Tumor Biol. DOI 10.1007/s13277-014-2658-6
REVIEW
The impact of hepatitis B virus x protein and microRNAs in hepatocellular carcinoma: a comprehensive analysis Li Lin & Xiaomao Yin & Xiumei Hu & Qian Wang & Lei Zheng
Received: 24 April 2014 / Accepted: 18 September 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Keywords microRNAs . Hepatitis B virus x protein . Hepatocellular carcinoma . Hepatitis B virus infection
protein, is an essential factor for HBV replication and is thought to play a key role in the molecular pathogenesis of HBV-related HCC (HBV-HCC) [3, 4]. microRNAs (miRNAs), small endogenous non-coding RNAs which regulate target gene expression at the posttranscription level [5–7], are also believed to have a role in hepatocarcinogenesis. Over 50 % of genes that encode miRNAs are located at cancer-associated genomic regions that are fragile or aberrantly expressed in a wide variety of tumors [8, 9]. Several studies have recently found that HBx can promote the proliferation, differentiation, migration, and inhibit apoptosis of hepatoma cells by regulating miRNA expression. Further understanding of the roles of miRNAs and HBx in HBV-related HCC could therefore provide insights into the mechanisms of tumor development and novel therapeutic strategies.
Introduction
miRNA expression correlates with HCC pathogenesis
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and one of the most common malignant tumors in China. Due to its late time to diagnosis and high recurrence rate, HCC is an aggressive human cancer and the second leading cause of death in the world [1]. Hepatocarcinogenesis is a complex and multistep biological process that involves multiple factors. One well-known risk factor for HCC is chronic infection with hepatitis B virus (HBV), which occurs in more than 80 % of primary liver cancers [2]. Hepatitis B virus x protein (HBx), a ∼17 kDa
Since the first identification of a miRNA, lin-4, in Caenorhabditis elegans in 1993, many additional miRNAs have been identified in a wide range of species such as animals, plants, and viruses [10, 11]. miRNAs are small and non-coding endogenous RNAs, approximately 22 nucleotides (nts) in length, that inhibit gene expression by preferentially interacting with the 3′-untranslated regions (3′-UTRs) of target mRNAs. These interactions may either inhibit the translation of targeted mRNAs or induce the degradation of these targets [12, 13]. Recent studies have demonstrated that abnormal miRNA expression plays an important role in the formation of a wide variety of tumors and is directly involved in the occurrence, development, diagnosis, and staging of HCC. In the early stages of tumorigenesis, miRNAs are aberrantly expressed in carcinoma and adjacent HCC tissue.
Abstract microRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs, approximately 22 nucleotides (nts) in length, widely found in animals, plants, and viruses. Mature miRNAs control gene expression at a posttranscriptional level through blocking protein translation or inducing mRNA degradation. Many recent studies have shown that hepatitis B virus x protein (HBx), a viral protein with a crucial role in hepatogenesis, is associated with the regulation of miRNAs. This interaction impacts fundamental tumor processes, such as cell proliferation, differentiation, and apoptosis. In this review, we summarized the recent literature on the roles of HBx-regulated miRNAs in the pathogenesis of hepatocellular carcinoma.
L. Lin : X. Yin : X. Hu : Q. Wang : L. Zheng (*) Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, No.1838 North of Guangzhou Road, Baiyun District, Guangzhou 510515, People’s Republic of China e-mail: [email protected]
Tumor Biol.
Murakami et al. analyzed miRNA expression profiles in HCC samples, adjacent non-tumorous tissue (NT) samples, and chronic hepatitis samples (CH) through microarray and Northern blot assays [14]. The results showed that 30 miRNAs are significantly and differentially expressed in HCC compared with the corresponding NT specimens. Furthermore, the expression level of some miRNAs was correlated with the extent of HCC differentiation. For example, three miRNAs (miR-20, miR-18, and pre-miR-18) were significantly higher in HCC samples that exhibited low differentiation compared to those with moderate or high differentiation. miRNA expression has also been correlated with gender; in a study involving 80 male and 36 female patients with HBV-related HCC, the expression of miR-22 was increased more in men than in women, which may partly explain the high incidence of HBV-associated HCC in the male population [15]. Subsequent studies have shown that miRNAs are involved in HCC proliferation and migration [16, 17]. For example, miR-122 is down-regulated in HBV-infected HCC, permitting viral replication and cell proliferation by targeting the transcripts encoding NDRG3, a member of the N-myc downregulated gene family and NDRG3-mediated pathways [18]. In another example, the development of HBV-HCC to portal vein tumor thrombus (PVTT) and elevated TGF-β activity suppresses the expression of miR-34a, leading to enhanced production of chemokine CCL22, which recruits regulatory T (Treg) cells to facilitate immune escape [19].
