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Copyright ©The Author(s) 2015.
World J Gastroenterol. Jun 28, 2015; 21(24): 7375-7399
Published online Jun 28, 2015. doi: 10.3748/wjg.v21.i24.7375
Table 1 Cell-based model systems for the study of hepatitis B virus
Model systemSourceCharacteristics
Cell lines
HepG2Human hepatoblastoma[59]Transformed cell line that is easy to grow and maintain. Serves as a model system for HBx-dependent HBV replication, although these cells cannot be directly infected by HBV
HepG2.2.15HepG2 with two integrated head-to-tail dimers of HBV genome[62]Stably replicates HBV and produces infectious virus; however, continuous passage since development has created a large separation from the parental HepG2 cells so that differences between HepG2 and HepG2.2.15 cells may not be HBV-specific
HepAD38HepG2 stably expressing HBV pgRNA that is controlled by a tetracycline-responsive promoter[63]Presence or absence of tetracycline (tet) allows cell line to act as its own baseline. HBV replication easily controlled, but only pgRNA expression is under tet-control, so some viral proteins maystill be made in the presence of tet
Huh7Human hepatoma[60]Transformed cell line that is easy to grow and maintain. Cells cannot be directly infected by HBV. For reasons that remain unknown, HBV replication in these cells is HBx-independent
PLC/PRF/5Human hepatoma[185]Transformed cell line that is easy to grow and maintain. These cells express HBsAg from integrated HBV DNA
HepaRGHuman hepatoma[186]Transformed cell line, but differentiation can be induced to promote primary hepatocyte-like characteristics. After differentiation, these cells can be directly infected with HBV, although the infection efficiency is low. Acquisition of a differentiated, hepatocyte-like phenotype requires two-week maintenance in 2% DMSO prior to induction of differentiation; this process generates a mixed culture of hepatocytes and cholangiocytes
Primary cells
Cultured primary human hepatocytesHepatocyte isolation from liver tissueNatural target of an HBV infection; however, quickly lose susceptibility to an HBV infection after isolation and culture. These cells can be difficult to acquire, difficult to maintain in culture, and begin to de-differentiate soon after isolation and plating. These cells can be difficult to transfect
Cultured primary mouse/rat hepatocytesPerfused liver and isolation of hepatocytesPrimary cells that can be cultured to maintain a "normal" phenotype and serve as a surrogate model for studies in primary human hepatocytes. Support HBV replication although not direct HBV infection. Isolation requires access to animals and ability to reliably isolate high quality hepatocytes
Liver tissue samplesHCC and adjacent normal liver tissue; HBV and non-HBV liver tissuePrimary cells can give a more accurate profile of the liver RNA transcripts and expressed proteins than transformed cell lines. Disease versus normal tissue comparisons facilitate analysis of disease-mediated differences; however, it is often difficult to determine if differences are a cause or consequence of the disease. Requires access to patient samples that is often hampered by a finite supply and limited access such that these samples are often only used for primary screens or final confirmation
Peripheral blood mononuclear cellsCirculationEasier to acquire than primary hepatocytes, but only support low levels of HBV infection and replication
Table 2 Methods for studying microRNA expression levels
StrategyAdvantagesDisadvantages
qRT-PCRCan determine absolute miRNA-expression level when compared to standard curve, requires minimal bioinformatics analysisAnalysis of single miRNA at a time; often used to determine expression of small numbers of miRNAs; requires prior identification of miRNAs of interest
miRNA microarrayCan be used to determine expression changes for a large group of miRNAsData are expressed as fold change relative to a baseline and not absolute expression values; can require prior identification of miRNAs of interest
Next-generation sequencingIdentifies the complete miRNome and determines absolute expression values in the context of all other cellular miRNAsExpensive and requires extensive bioinformatics analysis
Table 3 Hepatitis B virus-mediated regulation of cellular microRNAs
miRNARegulationSample typeProposed targetProposed targeted pathwayMethod of detectionRef.
