| Part ITaqman array analysis of miRNAs in hepatocellularcarcinoma and the roles of miR-138and miR-483-5p intumorigenesisHepatocellular carcinoma (HCC) is the leading cause of cancer deaths in theworld．More than half of those people are in China．The overall5-year survival rate forHCC patients is less than5％．Early detection of HCC is of supreme importance becausethe treatment options are severely limited by a late diagnosis．Late clinical presentationshave also led to poor prognosis for HCC patients．Thus,it is necessary to el ucidate themolecular mechanisms underlying HCC and identify novel therapeutic targets andbiomarkers for the early detection of HCC．Although serum a-fetoprotein(AFP)is a usefultumor marker for the detection and monitoring of HCC development,the false-negativerate by AFP level alone may be as high as40％for patients with a small HCC．So,if a newtumor marker has more sensitivity and can be detected in the primary stage of HCC,it ispossible to extrapolate the likelihood of a malignant liver tumor．Thus,discovery of newtarget molecules that are critically involved in the majority of cases and expressedspecifically in tumors will be essential to understand the mechanisms and improvetherapeutic intervention and prognosis of HCC．MicroRNA (miRNA) represent a class of small noncoding RNA that control geneexpression by targeting mRNA and triggering either translation repression or RNAdegradation．Mature miRNA are19-25nucleotides that can silence genes through eitherconsummate or defective binding to the3' untranslated region (3'UTR) of thetranscript．miRNA have been involved in the modulation of diverse cellular processesincluding cell differentiation, proliferation and apoptosis．An increasing number of studiessuggest that there all differential miRNA expression profiles between those in cancertissues and adjacent nontumorous tissue,these studies also show that miRNA profilescould distinguish malignant tumor of liver,breast,lung,pancreas and leukemia fromadjacent nontumorous tissue．The importance of miRNA in cancer is highlighted by the observation that50％of miRNA genes are located in cancer associated genomic regions orfragile sites．Importantly,miRNA expression is frequently deregulated in several cancersincluding B-cell chronic lymphocytic leukemia Burkitt' S lymphoma,colorectal cancer,lung cancer and HCC．Additionally,differential expression of miRNA have been found tobe associated with postoperativesurvival in lung cancer patients and all diagnostic andprognostic markers of lung cancer．miRNA have been implicated to play both tumorsuppressor and oncogenic roles．These studies indicate that miRNA profiling studies couldbe used for defining clinical phenotypes,as well as potentially useful molecular diagnosticmarkers．An understanding of the molecular mechanism by which mi RNA regulate HCCdevelopment potentially may provide new avenues of research that could aid earlydiagnosis and treatment of this highly malignant tumor．In this present study,we examined the expression profiles of667miRNA in3pairs ofHCC and adjacent non-tumorous tissue by a Taqman low density miRNA array(TLDA)．The biological functions and targets of some differentially expressed miRNAshave been detected by Real-time RT-PCR and Western-blotting analysis．This study mayhelp clarify the molecular mechanisms involved in the progression of HCC．1,Deregulated miRNAs in hepatocellular carcinoma667microRNA expression profiles of was performed on3pairs of surgically removedHCC and adjacent non-tumorous tissues by using TLDA, then23deregulated miRNAswere validated in an extended samples set of18paired HCC and adjacent non-tumorousliver tissues by realtime reverse transcription polymerase chain reaction (RT-PCR)analysis．Results:Only miRNAs altered by at least3-fold in all three pairs of the samples wereconsidered significant candidates. Under these strict criteria,11up-regulated miRNAs and12down-regulated miRNAs were identified. To validate the miRNA array data, qRT-PCRwas performed in18pairs of HCC tissues.10up-regulated and10down-regulatedmiRNAs showed consistent changes in more than50%tumorous tissues, of whichmiR-217, miR-520g, miR-522and miR-525-3p were up-regulated in more than70%tumorous tissues, and miR-199a-5p, miR-138, miR-483-5p and miR-511weredown-regulated in more than70%tumorous tissues.The targets of the above20deregulated miRNAs were predicted by TargetScan. Tojudge the most significant candidates and investigate the cellular function, the signaling pathway and GOs of these target genes were analyzed. The results showed that a widevariety