HBx plays an important role in HCC pathogenesis The X gene is the smallest of four overlapping open reading frames (S, C, Pol, and X) in the HBV genome and is the most common HBV gene integrated into the host genome. HBx, a regulatory protein encoded by X gene, is generally synthesized on the ribosomes of rough endoplasmic reticulum in the cytoplasm. The protein then enters nuclei via nuclear pores and regulates a variety of gene sequences in the molecular pathogenesis of HBV-related HCC [20–22]. The mechanism of HBx in the occurrence of HCC has been reported, including gene transcription, cell cycle regulation, and several signaling pathways [23]. After the X gene integrates into the host, HBx directly or indirectly transactivates gene promoters or enhancers. HBx thereby activates a wide range of key cell signaling pathways of cells in HCC [24, 25]. Another important mechanism of the X gene in HCC is its ability to influence the inactivation and mutation of the p53 tumor suppressor [26, 27]. As p53 influences cell growth by regulating DNA replication, the mutation or deactivation of p53 results in the acceleration of cell proliferation [28]. Subsequently, some new mechanism of HBx in connects with HBV infections to the development of HCC has been
reported. For example, in Gearhart TL’s study [29], the authors demonstrated that HBx could modulate hepatocyte proliferation pathways to stimulate viral replication. HBx induces normally quiescent hepatocyte to enter the G1 phase of the cell cycle, which is required for HBV replication. Although there is a preliminary understanding of HBx mechanisms leading to HCC, the precise actions of HBx remain to be elucidated. miRNAs are involved in multiple biological processes related to HCC, such as cell proliferation, apoptosis, and migration. As HBx can directly or indirectly lead to hepatocellular carcinoma, the role of HBx in HCC through the regulation of miRNA expression has been actively investigated. A study by Li [30] showed that HBx-induced miR-21 expression was highly dependent on the activation of IL-6STAT3 pathway, which suggested a unique viral oncogenic pathway that could aberrantly affect a network of gene expression.
HBx contributes to HCC pathogenesis through regulating miRNA expression Increasing evidence has demonstrated that HBx may affect cancer cell proliferation, differentiation, and migration through regulating miRNA expression in HBV-related HCC. The specific functions of HBx in these processes are described below (Fig. 1). Aberrant DNA methylation Recent reports have indicated that HBx potentially triggers epigenetic modifications in some genes during hepatocarcinogenesis and that DNA hypermethylation may especially play crucial roles in cancer initiation [31]. The aberrant expression of members in the DNA methyltransferase (DNMT) family, including DNMT1, DNMT3A, and DNMT3B, can increase global gene silencing, thereby permitting cancer cell growth [32]. DNMT activation is reportedly a main pathway for hypermethylation. Wei et al. [33] examined HBx-expressing HepG2 cells and found a significant decrease in miR-101 expression and a significant increase in its direct downstream target gene, DNA methylation transferase (DNMT3A). The overexpression of HBx was shown to down-regulate miR-101 expression and up-regulate DNMT3A expression. The up-regulation of DNMT3A by HBx was found to partly occur through suppressing miR101 expression, resulting in the silencing of many genes, such as glutathione S-transferase pi 1 (GSTP1), RASSF1, RUNX3, and PRDM2. In addition, a study by Huang [34] showed that HBx can inhibit miR-152 and up-regulate DNMT1, leading to a decrease in global DNA hypermethylation and an increase in the methylation levels of two tumor suppressor genes, GSTP1 and E-cadherin 1 (CDH1). Additionally, Tao Zhang’s study [35] showed that HBx is able to inhibit tumor suppressor miR-
Tumor Biol.
