hepatitis b virus x protein-induced serine protease...

Research ArticleHepatitis B Virus X Protein-Induced Serine ProteaseInhibitor Kazal Type 1 Is Associated with the Progression ofHBV-Related Diseases

Chengliang Zhu 1 Huan Han1 Jie Li2 Limin Xu2 Fang Liu3

KailangWu3 and Xinghui Liu 2

1Department of Clinical Laboratory Renmin Hospital of Wuhan University Wuhan Hubei 430060 China2Department of Clinical Laboratory Shanghai Gongli Hospital e Second Military Medical UniversityPudong New Area Shanghai China3e State Key Laboratory of Virology College of Life Sciences Wuhan University Wuhan Hubei 430072 China

Correspondence should be addressed to Xinghui Liu syliuxh163com

Received 23 January 2019 Revised 14 April 2019 Accepted 24 April 2019 Published 22 May 2019

Academic Editor Xin-yuan Guan

Copyright copy 2019 Chengliang Zhu 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

Objective Hepatitis B virus (HBV) causes inflammation of the liver and is the leading cause of both liver cirrhosis (LC) andhepatocellular carcinoma (HCC) Serine protease inhibitor Kazal type 1 (SPINK1) is an acute-phase response protein that isoverexpressed in liver cancer tissueThis study investigated the clinical value of SPINK1 with regard to the diagnosis of HBV-relateddiseases and its regulatory mechanismMethods Serum levels of SPINK1 in HBV-infected patients and healthy participants weredetected by enzyme-linked immunosorbent assay (ELISA) Reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR) andwestern blottingwere used todetect differential expression of SPINK1mRNAandprotein inHepG2 andHepG2215cells The HBV infectious clone pHBV13 and its individual genes were cotransfected into HepG2 cells with the SPINK1 promotercoupled to a luciferase reporter luciferase activity was measured and the expression levels of SPINK1 were examined ResultsSerum SPINK1 levels of HBV-infected patients were significantly higher than those of healthy participants and the serum levels ofSPINK1 in patients who tested positive for HBeAg were significantly higher than those in patients who tested negative for HBeAgThe serum SPINK1 levels of patients with LC or HCCwere markedly higher than those of patients with chronic hepatitisThe HBVX protein (HBx) activated the SPINK1 promoter to upregulate expression of SPINK1 at bothmRNA and protein levelsConclusionsHBV enhances expression of SPINK1 through X SPINK1 levels are increased during progression of HBV-relateddiseases andmightbe utilized as a biomarker for the diagnosis of HBV-related diseases

1 Introduction

Hepatitis B virus (HBV) infection is a serious public healthproblem worldwide Approximately 250 million people havechronic HBV infection of these cases approximately 20to 30 develop into liver cirrhosis (LC) Moreover approx-imately 2 to 5 of cirrhosis cases progress to liver cancer [12] The gene serine protease inhibitor Kazal type 1 (SPINK1)also known as tumor-associated trypsin inhibitor (TATI) orpancreatic secretory trypsin inhibitor (PSTI) is located onchromosome 5q32 approximately 75 kb long and containsfour exons [3] SPINK1 a secretory peptide composed of

56 amino acids belongs to the Kazal-type serine proteaseinhibitor family the main roles of which are to inhibit theactivity of numerous pancreatic serine proteases In additionSPINK1 appears to participate in tumorigenesis [4ndash7] HBVand hepatitis C virus (HCV) upregulate expression of serineprotease inhibitor Kazal (SPIK) and SPINK1 is overexpressedinHCV-positive hepatocellular carcinoma (HCC) and thus isa promising prognostic marker for this cancer [8ndash10]

In a preliminary study we screened for differentiallyexpressed genes between control HepG2 cells andHepG2215cells integrated with the entire HBV genome and found thatexpression of SPINK1 was increased by more than 40-fold in

HindawiBioMed Research InternationalVolume 2019 Article ID 9321494 7 pageshttpsdoiorg10115520199321494

2 BioMed Research International

HBV-infected HepG2215 cells (data not shown) Althoughit is known that HBV is a major cause of HCC [2 11]the mechanism by which HBV regulates SPINK1 expressionremains unclear This study investigated the effect of HBVinfection on SPINK1 expression analyzed the relationshipbetween serum SPINK1 levels and the progression of HBV-related diseases and explored the molecular mechanismunderlying the regulation of SPINK1 expression by HBx

