Hepatitis B virus X protein–elevated MSL2 modulates hepatitis B virus covalently closed circular DNA by inducing degradation of APOBEC3B to enhance hepatocarcinogenesis : Hepatology

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Original Articles: VIRAL HEPATITIS

Hepatitis B virus X protein–elevated MSL2 modulates hepatitis B virus covalently closed circular DNA by inducing degradation of APOBEC3B to enhance hepatocarcinogenesis

Gao, Yuen1,†; Feng, Jinyan1,†; Yang, Guang1; Zhang, Shuqin1; Liu, Yunxia1; Bu, Yanan1; Sun, Mingming1; Zhao, Man1; Chen, Fuquan1; Zhang, Weiying1; Ye, Lihong*,2; Zhang, Xiaodong*,1

Author Information
Hepatology 66(5):p 1413-1429, November 2017. | DOI: 10.1002/hep.29316

Abstract

Potential conflict of interest: Nothing to report.

Supported in part by the National Basic Research Program of China (973 Program, grant nos. 2015CB553703 and 2015CB553905), the National Natural Science Foundation of China (grant nos. 31670769 and 31470756), and the Project of Prevention and Treatment of Key Infectious Diseases (grant no. 2014ZX0002002‐005).

Hepatitis B virus (HBV) is a small 3.2‐kb DNA virus that selectively infects hepatocytes in human liver.1 The infection of HBV is closely related to the development of liver diseases.2 The global disease burden is large, with more than 350 million people chronically infected worldwide, of whom approximately one third develop severe HBV‐related complications.5 The HBV covalently closed circular DNA (cccDNA), which is organized into a minichromosome harboring a chromatin‐like structure in the nuclei of infected cells, serves as a template for transcription of all viral RNAs, including the pregenomic RNA as well as subgenomic RNAs.7 It has been reported that epigenetic regulation guard against HBV cccDNA that would otherwise hijack cellular functions for their own ends.9 DNA methylation, histone modification, nucleosome remodeling, and RNA‐mediated targeting regulate many biological processes.10 These modifications are interpreted by proteins that recognize a particular modification and facilitate the appropriate downstream biological effects.12

As a key viral oncoprotein, HBx plays crucial roles in the development of hepatocellular carcinoma (HCC),15 whose primary role is to enhance the transformation of liver cell because of its activities on cell cycle regulation, signaling pathways and DNA repair.17 It is rather remarkable that HBx is particularly required for HBV replication, which accomplishes this task by an unusual mechanism, enhancing transcription from extrachromosomal DNA templates through recruitment of HBx partners CBP, P300, and PCAF to cccDNA.19 Recently, it has been reported that APOBEC3A (A3A) and APOBEC3B (A3B) are responsible for the deamination of HBV cccDNA and overexpression of APOBEC3A or APOBEC3B can decrease the levels of HBV cccDNA in HepaRG cells infected by HBV particles.20

Male‐specific lethal 2 (MSL2), a dosage compensation gene of Drosophila, undergoes sex‐specific regulation and encodes a protein with a RING finger and a metallothionein‐like cysteine cluster.21 The human MSL2 ortholog is an E3 ubiquitin ligase, which is believed to ubiquitylate the tumor suppressor p53 as well as histone H2B for transcriptional control.22 It has been documented that HBx modulates the complex Smc5/6 for degradation through hijacking the cellular DDB1‐containing E3 ubiquitin ligase in the interaction of HBV with host cells.9 However, the significance of other E3 ubiquitin ligases, such as MSL2, in the interaction of HBV with host cells is intriguing.

In this study, we investigated the effect of key factors in host hepatoma cells on HBV replication. We first report that HBx‐elevated MSL2 is able to modulate HBV cccDNA in liver cancer cells, leading to hepatocarcinogenesis. Our findings provide insights into the mechanism by which MSL2 as a key factor affects HBV cccDNA. Therapeutically, MSL2 may serve as a target for the treatment of HBV‐related liver cancer.

Materials and Methods

CELL LINES, CELL CULTURE, AND CELL TRANSFECTION

The hepatoma cell lines HepG2, HepG2‐P, HepG2‐X, and HepG2.2.15 and PLC/PRF/5 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY). HepaRG, H7402, H7402‐P, and H7402‐X cell lines were cultured in Roswell Park Memorial Institute 1640 (Gibco) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin in 5% CO2 at 37°C. The HepAD38 cell line regulates HBV replication through the presence or absence of tetracycline in the culture medium. HepAD38 cells were cultured in DMEM/F12 medium (Life Technologies, Carlsbad, CA) supplemented with 10% heat‐inactivated fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μg/mL kanamycin, 400 μg/mL G418, and with 0.3 μg/mL tetracycline (for inhibition of HBV replication) or without any tetracycline (for induction of HBV replication). The cells were cultured in a 6‐, 24‐, or 96‐well plate for 12 hours and then transfected with plasmid or siRNAs. All transfections were performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol.