Fig. 1 The line diagram which contain all possible molecular players and their suggested connection. It briefly explain the role of HBx contributing to HCC pathogenesis such as DNA methylation, cell proliferation, and migration through regulating miRNA expression
205 and to enhance hepatocarcinogenesis through inducing hypermethylation of miR-205 promoter during their interaction. Similar results were also demonstrated in Xufu Wei’s study [36]. Altogether, these results suggest that the tumor suppressor role of miRNAs in HCC, partly controlled by HBx, can be accomplished by epigenetic changes. Cell proliferation and apoptosis Normal cells generally undergo apoptosis after continuous proliferation in culture due to the procedural regulation of body factors like contact inhibition, while tumor cells are not subject to this mechanism. miRNAs are key regulators of apoptosis and a variety of biological processes, including cell differentiation and proliferation [37]. The HBx protein can influence both cell proliferation and apoptosis by regulating the expression of at least 18 cellular miRNAs. One of these miRNAs, miR-29c, is a tumor-
related miRNA significantly expressed at low levels in HBV-related HCC. Overexpression of this miRNA in HepG2 cells effectively suppressed tumor necrosis factor alpha-induced protein 3 (TNFAIP3) expression and HBV DNA replication, inhibiting cell proliferation and inducing apoptosis [38]. Another miRNA, lethal-7 (let-7), is down-regulated in HCC patients [39]. Let-7a was found to negatively regulate cell proliferation, at least in part, by targeting STAT3 [40, 41]. Furthermore, when the let-7a precursor was introduced into HepG2 cells, there was a significant 30 % decrease in cell proliferation. However, cell proliferation significantly increased upon the introduction of a let-7a inhibitor. The let-7 miRNA has a significant inverse correlation with HBx expression, and HBx can influence both cell proliferation and apoptosis by regulating let-7a expression [42].
Tumor Biol.
The infinite proliferation of tumor cells is closely related to the expression of cyclins, and miRNA dysregulation promotes HCC cell proliferation by altering cyclin levels. For example, the miR-16 family (miR-15/16), which suppresses the tumorigenicity of HBx-expressing HepG2 cells by arresting and inducing apoptosis in the G1 phase, is down-regulated by HBx in HepG2 cells. CCND1 is a target of the miR-16 family, which functions in the G1/S transition of the cell cycle; thus, reduced expression of miR-16 accelerated the growth and cell cycle progression of HepG2 cells [43]. However, Wu’s study [44] suggested that HBx could suppress the expression of miR-15b, which directly targeted fucosyltransferase 2 (FUT2) and then increase levels of Globo H, a cancerassociated carbohydrate antigen, to enhance HCC cell proliferation. Other miRNAs, such as miR-138 and miR-199a-5p, whose direct targets are cyclin D3 (CCND3) and clathrin heavy chain (CHC), respectively, also play an important role in HBV-HCC tumorigenesis [45]. Tumor invasion and metastasis The high mortality of patients with HCC is mainly due to the high recurrence rate and metastasis of HCC. Researchers are currently attempting to identify specific miRNAs whose aberrant expression is related to HCC invasion and metastasis. One such microRNA, miR148a, is repressed by HBx, promoting cancer growth and metastasis in HCC [46, 47]. Overexpression of miR-148a in hepatoma cells reduces the expression of hematopoietic preB-cell leukemia transcription factor-interacting protein (HPIP), leading to AKT and ERK repression and subsequent inhibition of mTOR through the AKT/ERK/FOXO4/ATF5 pathway. miR-148a also reduced the epithelial-tomesenchymal transition as well as HCC growth, invasion, and metastasis through inhibition of HPIP-mediated mTOR signaling in cultured cells or in a mouse xenograft model [48]. Nuclear factor kappa B (NFκB) promotes the invasive activities of hepatoma cells by inhibiting the expression of fibronectin type III domain containing 3B (FNDC3B). miR143, which is transcribed by NFκB and up-regulated in hepatoma cells, directly targets FNDC3B and promotes cell invasion/migration and tumor metastasis in HBV-HCC. The intratumoral administration of miR-143 promotes tumor invasion in vivo, while blocking miR-143 inhibits HBV-HCC metastasis in p21-HBx transgenic mice [49]. Similarly, miR29a is involved in the HBx-mediated regulation of migration of hepatoma cells through its target gene PTEN in a cell culture model [50]. When a miR-29a-specific inhibitor was used to block miR-29a expression in HepG2-X cells, in vitro assays showed a significant reduction in the migration of HepG2-X cells. The ability of miR-29a to promote Akt phosphorylation may be relevant, as the knockdown of Akt expression was shown to abolish the increased migration ability mediated by miR-29a in HepG2 cells [45]. In a study by Jing Zhao [51], the authors found that HBx could elevate AEG-1
protein level without altering its mRNA level. When blocking AEG-1 expression with siRNA in HBx-transfected cells, the HBx-induced cell migration was significantly suppressed. While further study indicated that miR-375 and miR-136 that targeted AEG-1 were down-regulated with HBx expression. Moreover, overexpressing miR-375 and miR-136 could effectively attenuate HBx-mediated AEG-1 elevation and cell migration.