2 Materials and Methods

21 Participants A total of 248 patients with a clinicaldiagnosis of HBV infection from Renmin Hospital ofWuhanUniversity (Wuhan China) from January 2015 to November2018 were included in the present study The patients weredivided into three groups according to the results of clinicalbiochemistry tests computed tomography (CT) magneticresonance imaging (MRI) and pathology examinations asfollows 104 patients with chronic hepatitis B (CHB) definedby persistent HBsAg positivity for gt 6 months [12] (78 menand 26 women with a mean age of 405 plusmn 127 years) 82patients with LC (60 men and 22 women with a mean ageof 477 plusmn 153 years) and 62 patients with HCC (48 men and14 women with a mean age of 558 plusmn 175 years) None ofthe patients had other viral infections including HCV HDVor HIV A total of 124 healthy participants (90 men and 34women with a mean age of 422 plusmn 146 years) were includedas a control groupTheEthics Committee of RenminHospitalofWuhan University approved this study and all participantssigned an informed consent document

22 Cell Culture and Transfection HepG2 and HepG2215cells were cultured in Dulbeccorsquos Modified Eaglersquos Medium(DMEM Gibco) containing 10 FBS and 100 Umlpenicillin-streptomycin in an incubator at 37∘C and 5CO2 HepG2 cells were seeded in 6-well or 24-well plates

and were transfected with Lipofectamine 2000 transfectionreagent (Invitrogen) according to the userrsquos manual whenreaching 80 confluence Briefly HBV infectious clonepHBV13 eukaryotic expression plasmids containing all ofthe HBV genes (pCMV-S pCMV-E pCMV-C pCMV-Xand pCMV-P) and Lipofectamine 2000 were diluted inDMEM medium at room temperature for 20 min [13] Theprepared transfection solution was then added to the HepG2cell culture medium and the cells were cultured in a CO

2

incubator

23 Reverse Transcription Quantitative Real-Time PCR (RT-qPCR) HepG2 and HepG2215 cells were collected 1 mlof TrizolR reagent (Invitrogen) was added and total RNAwas extracted according to the userrsquos manual cDNA wassynthesized from 1 120583g of total RNA using reverse tran-scription with M-MLV (Promega) in a volume of 20120583Lthe cDNA was then diluted 10 times with DEPC waterand real-time fluorescence quantitative PCR amplificationwas performed using a Roche LightCycler 480 (Roche) ina reaction mixture of 20120583L containing 10120583L SYBR GreenqPCR Master Mix (DBI Bioscience) 05 120583M of each PCRprimer 3 120583L of diluted template and RNase-free water

to complete the 20 120583L volume GAPDH was used as theinternal referenceTheupstreamanddownstreamprimers forSPINK1 were 51015840-AACACTGGAGCTGACTCCCT-31015840 and 51015840-ATCAGTCCCACAGACAGGGT-31015840 respectively The PCRreaction was performed at 95∘C for 10 minutes followedby 35 cycles of 95∘C for 15 seconds and 58∘C for 30seconds The relative expression level of SPINK1 mRNAwas expressed as 2-Ct The experiment was repeated threetimes

24 Western Blotting Total protein was extracted from cellsusing RIPA lysis buffer (50 mM Tris-HCl 150 mM NaCl1 Triton X-100 1 mM EDTA 10 glycerol and proteaseinhibitor cocktail (Roche) pH 74) and quantified using BCAProtein Quantitation Assay Kit (Pierce) Samples of 50 120583gwere separated by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and the protein bandswere transferred to a nitrocellulose (NC) membrane Afterblocking with phosphate-buffered saline (PBS) containing01 Tween 20 (PBST) for 2 hours the primary anti-SPINK1antibody (Abcam ab188307 diluted 12000) or anti-beta actinantibody (Sigma diluted 15000) was added and incubatedat 4∘C overnight The membrane was washed three timeswith PBST buffer followed by the addition of a horseradishperoxidase-conjugated secondary antibody (Sigma diluted15000) incubation at room temperature for 1 hour andthree washes with PBST buffer avoiding light The enhancedchemiluminescence (ECL) method was used for develop-ment followed by fixation and photography

25 Luciferase Assay Transfected HepG2 cells were culturedfor 48 hours and then lysed using lysis buffer (Promega)A 20 120583L aliquot of the cell lysate and 100 120583L of luciferasesubstrate (Promega) were mixed well and the optical densitywas measured using a luminometer (Turner T2020) Theexperiment was repeated three times

26 Serology Tests HBsAg and HBeAg were detected usingthe double-antibody sandwich method with a COBAS e601electrochemiluminescence spectrometer (Roche) at a lowerlimit of 11 COI (cut off index) for HBsAg and 1 COI forHBeAg HBV DNA was examined by real-time fluorescencequantitative PCR using a Roche LightCycler 480 (Roche)The liver function indicators ALT and AST were measuredusing an enzymatic method with an ADVIA 2400 automatedbiochemistry analyzer (Siemens)

27 SPINK1 Test Using human serum samples stored at -80∘C SPINK1 was detected with an SPINK1 enzyme-linkedimmunosorbent assay (ELISA) kit (Abcam) according to themanufacturerrsquos instructions

