MicroRNA profile in HBV-induced infection and hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is among the most common of solid cancers with the third highest mortality worldwide [1]. Chronic hepatitis B virus (HBV) infection is a major risk factor for HCC [2]. Studies in literature indicate that several HBV-coded proteins promote malignant transformation in hepatocytes [3, 4]. HBV-related HCC has poor clinical recovery because a curative treatment is still lacking and the high rate of recurrence after treatment [5]. An understanding of the pathogenesis of HBV-associated HCC will provide insights for developing effective therapeutic and/or preventive strategies to combat this highly malignant form of cancer [6].

MicroRNAs (miRNA) constitute a recently discovered class of non-coding RNAs and are known to function in the regulation of gene expression [7, 8]. These molecules regulate the expression of as much as 30% of all mammalian protein-encoding genes. In addition to their important roles in healthy individuals, many studies have revealed that various miRNAs are involved in human carcinogenesis and other diseases. Consequently, miRNAs are being evaluated as candidates for diagnostic/prognostic biomarkers and predictors of drug response. The abnormal expression of miRNAs through transcriptional/post-transcriptional regulation or imperfect pairing with target messenger RNAs (mRNAs) of genes have been observed in disease processes [9‐12]. Several studies have shown that expression of miRNAs is dysregulated in HCC compared to non-tumor liver tissues [13]. For example, miR-122 is involved in liver development, differentiation, homeostasis and metabolic functions. MiR-122 targets CUTL1 and CCNG1, and loss of miR-122 results in blocked differentiation, genomic instability, and inflammation associated with liver disease and HCC [14]. MiR-199 targets hepatocyte growth factor receptor, mammalian target of rapamycin (mTOR), and hypoxia-inducible factor (HIF1α), thereby regulating receptor tyrosine kinase and mTOR activation [15]. MiR-21 targets programmed cell death protein 4 (PDCD4) and phosphatase and tensin homolog (PTEN), thereby modulating apoptosis resistance [16]. There is a paucity of information on miRNAs engaged in HBV-related HCC and the regulatory mechanisms of these miRNAs remain largely unknown.

The present study was undertaken to investigate the expression pattern and possible function of miRNAs to provide insights on molecular mechanisms of HBV-related HCC. Results of this study can be used to provide potential candidate biomarkers for HBV-related HCC detection. By miRNA expression profile, we found that miR-150, miR-342-3p, miR-663, miR-20b, miR-92a-3p, miR-376c-3p, and miR-92b are specifically altered in HBV-related HCC. Gene Ontology (GO) and KEGG analysis suggest that these miRNAs may be involved in transcription regulation, MAPK dependent protein phosphorylation, as well as modulation of focal adhesion and actin cytoskeleton. IPA analysis also suggests that these miRNAs act on AGO2, TP53, CCND1, and 11 other genes that are implicated in the occurrence of HCC and HBV infection.

HBV infection is a major health problem that leads to a significant rise in mortality and is reported to be closely related to HCC [20]. HBV contains four open reading frames (ORFs), namely, S, P, X, and pre C, whose products are HBeAg, HBcAg, HBsAg, and HBx, respectively [21]. Expression of HBV proteins was previously demonstrated to modulate the expression of some genes that likely contribute to the pathogenesis of HCC [3, 4]. Especially HBx plays a critical role in HBV-related HCC [21]. HBx can stop cell death mediated by p53, Fas, and transforming growth factor-β [22, 23], thereby implying the importance of regulation of apoptosis in the occurrence of HBV-related HCC. One report has shown that down-regulating the expressions of HBsAg, HBcAg, p21, and Rb proteins in HCC increases the propensity for HCC occurrence, indicating that these proteins are tumor suppressors and would influence the course of cell cycle and apoptosis [24]. All of the above information implied that HBV infection could affect cell cycle and survival and could eventually lead to HCC.