of cellular processes were featured significantly in signaling pathways. Many ofthese signaling pathways, such as insulin, MAPK, TGF-beta and Wnt signaling pathway,have been shown to participate in the tumorigenesis. However, some other signalingpathways have never been reported to play a role in tumorigenesis, e.g. axon guidance.Among all these differentially regulated signaling pathways,"regulation of actincytoskeleton" and "pathway in cancer" appeared to be the most enriched two in bothup-regulated and down-regulated miRNA groups. A similar phenomenon was observed inGOs analysis. Many cellular functions were featured significantly, of which the "signaltransduction" appeared to be the most enriched one. Based on these results, the miRNAswhich were involved in both "signal transduction" and "the regulation of actincytoskeleton or pathway in cancer" might be the most significant candidates. MiR-520g,miR-483-5p, miR-138, miR-199a-5p, miR-217and miR-518e were selected under thecriteria for further studies. Here, the studies on miRNA-138were presented.2,miR-138induces cell cycle arrest by targeting cycline D3The significant reduction of miR-138expression in HCC tissues indicated possiblebiological significance in tumorgenesis. At first, the effect of miR-138on cell growth wasevaluated in HepG2and Huh7cells transfected with or not, miR-138mimic, miR-138inhibitor or NC duplex. From2day (HepG2) or3day (Huh7) after the transfection, theviability of cells transfected with miR-138mimic significantly decreased compared withthat of NC duplex transfected or nontransfected cells, but the viability of cells transfectedwith miR-138inhibitor significantly increased. These results indicate miR-138couldinhibit cell growth. To validate the inhibitor effect of miR-138on cell growth, the colonyformation assay was performed in HepG2and Huh7transfected with or not, miR-138mimic, miR-138inhibitor or NC duplex. HepG2and Huh7cells transfected with20nMmiR-138mimic displayed much fewer and smaller colonies (218or161colonies)compared with NC duplex transfected (783or729colonies) and nontransfected cells (756or692colonies), but cells transfected with20nM miR-138inhibitor displayed much moreand larger colonies (1238or1349colonies).To further confirm the above findings, an in vivo mouse model was used. For theduration of the treatment with miR-138mimic or miR-138inhibitor for5weeks, tumorvolume curves revealed a significant decrease in growth rates at the3rd,4th and5th weekafter treatment with miR-138mimic and a significant increase in growth rates at the4th and5th week after treatment with miR-138inhibitor whereas no significant differences intumor growth rates were observed between the NC group and the ctrl group. These resultsindicate that introduction of miR-138significantly inhibits tumorigenicity of HepG2cellsin xenograft nude mouse model.To investigate the mechanism of inhibitor effect of miR-138, flow cytometry assayshowed that the percentages of miR-138mimic transfected HepG2and Huh7cells in theG0-G1phase were18%(HepG2) and11%(Huh7) higher than that of NC duplextransfected or nontransfected cells, which paralleled with a44%(HepG2) and38%(Huh7)decrease in the S phase. In miR-138inhibitor transfected cells, the percentages of cells inthe G0-G1phase were8%(HepG2) and6%(Huh7) less than that of NC duplex transfectedor nontransfected cells, which paralleled with a14%(HepG2) and19%(Huh7) increase inthe S phase. These results indicate miR-138could inhibit HepG2and Huh7proliferationby inducing cell cycle arrest at G1/S phase.It is generally accepted that miRNAs exert their function through regulating theexpression of their downstream target genes. Cycline D3(CCND3) was predicted as apotential target of miR-138by TargetScan and Miranda. To validate whether CCND3is adirect target of miR-138, a human CCND33'-UTR fragment containing wild-type ormutant miR-138binding sequence was cloned downstream of the firefly luciferase reportergene in pGL3. In HEK293cells cotransfected with the reporter plasmids and miR-138mimic or NC duplex, the luciferase activity of the reporter that contained wild-type3'-UTR was significantly suppressed by miR-138mimic, but the luciferase activity ofmutant reporter was unaffected, indicating that miR-138may suppress gene expressionthrough miR-138binding sequence at the3'-UTR of CCND3. Furthermore, transfection ofmiR-138mimic decreased CCND3expression and transfection of miR-138inhibitorincreased CCND3expression in HepG2cells at protein but not mRNA level, suggestingthat CCND3expression could be inhibited by