STATISTICAL ANALYSIS

Each experiment was repeated at least three times. Statistical significance was assessed by comparing the mean values (± standard deviation) using a Student t test for independent groups and was assumed for P < 0.05, P < 0.01, and P < 0.001. One‐way analysis of variance was performed to compare MSL2 expression in all individual HBV‐negative hepatoma cell lines with HBV‐related hepatoma cell lines. Pearson's correlation coefficient was used to determine the correlation between MSL2 and HBx messenger RNA (mRNA) levels in tumorous tissues. MSL2 expression in tumor tissues and matched adjacent nontumor tissues were compared using the Mann‐Whitney U test.

Results

MSL2 IS UP‐REGULATED IN HBV‐Tg MICE, CLINICAL HCC PATIENTS, AND HBV‐EXPRESSING HEPATOMA CELLS

It has been reported that epigenetic regulation guards against viral genomes that would otherwise hijack cellular functions for their own ends.9 To examine whether HBV transcription and replication were associated with histone modulation and to identify the host factors involved, we screened a panel of the known enzymes including lysine (K)‐specific demethylase 6A, lysine (K)‐specific demethylase 2A, lysine (K)‐specific methyltransferase 2C, male‐specific lethal 2 (MSL2), and histone deacetylase 6, involving modification of histone demethylation, methylation, ubiquitination, or deacetylation, by way of qRT‐PCR in the mixture of liver tissues from HBV‐transgenic (HBV‐Tg) mice13 (n = 30) (Fig. 1A). qRT‐PCR revealed that MSL2 levels were significantly up‐regulated in the liver tissues of 6‐month‐old HBV‐Tg mice (n = 30) relative to those of wild‐type mice (n = 30). Immunohistochemistry staining revealed that the positive rate (MSL2 immunoreactivity was graded as negative [score 0] and positive [scores 1 to 3] according to the previous study 24) of MSL2 was higher in liver tissues of HBV‐Tg mice (27/30; 9 with score 1, 7 with score 2, 11 with score 3) than that in wild‐type mice (5/30; 3 with score 1, 1 with score 2, 1 with score 3) (P < 0.001, chi‐square test) (Fig. 1B). The mRNA levels of MSL2 in the liver tissues of HBV‐Tg mice were validated further, which was consistent with our hypothesis (P < 0.01, Mann‐Whitney U test) (Fig. 1B). Viral particle concentration in the serum of mice was tested by way of qPCR (Supporting Fig. S1A). Then, we selected MSL2 for further study.

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Figure 1:
MSL2 is up‐regulated in HBV‐Tg mice, clinical HCC patients, and HBV‐expressing hepatoma cells. (A) The expression levels of epigenetic related genes (KDM6A, KDM2A, KMT2C, MSL2 and HDAC6) were examined by qRT‐PCR in the mixed liver tissues of HBV‐Tg mice compared to wild type mice; (B) Up panel showed the expression level of MSL2 examined by immunohistochemical staining in the liver tissues of wild type and HBV‐Tg mice. The bottom figure was the relative mRNA levels of MSL2 examined by qRT‐PCR in 30 liver tissues of wild‐type and HBV‐Tg mice (**P < 0.01, Mann‐Whitney U test). (C) The expression of MSL2 was examined by way of immunohistochemical staining in paired clinical tissues of HCC patients. (D) The relative mRNA levels of MSL2 were examined using qRT‐PCR in 55 paired HBV‐related HCC tissues and adjacent nontumorous liver tissues. *P < 0.05 (unpaired t test). (E) MSL2 expression was examined using RT‐qPCR and western blot analysis in HBV‐negative hepatoma cells and HBV‐related hepatoma cells (**P < 0.01). One‐way analysis of variance was used to compare MSL2 expression in all individual HBV‐negative hepatoma cell lines with HBV‐related hepatoma cell lines. (F) MSL2 expression was examined using qRT‐PCR and western blot analysis in HepG2 cells transiently transfected with control vector (2 μg/well) or pCH‐9/3091 (2 μg/well), HepaRG cells infected with 0, 500, or 1000 copies/cell HBV particles, and HepAD38 cells treated with 0, 0.1, or 0.3 μg/mL tetracycline. Error bars represent the mean ± standard deviation (n = 3). Statistically significant differences are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant (Student t test).