In our IPA results, the 6 selected miRNAs (miR-150, miR-342-3p, miR-20b, miR-92a-3p, miR-92b, and miR-376c-3p) are shown to comprise a network which linked themselves among AGO2, DICER1, BCL2L11, CCND1, CCND2, CCNE1, CDK7, E2F1, E2F3, TP53, and four other genes (Fig. 6). Argonaute RISC catalytic component 2 (AGO2) and dicer 1 (DICER1) were highlighted. AGO2 plays a role for RNA interference in regulating the chromatin structure. AGO2 may interact with dicer1 and play a role in short-interfering-RNA-mediated gene silencing. Previous reports have indicated that HBsAg and HBcAg co-localized with AGO2. Moreover, HBV-specific miRNAs, together with AGO2, play a role in the viral life cycle [25]. So far, no HBV-encoded miRNA has been identified [26]. Other genes shown in the IPA network were involved in regulation of cellular regulatory pathway. Thus, we can speculate that cellular miRNAs may function in HBV-induced HCC either by targeting cellular transcriptions factors required for HCC occurrence or by directly binding to HBV transcripts to affect HBV gene expression.

Many functional pathways are reportedly regulated during HBV infection. The deregulation of signaling pathways includes MAPKs, p53, Wnt/β-catenin, transforming growth factor β (TGF β), cytokines, and Jak-STAT [27, 28], and results to down-regulation of tumor suppressor gene expression and/or up-regulation of oncogene expression [29]. Coincidently, the pathway analysis results showed that the 7 miRNAs selected through the process were closely involved in MAPK signaling pathway, TGF-beta signaling pathway, cytokine-cytokine receptor interaction, wnt signaling pathway, Jak-STAT signaling pathway, and apoptosis (Fig. 5c, d). Thus, HBV infection may induce HCC mainly by acting on these miRNAs to influence liver cell states.

Products of HBV contribute to HBV-associated HCC; some of these can prompt cell cycle regulatory pathways [30]. For example, studies demonstrated that HBx, a product of HBV, triggered the occurrence of HCC by decreasing the levels of cell cycle proteins that prevent progression into G1 phase and increasing the levels of cell cycle proteins active in the G1 phase [31]. Cell cycle regulation plays an important role in HBV-induced HCC. In our study, genes of cell cycle proteins, namely, cyclin D1 (CCND1), cyclin D2 (CCND2), cyclin E1 (CCNE1), cyclin-dependent kinase 7 (CDK7), transcription factor 1 (E2F1), transcription factor 3 (E2F3), and tumor protein p53 (TP53) appeared in the IPA network (Fig. 6). The proteins encoded by these genes are obviously involved in cell cycle. Cyclin D1, cyclin D2 and cyclin E1 belong to the highly conserved cyclin family. Among these proteins, cyclin D1 and cyclin D2 form a complex with and function as a regulatory subunit of CDK4 or CDK6, whose activity is required for cell cycle G1/S transition. In addition, cyclin E1 functions with CDK2, whose activity is also necessary in G1/S transition [32]. Thus, mutations, amplification, and overexpression of these genes are frequently observed in HBV-related HCC [33]. Cyclin-dependent kinase 7 serves as a direct link between the regulation of transcription and the cell cycle [34]. E2F1 and E2F3 are members of the E2F family, their target genes function in DNA replication and repair, cell cycle regulation, cell cycle checkpoint, cell death, differentiation. E2F transcription factors are key targets of the retinoblastoma (Rb) tumor suppressor [35]. Previous research indicated that P53 was the most frequently altered pathway in HBV-related HCC, and TP53 was associated with shorter survival only in HBV-related HCC [36]. Coincidently, our target gene analysis showed that all the 4 up-regulated miRNAs (miR-20b, miR-92a-3p, miR-92b, and miR-376c-3p) target TP53, cyclin-dependent kinase inhibitor 1A (CDKN1A) and cyclin-dependent kinase inhibitor 2A (CDKN2A). Our study verified that the expression of TP53 and CDKN1A were decreased in HBV positive tumors compared with HBV positive non tumor tissues (Additional file 1: Figure S2). TP53 is a classical suppressor gene that is related to cell cycle and accumulation of genetic changes. The proteins encoded by CDKN1A and CDKN2A can inhibit the activity of cyclin-CDK and function as tumor suppressors to control cell cycle in HBV-related HCC [37, 38]. In addition, all down-regulated miRNAs (miR-150, miR-342-3p, and miR-663) target baculoviral IAP repeat containing 5 (BIRC5), CCND1, and protein tyrosine kinase 2 (PTK2). The expression of these three genes was increased in HBV positive tumors compared with HBV positive non-tumor tissues (Additional file 1: Figure S2). The product of PTK2 acts on cell cycle by regulating the tumor suppressor p53 [39]. Based on these results, aberrant regulation of cell cycle may be partially attributed to the occurrence of HBV-associated HCC.