miR-138at posttranscriptional level.Together, the results show that miR-138could regulate the expression of endogenoushuman CCND3by directly targeting the3'-UTR of CCND3mRNA and human CCND3isa new target of miR-138.To identify whether inhibition of CCND3, just like miR-138restoration, also resultedin HCC repression, the effects of knockdown of CCND3on cell growth were examined.Firstly, HepG2cells were transfected with or not, CCND3siRNA or control siRNA.Seventy two hours after transfection, a dose dependent knockdown of CCND3was observed in HepG2cells. In cell viability assay and cell cycle analysis, in vitro knockdownof CCND3repressed cell viability, induced cell cycle arrest and inhibited the colonyformation. The similar data were obtained in Huh7cells transfected with CCND3siRNA.These results indicate that CCND3is most likely involved in the induction of cell cyclearrest by miR-138.3,Retinoic acid induced16, regulated by miR-483-5p, is a novel oncogene and tumormarker for hepatocellular carcinomaThe significant reduction of miR-483-5p expression in HCC tissues indicatedpossible biological significance in tumorgenesis. At first, the effect of miR-483-5p on cellgrowth was evaluated in HepG2and Huh7cells transfected with or not, miR-483-5p mimic,miR-483-5p inhibitor or NC duplex. From2day (HepG2) or3day (Huh7) after thetransfection, the viability of cells transfected with miR-138mimic significantly decreasedcompared with that of NC duplex transfected or nontransfected cells, but the viability ofcells transfected with miR-483-5p inhibitor significantly increased. These results indicatemiR-138could inhibit cell growth.To investigate the mechanism of inhibitor effect of miR-483-5p, flow cytometry assayshowed that the percentages of miR-483-5p mimic transfected HepG2and Huh7cells inthe G0-G1phase were18%(HepG2) and11%(Huh7) higher than that of NC duplextransfected or nontransfected cells, which paralleled with a44%(HepG2) and38%(Huh7)decrease in the S phase. In miR-483-5p inhibitor transfected cells, the percentages of cellsin the G0-G1phase were8%(HepG2) and6%(Huh7) less than that of NC duplextransfected or nontransfected cells, which paralleled with a14%(HepG2) and19%(Huh7)increase in the S phase. These results indicate miR-483-5p could inhibit HepG2and Huh7proliferation by inducing cell cycle arrest at G1/S phase.Retinoic acid induced16(RAI16) was predicted as a potential target of miR-483-5pby TargetScan and Miranda. To validate whether RAI16is a direct target of miR-483-5p, ahuman RAI163'-UTR fragment containing wild-type or mutant miR-483-5p bindingsequence was cloned downstream of the firefly luciferase reporter gene in pGL3. InHEK293cells cotransfected with the reporter plasmids and miR-483-5p mimic or NCduplex, the luciferase activity of the reporter that contained wild-type3'-UTR wassignificantly suppressed by miR-483-5p mimic, but the luciferase activity of mutantreporter was unaffected, indicating that miR-483-5p may suppress gene expression throughmiR-483-5p binding sequence at the3'-UTR of RAI16. Furthermore, transfection of miR-483-5p mimic decreased RAI16expression and transfection of miR-138inhibitorincreased RAI16expression in HepG2cells at protein but not mRNA level, suggesting thatRAI16expression could be inhibited by miR-483-5p at posttranscriptional level. Together,the results show that miR-483-5p could regulate the expression of endogenous humanRAI16by directly targeting the3'-UTR of RAI16mRNA and human RAI16is a newtarget of miR-483-5p.In commercial HCC tissue arrays, most HCC tumor tissues showed moderate (score2) or strong (score3) RAI16expression (89of126cases). In contrast, most nontumortissues showed negative (score0) or weak (score1) RAI16expression (118of126cases).The sensitivity and specificity of RAI16expression for the diagnosis of HCC were70.6%and93.6%, respectively. Strikingly, the sensitivity and specificity increased to80.9%and92.0%when glypican-3was used with RAI16. These results indicate that RAI16may bepotential tumor marker for HCC diagnosis, especially combined with GPC3.The significant up-regulation of RAI16expression in HCC tissues indicated possiblebiological significance in tumorgenesis. At first, the viability of cells transfected withpcDNA3.1-RAI16significantly increased compared with that of empty vector pcDNA3.1transfected or nontransfected cells, but the viability of cells transfected with RAI16siRNAsignificantly decreased. HepG2and Huh7cells transfected with pcDNA3.1-RAI16displayed much more and larger colonies (1238or1349colonies) compared with emptyvector