Given that HBV was the most important risk factor for the development of HCC,25 we assumed that MSL2 might be involved in the process of hepatocarcinogenesis mediated by HBV. In 143 HCC liver tissues, 86.7% (124/143) showed increased expression levels of MSL2; however, in the normal liver tissues, 1 out of 8 yielded positive MSL2 signal (Fig. 1C; Supporting Table S1). Then, the mRNA levels of MSL2 in 55 paired HBV‐related HCC and adjacent nontumorous liver tissues (Supporting Table S2) were measured, confirming this result (*P < 0.05, unpaired t test) (Fig. 1D).

Next, we examined the event in hepatoma cells. Our data showed that the MSL2 levels were up‐regulated in HBV‐related hepatoma cell lines, such as HepG2.2.15, PLC/PRF/5, and HepAD38, relative to those in HBV‐free cell lines, such as HepG2, H7402, and HepaRG cells (Fig. 1E). Moreover, the MSL2 level was enhanced by HBV in HepG2 cells transfected with pCH‐9/3091 vectors containing full‐length HBV DNA or HepaRG cells infected with HBV particles (Fig. 1F), in which HBV DNA was quantified by qPCR (Supporting Fig. S1B,C). In addition, we showed that the expression of MSL2 was down‐regulated in a dose‐dependent manner when HBV DNA was decreased in HepAD38 cells treated with tetracycline (Fig. 1F; Supporting Fig. S1D). Thus, we conclude that MSL2 is highly expressed in HBV‐Tg mice, clinical HCC patients, and HBV‐expressing hepatoma cells.

MSL2 ACTIVATES HBV cccDNA IN HEPATOMA CELLS

To better understand the effect of MSL2 on gene expression, we examined the profiling of MSL2‐modulated genes using complementary DNA microarrays containing 28,264 genes. Our data revealed that 419 genes were up‐regulated and 223 genes were down‐regulated as the criterion of 1.5‐fold differences when MSL2 was knocked down by MSL2 small interfering RNAs (siRNAs) in HepG2.2.15 cells (Supporting Fig. S2A, Supporting Table S7). Gene ontology function enrichment analysis revealed the list of target genes of MSL2 (Supporting Fig. S2B,F,G). Strikingly, we observed that a set of virus‐responding genes were significantly enriched with specific transcription factors, such as NF‐κB, IRF1, and NFAT, using the transcription factor target analysis tool (Supporting Fig. S2C). KEGG pathway analysis demonstrated that MSL2 might function through MAPK signaling pathway and cytokine–cytokine receptor interaction (Supporting Fig. S2H). Heat maps illustrated that knockdown of MSL2 by siRNAs could down‐regulate the expression levels of TNF, JUN, and MAP3K14 in the MAPK pathway (Supporting Fig. S2D). In addition, we validated the expression levels of virus‐responding genes by way of qRT‐PCR in the cells (Supporting Fig. S2E), confirming that MSL2 may play pivotal roles in responding to viruses.

Next, we evaluated the levels of hepatitis B e antigen (HBeAg), hepatitis B surface antigen (HBsAg), HBV DNA, and cccDNA in the HepG2.2.15 cells and HBV‐infected HepaRG cells treated with overexpression (or interference) of MSL2.26 As expected, we found that the levels of HBeAg, HBsAg, and HBV DNA were dose‐dependently increased in the supernatant of MSL2‐overexpressed HepG2.2.15 cells and HBV‐infected HepaRG cells (Fig. 2A; Supporting Fig. S2I). The HBV cccDNA levels were also elevated in the nucleus of the treated cells (Fig. 2B; Supporting Fig. S2J). Conversely, treatment of si‐MSL2 reverted the event in HepG2.2.15 cells (Supporting Fig. S2K) and HBV‐infected HepaRG cells (Supporting Fig. S2M) where cccDNA levels were also decreased simultaneously (Supporting Fig. S2L,N), suggesting that MSL2 contributes to the HBV replication and can activate HBV cccDNA in hepatoma cells. Moreover, we further generated the MSL2‐mutant HepG2.2.15 cells by employing the CRISPR/Cas9 system. Accordingly,28 we designed two sgRNAs containing nonoverlapping sequences targeting the MSL2 exon and selected two alleles, MSL2sgRNA1 and MSL2sgRNA2, with an independent genetic background (Fig. 2C). We found a significant decrease in SgRNA1‐treated cells and complete absence of protein expression in the SgRNA2 selected mutant cells (Fig. 2D). Using Sanger sequencing, we recognized that MSL2 knockout in SgRNA1 was heterozygous editing and the clones in SgRNA2 had undergone dual allelic inactivation, followed by functional studies (Supporting Fig. S2O). The levels of HBeAg, HBsAg, and HBV DNA were significantly decreased in the supernatant of MSL2‐mutant HepG2.2.15 cells (Fig. 2E). cccDNA levels were also significantly reduced in the cells (Fig. 2F), suggesting that MSL2 is responsible for HBV replication in hepatoma cells. Therefore, we conclude that MSL2 activates cccDNA and accelerates the life cycle of the virus in hepatoma cells.