28 Statistical Analyses Data were analyzed using SPSS200 and the results are expressed as the mean plusmn standarddeviation (x plusmn s) Data comparisons between two groupswere conducted using t-tests Differences were consideredsignificant at P lt 005

BioMed Research International 3

Table 1 Clinical parameters and biochemical test results for the subjects enrolled in the study

Characteristic Healthy controls (n=124) CHB LC HCC(n=104) (n=82) (n=62)

Sex (malefemale) 9034 7826 6022 4814Age 422 plusmn 146 405 plusmn 127 477 plusmn 153 558 plusmn 175BMI (kgm2) 235plusmn14 253plusmn16 247plusmn15 244plusmn 15ALT (IUl) lt30 2064plusmn2837 527plusmn768lowast 599plusmn815lowastAST (IUl) lt30 1467plusmn2003 659plusmn651lowast 927plusmn886lowastn number of subjects CHB chronic hepatitis B LC liver cirrhosis HCC hepatocellular carcinoma BMI body mass index ALT alanine aminotransferaseAST aspartate aminotransferase lowast indicates Plt005 compared with healthy controls

0102030405060708090

100110

HBV individuals

SPIN

K1 (

gL)

Healthy controls

lowast

(a)

20

40

60

80

100

HBeAg positiveHBeAg negative

lowast

SPIN

K1 (

gL)

(b)

0

20

40

60

80

100

120

HCCLCCHB

lowast

lowast

SPIN

K1 (

gL)

(c)

Figure 1 Serum SPINK1 levels in patients infected with HBV and healthy control participants detected by ELISA (a) Comparison of serumSPINK1 levels between patients infected with HBV and healthy controls by ELISA (b) Comparison of serum SPINK1 levels between patientstesting positive and those testing negative for HBeAg (c) Comparison of serum SPINK1 levels among patients with CHB LC or HCC Boxplots show medians with 25 and 75 and whiskers with 5 and 95 percentiles lowast indicates Plt005

3 Results

31 Clinical Information for the Participants The248 patientsinfected with HBV were divided into three groups 104patients with CHB 82 patients with LC and 62 patientswith HCC 167HBV-infected patients were tested positive forHBeAg and 81 negative for HBeAg Table 1 shows the clinicalparameters and biochemical test results for the patientsinfected with HBV

32 Increased Serum SPINK1 Levels in Patients Infected withHBV Theserum levels of SPINK1were examined in theHBVgroup and the healthy control group using ELISAThe resultsshowed a serum SPINK1 level of 633 plusmn 155 120583gL for thehealthy control group and 254 plusmn 67 120583gL for the HBV groupa difference that was significant (P lt 005 see Figure 1(a))

Furthermore the relationship between the serumSPINK1level and HBeAg was analyzed The results showed that theSPINK1 level in HBV-infected patients who tested positive


0

02

04

06

08

1

12

HepG2 HepG2215

SPIN

K1G

APD

H

Fold

lowast

(a)

HepG2

SPINK1

HepG2215

-actin

(b)

0100200300400500600700800900

1000

pBlue-ks pHBV13

Luci

fAct

RUL

g

prot

ein

lowast

(c)

0

02

04

06

08

1

12

pBlue-ks pHBV13

SPIN

K1G

APD

H

Fold

lowast

(d)

SPINK1pBlue-ks pHBV13

-actin

(e)

Figure 2The effect of HBV on SPINK1 expression in cells (a)The expression level of SPINK1mRNA inHepG2215 andHepG2 cells detectedusing RT-qPCR (b) Expression of the SPINK1 protein in HepG2215 and HepG2 cells was detected using western blotting (c) Changes inluciferase activity at 48 hours after cotransfection of HepG2 cells with 02 120583g of SPINK1 promoter SPINK1-Luc and 06 120583g of HBV13 or itscontrol plasmid pBlue-ks the experiment was repeated three times (d) SPINK1 mRNA expression was detected using RT-qPCR at 48 hoursafter transfection of HepG2 cells with 4 120583g of HBV13 or its control plasmid pBlue-ks (e) SPINK1 protein expression detected using westernblotting at 48 hours after transfection of HepG2 cells with 4 120583g of HBV13 or its control plasmid pBlue-ks lowast indicates Plt005

for HBeAg (794 plusmn 94 120583gL) was significantly higher thanthat of HBV-infected patients who tested negative for HBeAg(379 plusmn 59 120583gL P lt 005 Figure 1(b)) No correlations werefound between serum SPINK1 level and HBV viral load ALTor AST (data not shown)

CHB infection gradually develops into cirrhosis and ulti-mately leads to liver cancer Thus serum SPINK1 levels werecompared between the groups of patients and according tothe results serum SPINK1 levels increased with continuousprogression of HBV-caused diseases Serum SPINK1 levelswere 416 plusmn 66 120583gL 573 plusmn 83 120583gL and 779 plusmn 138 120583gLin the CHB LC and HCC groups respectively and thesedifferences were significant (P lt 005 Figure 1(c))