In previous studies, a strong connection between apoptosis and HBV-related HCC has been shown [40, 41]. IPA result showed that the 6 co-regulated miRNAs function on TP53, E2F1, E2F3, and BCL2L11 directly or indirectly (Fig. 6). Thus, it is reasonable to speculate that these miRNAs play a role in occurrence of HCC through HBV infection regulating apoptosis. TP53 in the regulation of apoptosis was reportedly essential in HBV-induced HCC [42]. E2F1 and E2F3 can mediate cell proliferation and p53-dependent/independent apoptosis by binding to protein pRB [43, 44]. The protein encoded by BCL2-like 11 (BCL2L11) belongs to the BCL-2 protein family and acts as an apoptotic activator that serves a function in HCC occurrence [45]. Our study showed that high expression of miR-20b, miR-92a-3p, and miR-92b can down-regulate Fas-associated protein with death domain (FADD), which is known as an adaptor molecule that bridges the Fas-receptor [46] and is involved in apoptosis [47]. Moreover, the up-regulated miRNAs (miR-20b, miR-92a-3p, miR-92b, and miR-376c-3p) target BH3-interacting domain (BID), TP53 and PTEN. Consistently, the expression of gene ERK, TP53 and PTEN were decreased in HBV positive tumors compared with HBV positive non-tumor tissues (Additional file 1: Figure S2A). All down-regulated miRNAs (miR-150, miR-342-3p, and miR-663) target B-cell lymphoma/leukemia 2 (BCL2), BIRC5 and PTK2. The expression of these three genes was increased in HBV positive tumors compared with HBV positive non-tumor tissues (Additional file 1: Figure S2B). These genes were reportedly closely involved in apoptosis regulation in HBV-related HCC [37, 39, 47‐52]. Moreover, lower expression of miR-150 and miR-342-3p up-regulated Bcl-2–associated X protein and CASP8 and FADD-like apoptosis regulator (CFLAR), which have apoptotic activities [49, 53]. All these outcomes imply the importance of apoptosis regulation by miRNAs in HBV-related HCC.

From aforementioned results, we can speculate that a co-regulation exists among the 7 selected miRNAs, thereby promoting HBV-related HCC. First, 6 selected miRNAs comprised a network; these miRNAs linked themselves among AGO2, DICER1, BCL2L11, CCND1, CCND2, CCNE1, CDK7, E2F1, E2F3, TP53, and four other genes. AGO2 and DICER2, together with miRNAs and HBV products, affect viral life cycle. Other genes, however, mainly function in cellular regulatory pathways. So far, no HBV-encoded miRNA has been identified. Thus, we speculate that cellular miRNAs may function in HBV-induced HCC at the transcription level either by targeting cellular transcription factors required for HCC occurrence or by a directly binding to HBV transcripts to affect HBV gene expression. Furthermore, we found that all 7 HBV-specific miRNAs participate in cell cycle and apoptosis by regulating various genes that code for cell cycle and apoptosis regulators. All these miRNAs act together to regulate a variety of physiological functions, which ultimately lead to HCC. We may use these 7 miRNAs as possible biomarkers that can be applied to HBV-induced HCC detection, early intervention, and recurrence. However, all of these predictions are a good starting point for the involvement of miRNAs in HBV-related HCC, and the exact functions of the miRNAs identified in this article need experimental verification.

Aberrations in miRNA expression are correlated to cancer progression. In this study, we analyzed global miRNA expression profiles in HCC and adjacent matched non-HCC tissues from HBV-positive and HBV-negative patients. Our data indicates that the unique 7 miRNAs (miR-150, miR-342-3p, miR-663, miR-20b, miR-92a-3p, miR-376c-3p and miR-92b) expression signature could be involved in the development of HBV- related HCC, suggesting interesting potential novel therapeutic options. However, further functional studies are needed to clarify the role of these miRNAs in HBV-related HCC.