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Figure 2:
MSL2 activates HBV cccDNA in hepatoma cells. (A) HBeAg and HBsAg levels were measured in the supernatant of MSL2 overexpressed HepG2.2.15 cells by way of enzyme‐linked immunosorbent assays. HBV DNA levels were measured in the supernatant of MSL2 overexpressed HepG2.2.15 cells by qPCR. (B) The cccDNA level in the nucleus was measured in the MSL2 overexpressed HepG2.2.15 cells by qPCR. (C) Schematic representation of MSL2 mutant allele generation using the CRISPR/Cas9 system. The primer sequences of sgRNAs and information for MSL2 are indicated. (D) The expression of MSL2 was examined by way of western blot analysis in HepG2.2.15 cells. (E) HBeAg and HBsAg levels were measured in the supernatant of MSL2 mutant HepG2.2.15 cells by way of enzyme‐linked immunosorbent assay. HBV DNA levels were measured in the supernatant of MSL2 mutant HepG2.2.15 cells. (F) HBV cccDNA levels in the nucleus were measured by way of qPCR in MSL2 mutant HepG2.2.15 cells. Error bars represent the mean ± standard deviation (n = 3). Statistically significant differences are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001 (Student t test).

MSL2 REDUCES THE PROTEIN LEVELS OF APOBEC3B TO MAINTAIN HBV cccDNA STABILITY

Given that MSL2 in the MOF complex was an E3 ubiquitin ligase for H2BK34, which directly regulated H3K4 and K79 methylation through trans‐tail crosstalk in cells,29 we examined the effect of MSL2 on different histone modifications loaded on cccDNA using quantitative chromatin immunoprecipitation (ChIP‐qPCR) under treatment with or without the siMSL2#2 in HepG2.2.15 cells. The recruitment of H3K4me3 or H3K79me2 to cccDNA in HepG2.2.15 cells posttransfection was slightly but not significantly changed, whereas nothing could be obtained by anti‐H2BK34ub. As a positive control, the level of HBx associated with cccDNA was greatly decreased in the siMSL2#2‐treated cells, suggesting that transcriptional repression of cccDNA in HepG2.2.15 cells treated with siMSL2#2 is not associated with the deposition of histone modification marks on cccDNA‐associated nucleosomes (Supporting Fig. S3A). Interestingly, 3D PCR showed that knockdown of MSL2 in HepG2.2.15 cells led a low temperature of cccDNA denaturation, whereas the ectopic expression of MSL2 in HepaRG cells after infection could reverse the effect (Supporting Fig. S3B), suggesting that MSL2 is involved in cccDNA degradation. It has been reported that APOBEC3A and APOBEC3B are responsible for the degradation of HBV cccDNA.20 We supposed that APOBEC3A and APOBEC3B might be involved in the modulation of cccDNA mediated by MSL2. Our data revealed that overexpression of APOBEC3A or APOBEC3B could decrease the levels of HBV cccDNA in HepaRG cells infected by HBV particles collected from the supernatant of HepG2.2.15 cells (Supporting Fig. S3C). Conversely, knockdown of APOBEC3A or APOBEC3B by siRNAs increased the levels of HBV cccDNA in HepG2.2.15 cells (Supporting Fig. S3D). Moreover, 3D PCR assays revealed that APOBEC3A or APOBEC3B could induce the deamination of HBV cccDNA (Supporting Fig. S3E), supporting the idea that APOBEC3A and APOBEC3B contribute to the modulation of HBV cccDNA in hepatoma cells. Next, we evaluated the effect of MSL2 on APOBEC3A and APOBEC3B, respectively. Interestingly, we observed that MSL2 siRNAs#1 and siRNAs#2 resulted in the up‐regulation of APOBEC3B, rather than APOBEC3A, in HepG2.2.15 cells at the protein level, but failed to work at the mRNA level (Fig. 3A). We validated the event in HepaRG cells cotransfected with a flag tag labeled APOBEC3B and HA tag labeled MSL2 (Fig. 3B), suggesting that APOBEC3B but not APOBEC3A might be involved in MSL2‐mediated activation of HBV cccDNA. Next, we found that in HepaRG cells, overexpression of MSL2 could increase HBV cccDNA, intracellular HBV DNA, and HBeAg. Although the effect could be blocked by APOBEC3B, in which HBV cccDNA was reduced as a control, HBV DNA and HBeAg were reduced by 10.36% and 5.68%, respectively. Furthermore, the effect induced by overexpression of MSL2 could be accelerated by knocking down APOBEC3B, where HBV cccDNA was increased by 34.8%, and HBeAg 10.81%, but HBV DNA showed little change (Fig. 3C), suggesting that MSL2 affects HBV cccDNA through APOBEC3B and further modulates HBV duplication. In HepG2.2.15 cells knocking down MSL2 and up‐regulating APOBEC3B at the same time, HBV cccDNA was reduced to 46%, which was lower than the effect of siMSL2 (59%) (Fig. 3D), suggesting that MSL2 and APOBEC3B could synergistically affect HBV cccDNA.

