fold

chan

ge

(r = 053 P = 0009)

(b)

50 100 150 200 2500

20

40

60


miR

-122

fold

chan

ge

minus20

(r = 044 P = 0024)

(c)






Acknowledgment


References



6 Disease Markers



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















4 Disease Markers

P = 0001

Controls Patients00

05

10

15Re

lativ

e miR

-122

leve

l (lo

g10

)

(a)

00

02

04

06

08

10

00 02 04 06 08 10

Sens

itivi

ty

ROC curve

AUC = 0929

SE = 920

SP = 840

+PV = 8518

minusPV = 9130



(b)



4 Discussion





Disease Markers 5

0 20 40 60 800

10

20

30

40

50


miR

-122

fold

chan

ge

times106

(r = 052 P = 0006)

(a)

0 50 100 150 200 2500

10

20

30

40

50


miR

-122

fold

chan

ge

(r = 053 P = 0009)

(b)

50 100 150 200 2500

20

40

60


miR

-122

fold

chan

ge

minus20

(r = 044 P = 0024)

(c)






Acknowledgment


References



6 Disease Markers



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 5

0 20 40 60 800

10

20

30

40

50


miR

-122

fold

chan

ge

times106

(r = 052 P = 0006)

(a)

0 50 100 150 200 2500

10

20

30

40

50


miR

-122

fold

chan

ge

(r = 053 P = 0009)

(b)

50 100 150 200 2500

20

40

60


miR

-122

fold

chan

ge

minus20

(r = 044 P = 0024)

(c)






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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