Conclusion Many tumors are closely related to viral infections. Some examples include the relationship between HBV and hepatocellular carcinoma, Epstein-Barr virus and nasopharyngeal carcinoma, and HPV and cervical cancer. Growing evidence shows that some viruses influence the biological characteristics of their host cells through their own miRNAs or through changing host miRNAs. Thus, the interaction of host miRNAs, viral miRNAs, and corresponding target genes regulates numerous biological processes. As described above, HBx partly participates in the development of HCC by controlling the expression of miRNAs and plays a key role in HCC metastasis and invasion. Therefore, clarifying the role of HBx and miRNAs will contribute to a comprehensive molecular understanding of HCC formation, providing a theoretical basis for the treatment of HCC. Some studies found that HBx was able to induce abnormal lipid metabolism of hepatoma cells via up-regulating miR-205-targeted ACSL4 [52, 53]. Meanwhile, HBx mutants such as HBxD127 and HBx COOH-terminus could be involved in hepatocarcinogenesis too [54, 55]. Further studies are urgently required to clarify the role and mechanisms of HBx-mediated HCC occurrence and development.
Conflict of interest None
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REVIEW
The impact of hepatitis B virus x protein and microRNAs in hepatocellular carcinoma: a comprehensive analysis Li Lin & Xiaomao Yin & Xiumei Hu & Qian Wang & Lei Zheng
Received: 24 April 2014 / Accepted: 18 September 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Keywords microRNAs . Hepatitis B virus x protein . Hepatocellular carcinoma . Hepatitis B virus infection
protein, is an essential factor for HBV replication and is thought to play a key role in the molecular pathogenesis of HBV-related HCC (HBV-HCC) [3, 4]. microRNAs (miRNAs), small endogenous non-coding RNAs which regulate target gene expression at the posttranscription level [5–7], are also believed to have a role in hepatocarcinogenesis. Over 50 % of genes that encode miRNAs are located at cancer-associated genomic regions that are fragile or aberrantly expressed in a wide variety of tumors [8, 9]. Several studies have recently found that HBx can promote the proliferation, differentiation, migration, and inhibit apoptosis of hepatoma cells by regulating miRNA expression. Further understanding of the roles of miRNAs and HBx in HBV-related HCC could therefore provide insights into the mechanisms of tumor development and novel therapeutic strategies.
Introduction
miRNA expression correlates with HCC pathogenesis
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and one of the most common malignant tumors in China. Due to its late time to diagnosis and high recurrence rate, HCC is an aggressive human cancer and the second leading cause of death in the world [1]. Hepatocarcinogenesis is a complex and multistep biological process that involves multiple factors. One well-known risk factor for HCC is chronic infection with hepatitis B virus (HBV), which occurs in more than 80 % of primary liver cancers [2]. Hepatitis B virus x protein (HBx), a ∼17 kDa
Since the first identification of a miRNA, lin-4, in Caenorhabditis elegans in 1993, many additional miRNAs have been identified in a wide range of species such as animals, plants, and viruses [10, 11]. miRNAs are small and non-coding endogenous RNAs, approximately 22 nucleotides (nts) in length, that inhibit gene expression by preferentially interacting with the 3′-untranslated regions (3′-UTRs) of target mRNAs. These interactions may either inhibit the translation of targeted mRNAs or induce the degradation of these targets [12, 13]. Recent studies have demonstrated that abnormal miRNA expression plays an important role in the formation of a wide variety of tumors and is directly involved in the occurrence, development, diagnosis, and staging of HCC. In the early stages of tumorigenesis, miRNAs are aberrantly expressed in carcinoma and adjacent HCC tissue.