pcDNA3.1transfected (783or729colonies) and nontransfected cells (756or692colonies). Cell cycle analysis showed that the percentages of pcDNA3.1-RAI16transfectedHepG2and Huh7cells in the G0-G1phase were11.6%(HepG2) and9.4%(Huh7) lowerthan that of pcDNA3.1transfected or nontransfected cells, which paralleled with a35.6%(HepG2) and27.0%(Huh7) increase in the S phase. In RAI16siRNA transfected cells, thepercentages of cells in the G0-G1phase were10.9%(HepG2) and14.1%(Huh7) higherthan that of NC siRNA transfected or nontransfected cells, which paralleled with a25.8%(HepG2) and19.4%(Huh7) decrease in the S phase. These results indicate RAI16couldenhance HepG2and Huh7proliferation by promoting cell cycle from G0/G1to S phase.Furthermore, the percentage of apoptotic cells in pcDNA3.1-RAI16transfected HepG2andHuh7cells were36.4%(HepG2) and33.5%(Huh7) lower than that of pcDNA3.1transfected or nontransfected cells. In RAI16siRNA transfected cells, the percentages ofapoptotic cells were21.5%(HepG2) and26.3%(Huh7) higher than that of NC siRNAtransfected or nontransfected cells. To further confirm the above findings, an in vivo mouse model was used. For theduration of the treatment with HepG2cells stably expressing RAI16or transfected withRAI16siRNA, tumor volume curves revealed a significant increase in growth rates at the3rd,4th and5th week after treatment with HepG2cells stable expressing RAI16and asignificant decrease in growth rates at the4th and5th week after treatment with HepG2cells transfected with RAI16siRNA whereas no significant differences in tumor growthrates were observed between the NC siRNA transfected or non-transfected HepG2cellsgroups. These results indicate that introduction of RAI16significantly promotestumorigenesis in xenograft nude mouse model.To investigate the mechanism of RAI16enhancement of tumor growth in HCC,10major cancer related pathways were analyzed in transfected HepG2cells using adual-luciferase reporter system (Promega). The most affected pathways were transforminggrowth factor-β (TGF-β), mitogen-activated protein kinase (MAPK)/extracellularsignal-regulated kinase (ERK) and Wnt. The most affected pathways were TGF-β,MAPK/ERK and Nuclear factor κB (NFκB). The up-regulated MAPK/ERK and TGF-βpathways in RAI16overexpressed cells is consistent with the down-regulated MAPK/ERKand TGF-β pathways in RAI16knockdown cells These results indicate MAPK/ERK andTGF-β pathway may be involved in RAI16induction of tumorigenesis.To verify above findings, the key molecules in MAPK/ERK and TGF-β pathway weredetected by immunoblotting. RAI16overexpession increased the phosphorylation of MEKand ERK1/2significantly, the total ERK but not total MEK expression. Similar phenomenawere observed in TGF-β pathway, the phosphorylation of SMAD2/3and total SMAD2/3,together with SMAD4were increased by RAI16overexpression. RAI16knockdown ladeto the reverse results. Interesting, phosph-SMAD2/3and SAMD4in HCC tumor tissueswere increased much more significantly than that in RAI16transfected cell modal, butphosphorylation of MEK and ERK1/2seemed more significant in RAI16transfected cellmodal. These results suggest it is possible that MAPK/ERK and TGF-β pathway play therole in different stage of RAI16induction of tumorigenesis. Part IIApplication of HBx-induced anti-URGs as early warningbiomarker of liver cirrhosis and hepatocellularcarcinomaHepatocellular carcinoma (HCC) is one of the most common cancers, with morethan500,000deaths with over600,000new cases yearly worldwide. The clinical course ofHCC is mostly asymptomatic. Suspected liver changes are too large and too advanced forthe tumor to be subjected to potentially effective and radical therapy when they aredetected incidentally. Because of serious limitations of the surgical and oncologicaltreatment available, it seems necessary to concentrate on the earliest possible diagnosis,particularly sensitive detection of resectable focal liver changes-preferably when tumorsare less than2cm in diameter. Surveillance with immaging techniques and serumα-fetoprotein (AFP) analyses is recommended for all cirrhotic patients and other specificrisk groups every6months.AFP has to be considered "the golden standard" for HCC serum markers, however,the usefulness of AFP testing for the population at risk should be seriously questioned. AFPdiagnostic values for this assay are undoubtedly poor. AFP specificity varies from about76%to96%and increases with elevated cut-off value. Simultaneous sensitivity decreasesmuch more from about25%for potentially resectable tumors of less than3cm in