33 HBV Upregulates SPINK1 Expression at the Cellular LevelExpression of SPINK1 was examined in HepG2215 cells

with whole-HBV genome integration [14] and in controlHepG2 cells using RT-qPCR and western blotting ThemRNA (Figure 2(a)) and protein expression levels of SPINK1in HepG2215 cells were higher than those in HepG2cells (SPINK1120573-actin ratio of 091 in HepG2215 cells andSPINK1120573-actin ratio of 043 in HepG2 cells Figure 2(b))

In addition HepG2 cells were cotransfected with theHBV infectious clone pHBV13 and the SPINK1 promoterpSPINK1-LUC cells cotransfected with the empty vectorpBlue-ks and pSPINK1-LUC were used as the negative con-trol The results of the assay demonstrated that the activityof the SPINK1 promoter increased after transfection withpHBV13 compared with that in cells transfected with pBlue-ks luciferase activities were 1968 plusmn 227 RUL120583g protein and8643 plusmn 382 RUL120583g protein respectively (Figure 2(c)) indi-cating that HBV activates the SPINK1 promoter Moreover


0

200

400

600

800

1000

1200

pCM

V-ta

g2B

pCM

V-S

pCM

V-E

pCM

V-C

pCM

V-X

pCM

V-P

Luci

fAct

RU

L

g pr

otei

n

lowast

(a)

002040608

112

pCMV-tag2B pCMV-tag-HBx

SPIN

K1G

APD

H F

old lowast

(b)

HepG2215HepG2SPINK1

-actin

(c)

Figure 3The effect of HBx on SPINK1 expression (a) Changes in luciferase activity at 48 hours after cotransfection ofHepG2 cells with 06120583gof the eukaryotic expression plasmid containing each gene of the HBV genome (pCMV-S pCMV-E pCMV-C pCMV-X and pCMV-P) and02 120583g of the SPINK1 gene promoter SPINK1-Luc or its control plasmid pCMV-tag2B the experiment was repeated three times (b) SPINK1mRNA expression detected using RT-qPCR at 48 hours after transfection of HepG2 cells with 4 120583g of pCMV-X or its control plasmid pCMV-tag2B (c) SPINK1 protein expression detected using western blotting at 48 hours after transfection of HepG2 cells with 4 120583g of pCMV-X orits control plasmid pCMV-tag2B lowast indicates Plt005

RT-qPCR and western blotting revealed that the expressionlevels of SPINK1 mRNA (Figure 2(d)) and protein wereincreased after transfection with pHBV13 (SPINK1120573-actinratio of 105 with pHBV13 transfection and SPINK1120573-actinratio of 034 with pBlue-ks transfection Figure 2(e))

34 HBV Upregulates SPINK1 Expression through Its X GeneHepG2 cells were cotransfected with all of the individualplasmid eukaryotic expression vectors containing the HBVgenome and the plasmid pSPINK1-LUC containing theSPINK1 gene promoter cells transfected with the emptyvector pCMV-2B were used as the control HBx signifi-cantly upregulated the promoter activity of pSPINK1-LUC(Figure 3(a))

A eukaryotic expression plasmid for the X gene pCMV-X was transfected into HepG2 cells and cells transfectedwith the empty vector pCMV-tag2B were used as the con-trol Expression levels of SPINK1 mRNA and protein weresignificantly upregulated after transfection with pCMV-Xindicating that the HBV X protein enhances expression ofSPINK1 mRNA (Figure 3(b)) and protein (SPINK1120573-actinratio of 102 with pCMV-X transfection and SPINK1120573-actinratio of 031 with pCMV-tag2B transfection Figure 3(c))

4 Discussion

SPINK1 is overexpressed in various solid tumors such as gas-tric breast and colon cancers [15ndash18]Marshall et al screenedfor differentially expressed genes in normal liver tissue andHCC tissue using gene chips and found elevated SPINK1expression in the latter [19] Additionally Li et al examined

SPINK1 expression in diseased tissues from patients withLC and those with HCC using immunohistochemistry andwestern blotting and found that expression of SPINK1 inpatients with HCC was higher than that in patients with LC[6] The current study investigated the serum SPINK1 levelsof patients infected with HBV and healthy controls and foundelevated SPINK1 serum levels in the former Furthermorethe serum SPINK1 levels of patients who tested positivefor HBeAg were significantly higher than those of patientswho tested negative for HBeAg We also compared serumSPINK1 among patients with CHB LC or HCC and foundgradually increasing levels with the progression of HBV-related diseases To elucidate the regulatory effect of HBV onSPINK1 we confirmed DNA chip results using RT-qPCR andwestern blotting and demonstrated that the levels of SPINK1mRNA and protein expression were increased in HepG2215cells stably transfected with HBV