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Figure 3:
MSL2 reduces the protein levels of APOBEC3B to maintain HBV cccDNA stability. (A) Expression levels of A3A and A3B were measured by way of qRT‐PCR and western blot analysis, respectively, in HepG2.2.15 cells treated with MSL2 siRNAs. (B) The effect of exogenous MSL2 on flag‐labeled A3B was measured by way of western blot analysis in HepG2 cells. (C) HBV‐infected dHepaRG cells were treated at day 10 postinfection. Different regimens of treatment were applied as indicated. HBV markers including HBV DNA, HBV cccDNA, and HBeAg were tested. (D) HBV‐indicated markers were tested in HepG2.2.15 cells with different treatments. (E) The interaction between MSL2 and A3B was identified by way of co‐immunoprecipitation assays in HepG2 cells cotransfected or endogenously. (F) The direct interaction between bacterially expressed His‐APOBEC3B and GST‐MSL2 was examined by way of GST pull‐down assays in vitro. Error bars represent the mean ± standard deviation (n = 3). Statistically significant differences are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant (Student t test).

We then asked how MSL2 modulated HBV cccDNA. Immunoprecipitation assays revealed that MSL2 could endogenously or exogenously bind to APOBEC3B in HepG2 cells (Fig. 3E; Supporting Fig. S3F). Moreover, GST (Glutathione S transferase) pull‐down assays proved that MSL2 could directly bind to APOBEC3B, which shows a message in a way that MSL2 is able to directly interact with APOBEC3B to modulate HBV cccDNA stability (Fig. 3F). All in all, we conclude that MSL2 modulates the protein levels of APOBEC3B to maintain HBV cccDNA in hepatoma cells.

MSL2 INDUCES DEGRADATION OF APOBEC3B THROUGH UBIQUITYLATION

Given that MSL2 functioned as an E3 ubiquitin ligase22 and interacted with APOBEC3B directly as above, we speculated that MSL2 might induce degradation of APOBEC3B through ubiquitylation. Cycloheximide chase assays showed that the half‐life of APOBEC3B was significantly shortened in the presence of MSL2 in HepG2 cells, and MSL2 siRNAs reverted the effect in HepG2.2.15 cells (Fig. 4A). Moreover, using co‐immunoprecipitation and western blot analysis in HepG2 cells cotransfected with both exogenous HA‐MSL2 and flag‐APOBEC3B, we observed that MSL2 could mediate degradation of APOBEC3B by promoting its ubiquitylation. Conversely, knockdown of MSL2 by siRNAs resulted in the opposite effect in HepG2.2.15 cells by way of immunoprecipitation and western blot analysis (Fig. 4B), suggesting that APOBEC3B can be ubiquitylated by MSL2 in vivo. Next, in vitro ubiquitylation assays further validated that APOBEC3B could be ubiquitylated by MSL2. In addition, there was a clear increase at the level of ubiquitylation with time over the course of the reaction, and the nonubiquitylated APOBEC3B was largely depleted after 60 minutes under these conditions (Fig. 4C).