diameterto about50%for lesions of>3cm in diameter. Other serum biomarkers such as AFP-L3%,DCP, AFU, GGT, GP-73, MUC-1, SCCA and GPC-3have significant diagnosticlimitations, and in fact they are not particularly precise for the early diagnosis of HCC.Simultaneous determination of these markers in various combinations could improve theaccuracy in differentiating HCC from nonmalignant hepatopathy, but there still exists theunresolved problem of tiny 'grey' nodules in the 'black and white' diagnostic perspective.Previous studies have found that HBx contributes to the development of HCC byaltering patterns of host gene expression.5up-regulated genes (URGs: URG4, URG7,URG11, URG12and Sui1) associated with HBx triggered corresponding antibodies(anti-URGs) in the sera of patients prior to tumor development and/or at early stagetumorigenesis. 1,Establish of ELISA assay for anti-URGs detection using synthesized peptidesTo establish an ELISA method to detect the anti-URGs in the serum of patients withHCC,5groups of peptides (L4A/L4B, L7B, L11-1/L11-3/L11-4, L12A/L12B andSui1A/Sui1B) were synthesized. The sensitivity, specificity and precision of this assaywere evaluated.Results:(1)The procure of ELISA assay is as following:10ug/mlpeptides were coatedby0.01M NaHCO3buffer at4℃overnight, blocking by Tri-HCl buffer with3%BSA at37℃for more than2hours, diluted serum samples at1:10were incubatedat4℃overnight, after washing, HRP Goat anti-human IgG incubation at37℃for30minutes,washing, TMB development for10minutes,read the OD value after stopping reaction with2M H2SO4.(2)The Cut-off value of ELISA assay to detect anti-URG4,anti-URG7,anti-URG11, anti-URG12and anti-Sui1is:0.103,0.098,0.101,0.107and0.113respectively.(3)The developed ELISAmethod showed good sensitivity, specificity and precision.Intra-division is3.2-4.5%, inter-division is7.4-10.3%.2,Detection of anti-URGs in serum samples by ELISATo evaluate the anti-URGs as the potecial serological markers of HCC and thecorresponding ELISA assay. The anti-URGs were detected in serum samples from558patients and108blood donors by ELISA assay.Results:(1) The number of anti-URGs showed significantly difference among the558patient with HBV associated hepatitis, cirrhosis, HCC and108blood donor serum samples.In HBV associated live disease groups, the sensitivity of anti-URGs is35.8%,60.7%and55.2%respectively.(2)The spececificity of anti-URGs is99.1%,87.7%and90.0%in blood donorsgroup, HBV asymptomatic carriers and other tumors respectively.(3)In HBV associated cirrhosis and HCC groups, the sensitivity of anti-URGs is60.0%and53.3%; the sensitivity of AFP is33.3%and30.0%; the sensitivity of AFP-L3%is40.0%and33.3%; the sensitivity of GPC3is36.3%and23.3%; the sensitivity of GP73is53.3%and33.3%; In blood donors group, the spececificity of anti-URGs is100%, thespececificity of AFP, AFP-L3%, GPC3and GP73is93.4%,96.7%,93.4%and93.4% respectively. Combination anti-URGs with AFP, AFP-L3%, GPC3and GP73respectivelycould improve the sensitivity by10%.(4) Longitudinal studies in20HBV-cirrhosis patients with more than3anti-URGsor with less than1anti-URGs showed that patients with more anti-URGs were more likelyto develope tumor compared with those with fewer anti-URGs.3,ConclusionIn this study, ELISA to detect the anti-URGs in serum was established and used inHBV infected patients and blood donors. It was found that anti-URGs were predominantlypresent among patients with HBV-associated HCC (55.2%) and cirrhosis (60.7%), and at alower frequency among patients with chronic hepatitis (35.8%), with significant difference(P<0.001). The sensitivity of anti-URGs in patients with HBV-associated HCC andcirrhosis was58.3%and the specificity of anti-URGs in blood donors was99.1%.The innovation point of this study lies on:(1) anti-URGs were firstly as the earlywarning biomarker for liver cirrhosis and HCC with much better sensitivity and specificity.(2) The combination of anti-URGs with AFP, AFP-L3or GP73improved the sensitivity ofdetection at15%.(3) This study was focused on the whole progress of HBV infection fromhepatitis to HCC, the HBV hepatitis and cirrhosis patients with3or more than3anti-URGs would have more risk to develop HCC, and should be paid more attention. Asearly warning biomarker of HCC, anti-URGs may be useful in the identification of HBVpatients with chronic liver disease who are most likely to develop HCC, which wouldprovide the help to diagnosis of human small HCC, early-stage asymptomatic HCC and thescreening of HCC in high risk population.
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