HBV is a type of hepatotropic DNA virus that containsa partial double-stranded circular DNA molecule with fouropen-reading frames S C P and X [20] To study themolecular mechanism of HBV with regard to regulationof SPINK1 expression we employed the luciferase reportergene system to detect the impact of pHBV13 and eukary-otic expression plasmids harboring its genes (ie pCMV-S pCMV-E pCMV-C pCMV-X and pCMV-P) on theactivity of the SPINK1 promoter We found that pHBV13and its X gene upregulated the activity of the SPINK1promoter and expression of SPINK1 at themRNAandproteinlevels

HBx is a regulatory factor encoded by the HBV X geneinHBV-infected cells which transactivates gene transcription


and plays important roles in gene regulation signal trans-duction cell proliferation and transformation especiallywith regard to promoting the tumorigenesis of primaryliver cancer tumor cell invasion and metastasis [21ndash24]Studies have shown that SPINK1 participates in apoptosis bybinding to epidermal growth factor receptor (EGFR) pro-moting tumorigenesis and progression [25]Moreover serumSPINK1 levels are closely related to tumor prognoses such asfor ovarian breast bladder and colon cancers and SPINK1overexpression serves as a marker of poor tumor prognosis[4 5 26 27] Therefore HBV is likely to upregulate SPINK1expression through its X gene to promote the development ofliver cancer

Thus far serum markers such as alpha-fetoprotein iso-forms des-gamma-carboxy prothrombin (DCP) golgi pro-tein 73 (GP73) glypican-3 (GPC3) and small RNA (miRNA)have been used for the clinical diagnosis of HCC [28]The present study found SPINK1 to be associated with theprogression of HBV-related diseases and SPINK1 mightbecome a new serum marker for HCC diagnosis Becauseof the small number of specimens however the sensitivityand specificity of SPINK1with regard toHCC diagnosis awaitfurther study

In summary we demonstrate that HBV upregulates thesynthesis and secretion of SPINK1 These findings lay thefoundation for elucidating the pathogenic and tumorigenicmechanisms of HBV to identify serum markers for HCC

Data Availability

The data used to support the findings of this study areincluded within the article

Conflicts of Interest

The authors have no conflicts of interest to declare

Acknowledgments

This study was supported by the Discipline Group Construc-tion Project of Pudong Health Bureau of Shanghai (Grant noPWZxq2017-15) the Outstanding Leaders Training Programof Pudong Health Bureau of Shanghai (no PWR12018-05)the Pudong New Area Science and Technology DevelopmentFund (Grant no PKJ2016-Y56) the Key Specialty Construc-tion Project of Shanghai Municipal Health Bureau (Grantno ZK2015B16) the National Natural Science Foundation ofChina (81672079) and the Open Research Program of theState Key Laboratory of Virology of China (2019KF004)

References

[1] GLOBOCAN httpwww-depiarcfr[2] A Petruzziello ldquoEpidemiology of hepatitis B virus (HBV) and

hepatitis C virus (HCV) related hepatocellular carcinomardquoeOpen Virology Journal vol 12 no 1 pp 26ndash32 2018

[3] A Horii T Kobayashi N Tomita et al ldquoPrimary structureof human pancreatic secretory trypsin inhibitor (PSTI) generdquo

Biochemical and Biophysical Research Communications vol 149no 2 pp 635ndash641 1987

[4] Y-T Chen S-C Tsao H-P Tsai J-Y Wang and C-Y ChaildquoSerine protease inhibitor Kazal type 1 (SPINK1) as a prognosticmarker in stage IV colon cancer patients receiving cetuximabbased targeted therapyrdquo Journal of Clinical Pathology vol 69no 11 pp 974ndash978 2016

[5] B Kozakiewicz M Chadzynska E Dmoch-Gajzlerska and MStefaniak ldquoTumor-associated trypsin inhibitor in patients withendometrial cancerrdquoTUMORI vol 102 no 5 pp 527ndash532 2016

[6] F Li T Liu C-Y Xiao J-X Yu L-G Lu andM-Y Xu ldquoFOXP1and SPINK1 reflect the risk of cirrhosis progression to HCCwith HBV infectionrdquo Biomedicine amp Pharmacotherapy vol 72pp 103ndash108 2015

[7] K Rasanen O Itkonen H Koistinen and U-H StenmanldquoEmerging roles of SPINK1 in cancerrdquo Clinical Chemistry vol62 no 3 pp 449ndash457 2016

[8] J Lamontagne M Pinkerton T M Block and X Lu ldquoHep-atitis B and hepatitis C virus replication upregulates serineprotease inhibitor Kazal resulting in cellular resistance to serineprotease-dependent apoptosisrdquo Journal of Virology vol 84 no2 pp 907ndash917 2010