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Figure 4:
MSL2 promotes A3B ubiquitylation and degradation. (A) Exogenous overexpression of MSL2 in HepG2 cells or MSL2‐knockdown HepG2.2.15 cells were treated with 100 μg/mL cycloheximide (CHX), collected at the indicated time points, and then immunoblotted with the indicated antibodies. (B) The A3B ubiquitylation levels in vivo were measured by ectopic expression of MSL2 in HepG2 cells or MSL2 siRNAs in HepG2.2.15 cells. (C) Left: Ubiquitylation of A3B by purified MSL2 in vitro. Right: In vitro ubiquitylation of A3B by purified MSL2 at time points of 10, 30, 60, and 90 minutes as analyzed using western blot analysis with an anti‐ubiquitin antibody. (D) Evaluation of the lysine residues for response to MSL2‐induced degradation through single lysine mutation. Degradation responses were evaluated by way of cotransfection and western blot analysis in HepG2 cells. (E) Ubiquitylation of immunoprecipitated A3B and its lysine mutants by MSL2 exogenously were showed in HepG2 cells in vivo. (F) Ubiquitylation of A3B and its lysine mutants by purified MSL2 in vitro.

To identify the potential sites of ubiquitylation, we predicted the lysine residues of MSL2 using the online tool PhosphoSite Plus.30 By mutating each lysine to arginine, we found that the mutation of Lys 243 or Lys 320 rendered APOBEC3B less responsive to MSL2‐mediated down‐regulation (Fig. 4D), and the mutation of Lys 320 nearly completely unresponsive to MSL2‐mediated degradation and ubiquitylation in HepG2 cells (Fig. 4E). The same results were further validated by in vitro ubiquitylation assays (Fig. 4F), suggesting that Lys 320 of APOBEC3B is a key ubiquitylation site. Functionally, we measured that the MSL2‐elevated levels of HBV cccDNA could be rescued by overexpression of APOBEC3B in HepG2.2.15 cells. Meanwhile, ectopic expression of APOBEC3B‐K320R could also reduce the MSL2‐elevated HBV cccDNA levels (Supporting Fig. S4A), suggesting that the mutation of APOBEC3B on the ubiquitylation site does not affect its enzymatic activity, which is still available to destroy HBV cccDNA. To summarize, MSL2 activates HBV cccDNA by inducing degradation of APOBEC3B by ubiquitylation to maintain HBV cccDNA stability in hepatoma cells.

HBx CONTRIBUTES TO THE UP‐REGULATION OF MSL2 IN HEPATOMA CELLS

Next, we endeavored to identify which product of HBV, including HBc, HBs, or HBx, was ministered to the up‐regulation of MSL2 in hepatoma cells; to knock them down in HepG2.2.15 cells, we used siRNAs of HBc, HBs, or HBx, respectively. Our data revealed that the expression levels of MSL2 were affected by HBx, rather than HBs and HBc (Fig. 5A), implying that HBV may up‐regulate MSL2 through HBx. Next, we concentrated on the relationship between MSL2 and HBx. qRT‐PCR and western blot analysis revealed that silence of HBx in HepG2.2.15 or PLC/PRF/5 cells could down‐regulate MSL2 in a dose‐dependent manner (Fig. 5B). On the contrary, MSL2 were up‐regulated dose‐dependently by transiently overexpressing HBx in HepG2 and H7402 cells (Fig. 5C). We further performed assays in HepG2‐X/H7402‐X cells stably transfected with HBx and found that MSL2 was positively expressed with HBx in the engineered cells (Supporting Fig. S5A‐D, which was validated when si‐HBx was transfected into HepG2‐X/H7402‐X cells (Fig. 5D). Next, we employed an HBx‐transgenic (HBx‐Tg) mouse model accordingly31 to prove the result, which showed that the MSL2 levels were markedly increased in the tumor tissues from 24‐month‐old HBx‐Tg mice relative to those from 24‐month‐old wild‐type mice by way of qRT‐PCR and western blot analysis (Fig. 5E), suggesting that HBx is able to up‐regulate MSL2 in the HBx‐Tg mouse model. In clinical HCC samples with high levels of HBx mRNA/pgRNA (Supporting Fig. S5E), MSL2 levels were significantly elevated, offering robust evidence for our hypothesis (P < 0.001, r = 0.733; Pearson's correlation coefficient) (Fig. 5F). Thus, we made a tentative assumption that HBx contributes to the up‐regulation of MSL2.

