Hepatitis B virus and microRNAs: Complex interactions affecting hepatitis B virus replication and hepatitis B virus-associated diseases

Table 1 Cell-based model systems for the study of hepatitis B virus

Model system	Source	Characteristics
Cell lines
HepG2	Human 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
HepG2.2.15	HepG2 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
HepAD38	HepG2 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
Huh7	Human 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/5	Human hepatoma[185]	Transformed cell line that is easy to grow and maintain. These cells express HBsAg from integrated HBV DNA
HepaRG	Human 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 hepatocytes	Hepatocyte isolation from liver tissue	Natural 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 hepatocytes	Perfused liver and isolation of hepatocytes	Primary 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 samples	HCC and adjacent normal liver tissue; HBV and non-HBV liver tissue	Primary 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 cells	Circulation	Easier to acquire than primary hepatocytes, but only support low levels of HBV infection and replication

HBV: Hepatitis B virus; HBx: Hepatitis B virus -X protein.

Table 2 Methods for studying microRNA expression levels

Strategy	Advantages	Disadvantages
qRT-PCR	Can determine absolute miRNA-expression level when compared to standard curve, requires minimal bioinformatics analysis	Analysis of single miRNA at a time; often used to determine expression of small numbers of miRNAs; requires prior identification of miRNAs of interest
miRNA microarray	Can be used to determine expression changes for a large group of miRNAs	Data are expressed as fold change relative to a baseline and not absolute expression values; can require prior identification of miRNAs of interest
Next-generation sequencing	Identifies the complete miRNome and determines absolute expression values in the context of all other cellular miRNAs	Expensive and requires extensive bioinformatics analysis

miRNA: microRNA; qRT-PCR: Quantitative reverse transcription polymerase chain reaction.