[9] HGHass J JobstUVogelM Scheurlen andONehls ldquoOver-expression of tumor-associated trypsin inhibitor (SPINK1TATI) in hepatitis C-associated hepatocellular carcinomaPotential implications for viral hepatocarcinogenesisrdquoOncologyResearch and Treatment vol 37 no 12 pp 732ndash738 2014

[10] I Lyytinen M Lempinen A Nordin H Makisalo U-HStenman and H Isoniemi ldquoPrognostic significance of tumor-associated trypsin inhibitor (TATI) and human chorionicgonadotropin-120573 (hCG120573) in patients with hepatocellular carci-nomardquo Scandinavian Journal of Gastroenterology vol 48 no 9pp 1066ndash1073 2013

[11] E Waidely A-R O Al-Yuobi A S Bashammakh M S El-Shahawi and R M Leblanc ldquoSerum protein biomarkers rele-vant to hepatocellular carcinoma and their detectionrdquo Analystvol 141 no 1 pp 36ndash44 2016

[12] E M Sokal M Paganelli S Wirth et al ldquoManagement ofchronic hepatitis B in childhood ESPGHAN clinical practiceguidelines consensus of an expert panel on behalf of theEuropean Society of Pediatric Gastroenterology HepatologyandNutritionrdquo Journal of Hepatology vol 59 no 4 pp 814ndash8292013

[13] X Liu C Zhu J Li et al ldquoHBV upregulates CtBP2 expressionvia the X generdquoBioMed Research International vol 2018 ArticleID 6960573 6 pages 2018

[14] M A Sells M-L Chen and G Acs ldquoProduction of hepatitis Bvirus particles in Hep G2 cells transfected with cloned hepatitisB virusDNArdquoProceedings of theNational Acadamy of Sciences ofthe United States of America vol 84 no 4 pp 1005ndash1009 1987

[15] A Gaber M Johansson U-H Stenman et al ldquoHigh expressionof tumour-associated trypsin inhibitor correlates with livermetastasis and poor prognosis in colorectal cancerrdquo BritishJournal of Cancer vol 100 no 10 pp 1540ndash1548 2009

[16] WW Soon L DMiller M A Black et al ldquoCombined genomicand phenotype screening reveals secretory factor SPINK1 as aninvasion and survival factor associated with patient prognosisin breast cancerrdquo EMBO Molecular Medicine vol 3 no 8 pp451ndash464 2011


[17] U-H Stenman E Koivunen and O Itkonen ldquoBiology andfunction of tumor-associated trypsin inhibitor tatirdquo Scandina-vian Journal of Clinical amp Laboratory Investigation vol 51 no207 pp 5ndash9 1991

[18] J-P Wiksten J Lundin S Nordling A Kokkola U-H Sten-man and C Haglund ldquoHigh tissue expression of tumour-associated trypsin inhibitor (TATI) associates with a morefavourable prognosis in gastric cancerrdquoHistopathology vol 46no 4 pp 380ndash388 2005

[19] AMarshallM Lukk C Kutter S Davies G Alexander andDT Odom ldquoGlobal gene expression profiling reveals SPINK1 as apotential hepatocellular carcinoma markerrdquo PLoS ONE vol 8no 3 Article ID e59459 2013

[20] C Zhu G Gao H Song F Xu K Wu and X Liu ldquoHepatitisB virus inhibits apolipoprotein A5 expression through its coregenerdquo Lipids in Health and Disease vol 15 no 1 p 178 2016

[21] H Liu L Xu H He et al ldquoHepatitis B virus X proteinpromotes hepatoma cell invasion and metastasis by stabilizingSnail proteinrdquo Cancer Science vol 103 no 12 pp 2072ndash20812012

[22] S K Shukla and V Kumar ldquoHepatitis B virus X protein and c-Myc cooperate in the upregulation of ribosome biogenesis andin cellular transformationrdquo FEBS Journal vol 279 no 20 pp3859ndash3871 2012

[23] K M F Sze G K Y Chu J M F Lee and I O L Ng ldquoC-terminal truncated hepatitis B virus x protein is associated withmetastasis and enhances invasiveness by C-Junmatrix metal-loproteinase protein 10 activation in hepatocellular carcinomardquoHepatology vol 57 no 1 pp 131ndash139 2013

[24] G Yu X Chen S ChenW Ye K Hou andM Liang ldquoMiR-19amiR-122 and miR-223 are differentially regulated by hepatitis Bvirus X protein and involve in cell proliferation in hepatomacellsrdquo Journal of Translational Medicine vol 14 no 1 p 1222016