Abstract microRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs, approximately 22 nucleotides (nts) in length, widely found in animals, plants, and viruses. Mature miRNAs control gene expression at a posttranscriptional level through blocking protein translation or inducing mRNA degradation. Many recent studies have shown that hepatitis B virus x protein (HBx), a viral protein with a crucial role in hepatogenesis, is associated with the regulation of miRNAs. This interaction impacts fundamental tumor processes, such as cell proliferation, differentiation, and apoptosis. In this review, we summarized the recent literature on the roles of HBx-regulated miRNAs in the pathogenesis of hepatocellular carcinoma.
L. Lin : X. Yin : X. Hu : Q. Wang : L. Zheng (*) Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, No.1838 North of Guangzhou Road, Baiyun District, Guangzhou 510515, People’s Republic of China e-mail: [email protected]
Tumor Biol.
Murakami et al. analyzed miRNA expression profiles in HCC samples, adjacent non-tumorous tissue (NT) samples, and chronic hepatitis samples (CH) through microarray and Northern blot assays [14]. The results showed that 30 miRNAs are significantly and differentially expressed in HCC compared with the corresponding NT specimens. Furthermore, the expression level of some miRNAs was correlated with the extent of HCC differentiation. For example, three miRNAs (miR-20, miR-18, and pre-miR-18) were significantly higher in HCC samples that exhibited low differentiation compared to those with moderate or high differentiation. miRNA expression has also been correlated with gender; in a study involving 80 male and 36 female patients with HBV-related HCC, the expression of miR-22 was increased more in men than in women, which may partly explain the high incidence of HBV-associated HCC in the male population [15]. Subsequent studies have shown that miRNAs are involved in HCC proliferation and migration [16, 17]. For example, miR-122 is down-regulated in HBV-infected HCC, permitting viral replication and cell proliferation by targeting the transcripts encoding NDRG3, a member of the N-myc downregulated gene family and NDRG3-mediated pathways [18]. In another example, the development of HBV-HCC to portal vein tumor thrombus (PVTT) and elevated TGF-β activity suppresses the expression of miR-34a, leading to enhanced production of chemokine CCL22, which recruits regulatory T (Treg) cells to facilitate immune escape [19].
HBx plays an important role in HCC pathogenesis The X gene is the smallest of four overlapping open reading frames (S, C, Pol, and X) in the HBV genome and is the most common HBV gene integrated into the host genome. HBx, a regulatory protein encoded by X gene, is generally synthesized on the ribosomes of rough endoplasmic reticulum in the cytoplasm. The protein then enters nuclei via nuclear pores and regulates a variety of gene sequences in the molecular pathogenesis of HBV-related HCC [20–22]. The mechanism of HBx in the occurrence of HCC has been reported, including gene transcription, cell cycle regulation, and several signaling pathways [23]. After the X gene integrates into the host, HBx directly or indirectly transactivates gene promoters or enhancers. HBx thereby activates a wide range of key cell signaling pathways of cells in HCC [24, 25]. Another important mechanism of the X gene in HCC is its ability to influence the inactivation and mutation of the p53 tumor suppressor [26, 27]. As p53 influences cell growth by regulating DNA replication, the mutation or deactivation of p53 results in the acceleration of cell proliferation [28]. Subsequently, some new mechanism of HBx in connects with HBV infections to the development of HCC has been
reported. For example, in Gearhart TL’s study [29], the authors demonstrated that HBx could modulate hepatocyte proliferation pathways to stimulate viral replication. HBx induces normally quiescent hepatocyte to enter the G1 phase of the cell cycle, which is required for HBV replication. Although there is a preliminary understanding of HBx mechanisms leading to HCC, the precise actions of HBx remain to be elucidated. miRNAs are involved in multiple biological processes related to HCC, such as cell proliferation, apoptosis, and migration. As HBx can directly or indirectly lead to hepatocellular carcinoma, the role of HBx in HCC through the regulation of miRNA expression has been actively investigated. A study by Li [30] showed that HBx-induced miR-21 expression was highly dependent on the activation of IL-6STAT3 pathway, which suggested a unique viral oncogenic pathway that could aberrantly affect a network of gene expression.