Table 3 Hepatitis B virus-mediated regulation of cellular microRNAs

miRNA	Regulation	Sample type	Proposed target	Proposed targeted pathway	Method of detection	Ref.
HBV-mediated regulation
miR-122	Down	HepG2.2.15	HO-1	Inflammation and apoptosis	qRT-PCR	[151]
	Down	HBV-transfected HepG2/Huh7/SK-Hep-01, HBV-transgenic mice, HBV-infected liver tissue	PBF	Cell proliferation	qRT-PCR	[69]
	Down	HepG2.2.15	Cyclin G1	Cell cycle regulation; p53-mediated repression of HBV transcription	In-situ hybridization, qRT-PCR	[98]
	Down	HepG2.2.15	NDGR3	Cell proliferation	qRT-PCR	[95]
	Up	HBV-infected primary treeshrew hepatocytes, HBV patient serum	-	-	Next-generation sequencing, qRT-PCR	[66]
	Up	HBV patient serum	-	-	microarray	[77,159]
let-7a/g	Down	HBV-transfected HepG2	-	-	microarray	[84]
let-7a	Down	HBV-transfected HepG2	-	-	qRT-PCR	[106]
let-7 family	Up	HepG2.2.15	-	-	microarray	[84]
let-7a	Up	HepG2.2.15	-	-	microarray	[81]
miR-15/16 family	Down	HBV-transfected HepG2	-	-	microarray	[84]
	Down	HBV-transfected HepG2, HepG2.2.15, HBV-transgenic mice, HBV-associated HCC	Bcl-2	Apoptosis	qRT-PCR	[70]
	Down	HepG2.2.15	-	-	microarray	[83]
miR-22	Down	HepG2.2.15	CDKN1A	Cell proliferation	qRT-PCR	[167]
miR-29c	Down	HepG2.2.15	TNFAIP3	Cell proliferation	qRT-PCR	[166]
miR-125a-5p	Up	HBV-infected liver tissue	-	-	qRT-PCR	[121]
	Up	HepG2.2.15	-	-	microarray	[82,83]
	Up	Transfected Cell Line	-	-	qRT-PCR	[106]
miR-125b	Down	HepG2.2.15, HBV-transfected HepG2	SCCND1	-	microarray, qRT-PCR	[123]
miR-181a	Up	HepG2.2.15, AdHBV-infected HepG2	E2F5	Cell proliferation	qRT-PCR	[138]
mIR-199b	Down	HBsAg+ liver tissue	-	-	microarray	[82]
miR-372/373	Up	HepG2.2.15	NFIB	HBV replication	microarray	[82]
miR-501	Up	HepG2.2.15	HBXIP	HBV replication	microarray, qRT-PCR	[81]
miR-602	Up	HepG2.2.15	RASSF1A	Apoptosis, tumor suppressor	microarray, qRT-PCR	[99]
HBx-mediated regulation
miR-122	Down	HBx-expressing HepG2	PPARγ	miR-122 transcription	qRT-PCR	[97]
let-7 family	Down	HBx over-expressing HepG2, HCC	Stat3	Cell proliferation	microarray, qRT-PCR	[107]
miR-15/16 family	Down	HBx-expressing HepG2/Huh7/SK-HEP-1 cells	CyclinD1	Cell cycle	microarray, qRT-PCR	[129]
miR-21	Up	HBx-expressing HepG2/Huh7	PDCD4, PTEN	Akt, cell proliferation	qRT-PCR	[145]
miR-21	Up	HBx-expressing MIHA	-	Cell migration	qRT-PCR	[187]
miR-29a	Up	HepG2.2.15, HBx-expressing HepG2, HBx-transgenic mice	PTEN	Akt, cell migration	qRT-PCR	[67]
miR-101	Down	HepG2.2.15, HBx-expressing HepG2	DNMT3A	DNA methylation	qRT-PCR	[147]
miR-143	Up	HBx-expressing HepG2, HBx-transgenic mice	FNDC3B	Cell proliferation	qRT-PCR	[168]
miR-148a	Down	HBx-expressing LO2/HepG2 cells	HPIP	mTOR	qRT-PCR	[144]
miR-152	Down	HepG2.2.15, HBx-expressing HepG2, HBx-transgenic mice	DNMT`1	DNA methylation	microarray, qRT-PCR	[188]
miR-199a	Up	HBx-expressing HepG2, HepG2.2.15, HBV infected HCC	-	-	microarray, qRT-PCR	[107]
miR-205	Down	HBx-expressing HepG2, HBV-associated HCC		DNA methylation, cell proliferation	qRT-PCR	[72]
miR-224	Up	HBx-transgenic mice	Smad4	Cell proliferation	in-situ hybridization, qRT-PCR	[68]
HBV-associated HCC
miR-122	Down	HBV-associated HCC	PBF	Cell proliferation	qRT-PCR	[69]
	Down	HBV-associated HCC	NDGR3	Cell proliferation	qRT-PCR	[95]
let-7 family	Down	HCC	Stat3	Cell proliferation	microarray, qRT-PCR	[107]
miR-199a/b-3p	Down	HCC liver samples (HBV and non-HBV)	PAK4	ERK, cell proliferation	NGS, qRT-PCR	[30]
miR-199b	Down	HBV-associated HCC	-	-	qRT-PCR	[74]
miR-22	Down	HBV-associated HCC	CDKN1A	Cell proliferation	qRT-PCR	[167]
miR-29c	Down	HBV-associated HCC	TNFAIP3	Cell proliferation	qRT-PCR	[166]
miR-101	Down	HBV-associated HCC	DNMT3A	DNA methylation	qRT-PCR	[147]
miR-143	Up	HBV-associated HCC	FNDC3B	Cell proliferation	qRT-PCR	[168]
miR-145	Down	HBV-associated HCC	-	-	qRT-PCR	[74]
miR-224	Up	HBV-associated HCC	-	-	qRT-PCR	[74]
miR-224	Up	HBV-associated HCC	Smad4	cell proliferation	in-situ hybridization, qRT-PCR	[68]
miR-501	Up	HBV-associated HCC	HBXIP	HBV replication	microarray, qRT-PCR	[81]
miR-602	Up	HCC liver samples (HBV and non-HBV)	RASSF1A	Apoptosis, tumor suppressor	microarray, qRT-PCR	[99]

HBV: Hepatitis B virus; HBx: Hepatitis B virus-X protein; HCC: Hepatocellular carcinoma; miRNA: microRNA; qRT-PCR: Quantitative reverse transcription polymerase chain reaction.