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Figure 5:
HBx contributes to the up‐regulation of MSL2 in hepatoma cells. (A) Expression level of MSL2 was measured using qRT‐PCR in HepG2.2.15 cells when HBc, HBs, or HBx was knocked down by siRNAs, respectively. (B) MSL2 expression was determined by way of qRT‐PCR and western blot analysis in HepG2.2.15 or PLC/PRF/5 cells treated with si‐HBx. (C) MSL2 expression was examined by way of qRT‐PCR and western blot analysis in HepG2/H7402 cells treated with pcDNA3.1‐HBx. (D) MSL2 expression was examined by way of qRT‐PCR and western blot analysis in stably transfected HepG2‐X/H7402‐X cells treated with si‐HBx. (E) Expression of MSL2 at the levels of mRNA and protein was examined by way of qRT‐PCR in tumor tissues of 24‐month‐old HBx‐Tg mice versus the liver tissues of 24‐month‐old wild‐type mice. (F) Correlation between MSL2 mRNA levels and HBx mRNA levels was examined using qRT‐PCR in 55 cases of HCC tissues. P < 0.001, r = 0.733 (Pearson's correlation coefficient). The data are from three independent experiments. Error bars represent the mean ± standard deviation (n = 3). Statistically significant differences are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant (Student t test).

HBx ACTIVATES MSL2 PROMOTER THROUGH TRANSCRIPTIONAL FACTOR FoxA1

Then, we managed to clarify the mechanism by which HBx up‐regulated MSL2. The MSL2 promoter core region was identified. Various lengths of the MSL2 5′ flanking region, including −1317/+151 (pGL3‐1468), −1317/−316 (pGL3‐1002), −339/+151 (pGL3‐490), −1317/−830 (pGL3‐488), −901/−316 (pGL3‐586), −1317/−1167 (pGL3‐151), and −1184/−830 (pGL3‐355), were cloned and transiently transfected into the HepG2‐X (or H7402‐X) cells, respectively. The luciferase reporter gene assays indicated that the fragment of pGL3‐151 exhibited the maximum luciferase activities than the others (Fig. 6A), indicating that the region of −1317/−1167 might be the core region of MSL2 promoter regulated by HBx. Our data next revealed that the relative luciferase activities of full‐length pGL3‐1468 and fragment pGL3‐151 were remarkably increased by HBx in HepG2/H7402 cells in a dose‐dependent manner (Supporting Fig. S6A,B). Conversely, the effect was reverted dose‐dependently when HBx was knocked down (Supporting Fig. S6C,D). However, the pGL3‐Basic control construct showed no activation by HBx.2 Previous studies found that HBx functioned as a coactivator to modulate gene transcription2; accordingly, we proved that HBx failed to interact with the MSL2 promoter for coactivating its transcription (Supporting Fig. S6E), suggesting that HBx may activate MSL2 promoter by up‐regulating transcription factors.

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Figure 6:
HBx activates MSL2 promoter through transcriptional factor FoxA1. (A) HepG2‐X and H7402‐X cells were transiently transfected with pGL3‐Basic or the reporter constructs containing various lengths of the 5′ flanking region of the MSL2 gene as indicated. The results were obtained as the relative luciferase activity against the activity of pGL3‐Basic. Data are shown as the mean ± standard deviation of three independent experiments. (B) Luciferase activities of pGL3‐151 were measured in HepG2 and H7402 cell lines transfected with control vector or pcDNA3.1‐FoxA1(left) and HepG2‐X and H7402‐X cell lines transfected with si‐control or si‐FoxA1 (right). (C) Luciferase activities of pGL3‐151 and pGL3‐151‐mut were tested in the HepG2 and H7402 cell lines transfected with control vector or pcDNA3.1‐FoxA1. (D) Luciferase activities of pGL3‐151 were examined in the HepG2 and H7402 cell lines with single treatment of pcDNA3.1‐HBx or cotreatment of pcDNA3.1‐HBx and FoxA1 siRNAs, respectively. (E) ChIP assay in HepG2 cells immunoprecipitated with anti‐FoxA1 antibody or control immunoglobulin G after transient transfection with control vector or pcDNA3.1‐FoxA1. The bottom panel shows the enrichment data in the MSL2 promoter analyzed by way of qPCR. Error bars represent the mean ± standard deviation. ***P < 0.001 (Student t test). (F) Same assay as in panel D but immunoprecipitated in HepG2‐X cell lines after transient transfection with si‐control or si‐HBx. Error bars represent the mean ± standard deviation (n = 3). Statistically significant differences are indicated as follows: **P < 0.01; ***P < 0.001 (Student t test).