[25] C Wang L Wang B Su et al ldquoSerine protease inhibitor Kazaltype 1 promotes epithelial-mesenchymal transition throughEGFR signaling pathway in prostate cancerrdquo e Prostate vol74 no 7 pp 689ndash701 2014

[26] O Kemik A Kemik A Sumer et al ldquoThe relationshipbetween serum tumor-associated trypsin inhibitor levels andclinicopathological parameters in patients with gastric cancerrdquoEuropean Review for Medical and Pharmacological Sciences vol17 no 21 pp 2923ndash2928 2013

[27] X Pan X Zhang J Gong et al ldquoThe expression profileand prognostic value of SPINK1 in initially diagnosed bonemetastatic prostate cancerrdquoe Prostate vol 76 no 9 pp 823ndash833 2016

[28] S Yao J Zhang H Chen et al ldquoDiagnostic Value of Immuno-histochemical Staining of GP73 GPC3 DCP CD34 CD31and Reticulin Staining in Hepatocellular Carcinomardquo Journalof Histochemistry amp Cytochemistry vol 61 no 9 pp 639ndash6482013

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HBV-infected HepG2215 cells (data not shown) Althoughit is known that HBV is a major cause of HCC [2 11]the mechanism by which HBV regulates SPINK1 expressionremains unclear This study investigated the effect of HBVinfection on SPINK1 expression analyzed the relationshipbetween serum SPINK1 levels and the progression of HBV-related diseases and explored the molecular mechanismunderlying the regulation of SPINK1 expression by HBx

2 Materials and Methods

21 Participants A total of 248 patients with a clinicaldiagnosis of HBV infection from Renmin Hospital ofWuhanUniversity (Wuhan China) from January 2015 to November2018 were included in the present study The patients weredivided into three groups according to the results of clinicalbiochemistry tests computed tomography (CT) magneticresonance imaging (MRI) and pathology examinations asfollows 104 patients with chronic hepatitis B (CHB) definedby persistent HBsAg positivity for gt 6 months [12] (78 menand 26 women with a mean age of 405 plusmn 127 years) 82patients with LC (60 men and 22 women with a mean ageof 477 plusmn 153 years) and 62 patients with HCC (48 men and14 women with a mean age of 558 plusmn 175 years) None ofthe patients had other viral infections including HCV HDVor HIV A total of 124 healthy participants (90 men and 34women with a mean age of 422 plusmn 146 years) were includedas a control groupTheEthics Committee of RenminHospitalofWuhan University approved this study and all participantssigned an informed consent document

22 Cell Culture and Transfection HepG2 and HepG2215cells were cultured in Dulbeccorsquos Modified Eaglersquos Medium(DMEM Gibco) containing 10 FBS and 100 Umlpenicillin-streptomycin in an incubator at 37∘C and 5CO2 HepG2 cells were seeded in 6-well or 24-well plates

and were transfected with Lipofectamine 2000 transfectionreagent (Invitrogen) according to the userrsquos manual whenreaching 80 confluence Briefly HBV infectious clonepHBV13 eukaryotic expression plasmids containing all ofthe HBV genes (pCMV-S pCMV-E pCMV-C pCMV-Xand pCMV-P) and Lipofectamine 2000 were diluted inDMEM medium at room temperature for 20 min [13] Theprepared transfection solution was then added to the HepG2cell culture medium and the cells were cultured in a CO

2

incubator

23 Reverse Transcription Quantitative Real-Time PCR (RT-qPCR) HepG2 and HepG2215 cells were collected 1 mlof TrizolR reagent (Invitrogen) was added and total RNAwas extracted according to the userrsquos manual cDNA wassynthesized from 1 120583g of total RNA using reverse tran-scription with M-MLV (Promega) in a volume of 20120583Lthe cDNA was then diluted 10 times with DEPC waterand real-time fluorescence quantitative PCR amplificationwas performed using a Roche LightCycler 480 (Roche) ina reaction mixture of 20120583L containing 10120583L SYBR GreenqPCR Master Mix (DBI Bioscience) 05 120583M of each PCRprimer 3 120583L of diluted template and RNase-free water

to complete the 20 120583L volume GAPDH was used as theinternal referenceTheupstreamanddownstreamprimers forSPINK1 were 51015840-AACACTGGAGCTGACTCCCT-31015840 and 51015840-ATCAGTCCCACAGACAGGGT-31015840 respectively The PCRreaction was performed at 95∘C for 10 minutes followedby 35 cycles of 95∘C for 15 seconds and 58∘C for 30seconds The relative expression level of SPINK1 mRNAwas expressed as 2-Ct The experiment was repeated threetimes