HBx contributes to HCC pathogenesis through regulating miRNA expression Increasing evidence has demonstrated that HBx may affect cancer cell proliferation, differentiation, and migration through regulating miRNA expression in HBV-related HCC. The specific functions of HBx in these processes are described below (Fig. 1). Aberrant DNA methylation Recent reports have indicated that HBx potentially triggers epigenetic modifications in some genes during hepatocarcinogenesis and that DNA hypermethylation may especially play crucial roles in cancer initiation [31]. The aberrant expression of members in the DNA methyltransferase (DNMT) family, including DNMT1, DNMT3A, and DNMT3B, can increase global gene silencing, thereby permitting cancer cell growth [32]. DNMT activation is reportedly a main pathway for hypermethylation. Wei et al. [33] examined HBx-expressing HepG2 cells and found a significant decrease in miR-101 expression and a significant increase in its direct downstream target gene, DNA methylation transferase (DNMT3A). The overexpression of HBx was shown to down-regulate miR-101 expression and up-regulate DNMT3A expression. The up-regulation of DNMT3A by HBx was found to partly occur through suppressing miR101 expression, resulting in the silencing of many genes, such as glutathione S-transferase pi 1 (GSTP1), RASSF1, RUNX3, and PRDM2. In addition, a study by Huang [34] showed that HBx can inhibit miR-152 and up-regulate DNMT1, leading to a decrease in global DNA hypermethylation and an increase in the methylation levels of two tumor suppressor genes, GSTP1 and E-cadherin 1 (CDH1). Additionally, Tao Zhang’s study [35] showed that HBx is able to inhibit tumor suppressor miR-
Tumor Biol.
Fig. 1 The line diagram which contain all possible molecular players and their suggested connection. It briefly explain the role of HBx contributing to HCC pathogenesis such as DNA methylation, cell proliferation, and migration through regulating miRNA expression
205 and to enhance hepatocarcinogenesis through inducing hypermethylation of miR-205 promoter during their interaction. Similar results were also demonstrated in Xufu Wei’s study [36]. Altogether, these results suggest that the tumor suppressor role of miRNAs in HCC, partly controlled by HBx, can be accomplished by epigenetic changes. Cell proliferation and apoptosis Normal cells generally undergo apoptosis after continuous proliferation in culture due to the procedural regulation of body factors like contact inhibition, while tumor cells are not subject to this mechanism. miRNAs are key regulators of apoptosis and a variety of biological processes, including cell differentiation and proliferation [37]. The HBx protein can influence both cell proliferation and apoptosis by regulating the expression of at least 18 cellular miRNAs. One of these miRNAs, miR-29c, is a tumor-
related miRNA significantly expressed at low levels in HBV-related HCC. Overexpression of this miRNA in HepG2 cells effectively suppressed tumor necrosis factor alpha-induced protein 3 (TNFAIP3) expression and HBV DNA replication, inhibiting cell proliferation and inducing apoptosis [38]. Another miRNA, lethal-7 (let-7), is down-regulated in HCC patients [39]. Let-7a was found to negatively regulate cell proliferation, at least in part, by targeting STAT3 [40, 41]. Furthermore, when the let-7a precursor was introduced into HepG2 cells, there was a significant 30 % decrease in cell proliferation. However, cell proliferation significantly increased upon the introduction of a let-7a inhibitor. The let-7 miRNA has a significant inverse correlation with HBx expression, and HBx can influence both cell proliferation and apoptosis by regulating let-7a expression [42].
Tumor Biol.