HBV-mediated regulation
miR-122DownHepG2.2.15HO-1Inflammation and apoptosisqRT-PCR[151]
DownHBV-transfected HepG2/Huh7/SK-Hep-01, HBV-transgenic mice, HBV-infected liver tissuePBFCell proliferationqRT-PCR[69]
DownHepG2.2.15Cyclin G1Cell cycle regulation; p53-mediated repression of HBV transcriptionIn-situ hybridization, qRT-PCR[98]
DownHepG2.2.15NDGR3Cell proliferationqRT-PCR[95]
UpHBV-infected primary treeshrew hepatocytes, HBV patient serum--Next-generation sequencing, qRT-PCR[66]
UpHBV patient serum--microarray[77,159]
let-7a/gDownHBV-transfected HepG2--microarray[84]
let-7aDownHBV-transfected HepG2--qRT-PCR[106]
let-7 familyUpHepG2.2.15--microarray[84]
let-7aUpHepG2.2.15--microarray[81]
miR-15/16 familyDownHBV-transfected HepG2--microarray[84]
DownHBV-transfected HepG2, HepG2.2.15, HBV-transgenic mice, HBV-associated HCCBcl-2ApoptosisqRT-PCR[70]
DownHepG2.2.15--microarray[83]
miR-22DownHepG2.2.15CDKN1ACell proliferationqRT-PCR[167]
miR-29cDownHepG2.2.15TNFAIP3Cell proliferationqRT-PCR[166]
miR-125a-5pUpHBV-infected liver tissue--qRT-PCR[121]
UpHepG2.2.15--microarray[82,83]
UpTransfected Cell Line--qRT-PCR[106]
miR-125bDownHepG2.2.15, HBV-transfected HepG2SCCND1-microarray, qRT-PCR[123]
miR-181aUpHepG2.2.15, AdHBV-infected HepG2E2F5Cell proliferationqRT-PCR[138]
mIR-199bDownHBsAg+ liver tissue--microarray[82]
miR-372/373UpHepG2.2.15NFIBHBV replicationmicroarray[82]
miR-501UpHepG2.2.15HBXIPHBV replicationmicroarray, qRT-PCR[81]
miR-602UpHepG2.2.15RASSF1AApoptosis, tumor suppressormicroarray, qRT-PCR[99]
HBx-mediated regulation
miR-122DownHBx-expressing HepG2PPARγmiR-122 transcriptionqRT-PCR[97]
let-7 familyDownHBx over-expressing HepG2, HCCStat3Cell proliferationmicroarray, qRT-PCR[107]
miR-15/16 familyDownHBx-expressing HepG2/Huh7/SK-HEP-1 cellsCyclinD1Cell cyclemicroarray, qRT-PCR[129]
miR-21UpHBx-expressing HepG2/Huh7PDCD4, PTENAkt, cell proliferationqRT-PCR[145]
miR-21UpHBx-expressing MIHA-Cell migrationqRT-PCR[187]
miR-29aUpHepG2.2.15, HBx-expressing HepG2, HBx-transgenic micePTENAkt, cell migrationqRT-PCR[67]
miR-101DownHepG2.2.15, HBx-expressing HepG2DNMT3ADNA methylationqRT-PCR[147]
miR-143UpHBx-expressing HepG2, HBx-transgenic miceFNDC3BCell proliferationqRT-PCR[168]
miR-148aDownHBx-expressing LO2/HepG2 cellsHPIPmTORqRT-PCR[144]
miR-152DownHepG2.2.15, HBx-expressing HepG2, HBx-transgenic miceDNMT`1DNA methylationmicroarray, qRT-PCR[188]
miR-199aUpHBx-expressing HepG2, HepG2.2.15, HBV infected HCC--microarray, qRT-PCR[107]
miR-205DownHBx-expressing HepG2, HBV-associated HCCDNA methylation, cell proliferationqRT-PCR[72]
miR-224UpHBx-transgenic miceSmad4Cell proliferationin-situ hybridization, qRT-PCR[68]
HBV-associated HCC
miR-122DownHBV-associated HCCPBFCell proliferationqRT-PCR[69]
DownHBV-associated HCCNDGR3Cell proliferationqRT-PCR[95]
let-7 familyDownHCCStat3Cell proliferationmicroarray, qRT-PCR[107]
miR-199a/b-3pDownHCC liver samples (HBV and non-HBV)PAK4ERK, cell proliferationNGS, qRT-PCR[30]
miR-199bDownHBV-associated HCC--qRT-PCR[74]
miR-22DownHBV-associated HCCCDKN1ACell proliferationqRT-PCR[167]
miR-29cDownHBV-associated HCCTNFAIP3Cell proliferationqRT-PCR[166]
miR-101DownHBV-associated HCCDNMT3ADNA methylationqRT-PCR[147]
miR-143UpHBV-associated HCCFNDC3BCell proliferationqRT-PCR[168]
miR-145DownHBV-associated HCC--qRT-PCR[74]
miR-224UpHBV-associated HCC--qRT-PCR[74]
miR-224UpHBV-associated HCCSmad4cell proliferationin-situ hybridization, qRT-PCR[68]
miR-501UpHBV-associated HCCHBXIPHBV replicationmicroarray, qRT-PCR[81]
miR-602UpHCC liver samples (HBV and non-HBV)RASSF1AApoptosis, tumor suppressormicroarray, qRT-PCR[99]