24 Western Blotting Total protein was extracted from cellsusing RIPA lysis buffer (50 mM Tris-HCl 150 mM NaCl1 Triton X-100 1 mM EDTA 10 glycerol and proteaseinhibitor cocktail (Roche) pH 74) and quantified using BCAProtein Quantitation Assay Kit (Pierce) Samples of 50 120583gwere separated by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and the protein bandswere transferred to a nitrocellulose (NC) membrane Afterblocking with phosphate-buffered saline (PBS) containing01 Tween 20 (PBST) for 2 hours the primary anti-SPINK1antibody (Abcam ab188307 diluted 12000) or anti-beta actinantibody (Sigma diluted 15000) was added and incubatedat 4∘C overnight The membrane was washed three timeswith PBST buffer followed by the addition of a horseradishperoxidase-conjugated secondary antibody (Sigma diluted15000) incubation at room temperature for 1 hour andthree washes with PBST buffer avoiding light The enhancedchemiluminescence (ECL) method was used for develop-ment followed by fixation and photography

25 Luciferase Assay Transfected HepG2 cells were culturedfor 48 hours and then lysed using lysis buffer (Promega)A 20 120583L aliquot of the cell lysate and 100 120583L of luciferasesubstrate (Promega) were mixed well and the optical densitywas measured using a luminometer (Turner T2020) Theexperiment was repeated three times

26 Serology Tests HBsAg and HBeAg were detected usingthe double-antibody sandwich method with a COBAS e601electrochemiluminescence spectrometer (Roche) at a lowerlimit of 11 COI (cut off index) for HBsAg and 1 COI forHBeAg HBV DNA was examined by real-time fluorescencequantitative PCR using a Roche LightCycler 480 (Roche)The liver function indicators ALT and AST were measuredusing an enzymatic method with an ADVIA 2400 automatedbiochemistry analyzer (Siemens)

27 SPINK1 Test Using human serum samples stored at -80∘C SPINK1 was detected with an SPINK1 enzyme-linkedimmunosorbent assay (ELISA) kit (Abcam) according to themanufacturerrsquos instructions

28 Statistical Analyses Data were analyzed using SPSS200 and the results are expressed as the mean plusmn standarddeviation (x plusmn s) Data comparisons between two groupswere conducted using t-tests Differences were consideredsignificant at P lt 005





0102030405060708090

100110

HBV individuals

SPIN

K1 (

gL)

Healthy controls

lowast

(a)

20

40

60

80

100


lowast

SPIN

K1 (

gL)

(b)

0

20

40

60

80

100

120

HCCLCCHB

lowast

lowast

SPIN

K1 (

gL)

(c)


3 Results





0

02

04

06

08

1

12

HepG2 HepG2215

SPIN

K1G

APD

H

Fold

lowast

(a)

HepG2

SPINK1

HepG2215

-actin

(b)

0100200300400500600700800900

1000

pBlue-ks pHBV13

Luci

fAct

RUL

g

prot

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(c)

0

02

04

06

08

1

12

pBlue-ks pHBV13

SPIN

K1G

APD

H

Fold

lowast

(d)


-actin

(e)








0

200

400

600

800

1000

1200

pCM

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pCM

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112


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HepG2215HepG2SPINK1

-actin

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4 Discussion









Data Availability




Acknowledgments


References

































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018






0102030405060708090

100110

HBV individuals

SPIN

K1 (

gL)

Healthy controls

lowast

(a)

20

40

60

80

100


lowast

SPIN

K1 (

gL)

(b)

0

20

40

60

80

100

120

HCCLCCHB

lowast

lowast

SPIN

K1 (

gL)

(c)


3 Results





0

02

04

06

08

1

12

HepG2 HepG2215

SPIN

K1G

APD

H

Fold

lowast

(a)

HepG2

SPINK1

HepG2215

-actin

(b)

0100200300400500600700800900

1000

pBlue-ks pHBV13

Luci

fAct

RUL

g

prot

ein

lowast

(c)

0

02

04

06

08

1

12

pBlue-ks pHBV13

SPIN

K1G

APD

H

Fold

lowast

(d)


-actin

(e)








0

200

400

600

800

1000

1200

pCM

V-ta

g2B

pCM

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V-E

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Luci

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(b)

HepG2215HepG2SPINK1

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(c)





4 Discussion









Data Availability




Acknowledgments


References

































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



0

02

04

06

08

1

12

HepG2 HepG2215

SPIN

K1G

APD

H

Fold

lowast

(a)

HepG2

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0100200300400500600700800900

1000

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fAct

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0

02

04

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H

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0

200

400

600

800

1000

1200

pCM

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(b)

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(c)





4 Discussion









Data Availability




Acknowledgments


References

































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



0

200

400

600

800

1000

1200

pCM

V-ta

g2B

pCM

V-S

pCM

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-actin

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4 Discussion









Data Availability




Acknowledgments


References

































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018






Data Availability




Acknowledgments


References

































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018

















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


hepatitis b virus x protein-induced serine protease...

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