The infinite proliferation of tumor cells is closely related to the expression of cyclins, and miRNA dysregulation promotes HCC cell proliferation by altering cyclin levels. For example, the miR-16 family (miR-15/16), which suppresses the tumorigenicity of HBx-expressing HepG2 cells by arresting and inducing apoptosis in the G1 phase, is down-regulated by HBx in HepG2 cells. CCND1 is a target of the miR-16 family, which functions in the G1/S transition of the cell cycle; thus, reduced expression of miR-16 accelerated the growth and cell cycle progression of HepG2 cells [43]. However, Wu’s study [44] suggested that HBx could suppress the expression of miR-15b, which directly targeted fucosyltransferase 2 (FUT2) and then increase levels of Globo H, a cancerassociated carbohydrate antigen, to enhance HCC cell proliferation. Other miRNAs, such as miR-138 and miR-199a-5p, whose direct targets are cyclin D3 (CCND3) and clathrin heavy chain (CHC), respectively, also play an important role in HBV-HCC tumorigenesis [45]. Tumor invasion and metastasis The high mortality of patients with HCC is mainly due to the high recurrence rate and metastasis of HCC. Researchers are currently attempting to identify specific miRNAs whose aberrant expression is related to HCC invasion and metastasis. One such microRNA, miR148a, is repressed by HBx, promoting cancer growth and metastasis in HCC [46, 47]. Overexpression of miR-148a in hepatoma cells reduces the expression of hematopoietic preB-cell leukemia transcription factor-interacting protein (HPIP), leading to AKT and ERK repression and subsequent inhibition of mTOR through the AKT/ERK/FOXO4/ATF5 pathway. miR-148a also reduced the epithelial-tomesenchymal transition as well as HCC growth, invasion, and metastasis through inhibition of HPIP-mediated mTOR signaling in cultured cells or in a mouse xenograft model [48]. Nuclear factor kappa B (NFκB) promotes the invasive activities of hepatoma cells by inhibiting the expression of fibronectin type III domain containing 3B (FNDC3B). miR143, which is transcribed by NFκB and up-regulated in hepatoma cells, directly targets FNDC3B and promotes cell invasion/migration and tumor metastasis in HBV-HCC. The intratumoral administration of miR-143 promotes tumor invasion in vivo, while blocking miR-143 inhibits HBV-HCC metastasis in p21-HBx transgenic mice [49]. Similarly, miR29a is involved in the HBx-mediated regulation of migration of hepatoma cells through its target gene PTEN in a cell culture model [50]. When a miR-29a-specific inhibitor was used to block miR-29a expression in HepG2-X cells, in vitro assays showed a significant reduction in the migration of HepG2-X cells. The ability of miR-29a to promote Akt phosphorylation may be relevant, as the knockdown of Akt expression was shown to abolish the increased migration ability mediated by miR-29a in HepG2 cells [45]. In a study by Jing Zhao [51], the authors found that HBx could elevate AEG-1
protein level without altering its mRNA level. When blocking AEG-1 expression with siRNA in HBx-transfected cells, the HBx-induced cell migration was significantly suppressed. While further study indicated that miR-375 and miR-136 that targeted AEG-1 were down-regulated with HBx expression. Moreover, overexpressing miR-375 and miR-136 could effectively attenuate HBx-mediated AEG-1 elevation and cell migration.
Conclusion Many tumors are closely related to viral infections. Some examples include the relationship between HBV and hepatocellular carcinoma, Epstein-Barr virus and nasopharyngeal carcinoma, and HPV and cervical cancer. Growing evidence shows that some viruses influence the biological characteristics of their host cells through their own miRNAs or through changing host miRNAs. Thus, the interaction of host miRNAs, viral miRNAs, and corresponding target genes regulates numerous biological processes. As described above, HBx partly participates in the development of HCC by controlling the expression of miRNAs and plays a key role in HCC metastasis and invasion. Therefore, clarifying the role of HBx and miRNAs will contribute to a comprehensive molecular understanding of HCC formation, providing a theoretical basis for the treatment of HCC. Some studies found that HBx was able to induce abnormal lipid metabolism of hepatoma cells via up-regulating miR-205-targeted ACSL4 [52, 53]. Meanwhile, HBx mutants such as HBxD127 and HBx COOH-terminus could be involved in hepatocarcinogenesis too [54, 55]. Further studies are urgently required to clarify the role and mechanisms of HBx-mediated HCC occurrence and development.
Conflict of interest None
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