Next, we performed a search for possible transcription factor‐binding sites in the promoter region −1317/−1167 using MatInspector (www.genomatix.de/online_help/help_matinspector/matinspector_help) and observed that the promoter region −1317/−1167 contained several transcription factor‐binding elements, such as FoxA1, Sox5, and MYT1L (Supporting Fig. S6F). We previously reported that HBx could up‐regulate YAP and others showed that YAP elevated FoxA1.2 Thus, we speculated that HBx might activate promoter of MSL2 through FoxA1. Luciferase reporter gene assays showed that the ectopic expression of FoxA1 activated full‐length MSL2 promoter and its core region in HepG2 (or H7402) cells, but FoxA1 siRNAs reverted the effect in HepG2‐X (or H7402‐X) cells (Fig. 6B; Supporting Fig. S6G). To further validate the result, we constructed a mutant of the MSL2 promoter core region at the FoxA1 binding site. Compared with the wild‐type region, the mutant could not be regulated by FoxA1 in the cells (Fig. 6C; Supporting Fig. S6H), suggesting that FoxA1 may be involved in the activation of MSL2 promoter mediated by HBx. As expected, Fig. 6D and Supporting Fig. S6I yielded strong evidence that HBx modulated the MSL2 promoter through FoxA1. Interestingly, ChIP assays revealed that overexpression of HBx enhanced the interaction of FoxA1 with MSL2 promoter in HepG2 cells but that the knockdown of HBx diminished the effect in HepG2‐X cells, suggesting that HBx may up‐regulate FoxA1 to promote its activation for MSL2 (Fig. 6E,F). In conclusion, HBx activates MSL2 promoter through transcriptional factor FoxA1.

HBx UP‐REGULATES MSL2 VIA ACTIVATING YAP/FoxA1 SIGNALING

Accordingly, we next validated that FoxA1 was able to up‐regulate the expression of MSL2 in HepG2 (or H7402) cells in a dose‐dependent manner (Fig. 7A). Moreover, we observed that FoxA1 knockdown by siRNAs abolished the up‐regulation of MSL2 in a dose dependent manner in stable HBx‐transfected HepG2 (or H7402) cells (Fig. 7B). Furthermore, our data demonstrated that HBx‐upregulated MSL2 could be mostly blocked by siFoxA1 in HepG2 (or H7402) cells (Fig. 7C). On the contrary, in HepG2‐X (or H7402‐X) cells, MSL2 was decreased by siHBx, which was sharply rescued by the ectopic expression of FoxA1 (Fig. 7D), suggesting that FoxA1 is required for the activation of MSL2 regulated by HBx. Moreover, the expression levels of MSL2 were positively correlated with those of FoxA1 in clinical HCC tissues (P < 0.001, r = 0.655; Pearson's correlation coefficient), whereas FoxA1 mRNA levels were remarkably higher in clinical HCC tissues than those in peritumor tissues (Fig. 7E,F). In addition, ectopic expression of YAP could up‐regulate FoxA1 and MSL2 in a dose‐dependent manner at both the mRNA and protein level in HepG2 (or H7402) cells (Supporting Fig. S7A). In stable HBx‐transfected HepG2 (or H7402) cells, siYAP could efficiently abolish the effect of HBx on FoxA1 and MSL2 (Supporting Fig. S7B). Taken together, we conclude that HBx modulates MSL2 by activating YAP/FoxA1 signaling in hepatoma cells.

hep29316-fig-0007
Figure 7:
HBx up‐regulates MSL2 by activating YAP/FoxA1 signaling. (A,B) Expression of MSL2 and FoxA1 was detected by way of qRT‐PCR and western blot analysis in HepG2 and H7402 cell lines transfected with control vector or pcDNA3.1‐FoxA1(A) and HepG2‐X and H7402‐X cell lines transfected with si‐control or si‐ FoxA1 (B). (C,D) Expression of MSL2, FoxA1, and HBx was detected by way of qRT‐PCR and western blot analysis in HepG2 and H7402 cell lines with the single treatment of control vector, pcDNA3.1‐HBx or the cotreatment of pcDNA3.1‐HBx and si‐FoxA1, respectively (C) and HepG2‐X and H7402‐X cell lines with the single treatment of si‐control, si‐HBx, or cotreatment of si‐HBx and pcDNA3.1‐FoxA1, respectively (D). (E) Correlation between MSL2 mRNA level and FoxA1 mRNA level was examined using qRT‐PCR in 55 cases of HCC tissues. P < 0.001, r = 0.655 (Pearson's correlation coefficient). (F) Relative mRNA levels of FoxA1 were examined by way of qRT‐PCR in 30 paired HCC tissues and adjacent nontumorous liver tissues. ***P < 0.001 (Wilcoxon signed rank test). Error bars represent the mean ± standard deviation (n = 3). Statistically significant differences are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant (Student t test).

MSL2 ENHANCES THE GROWTH OF HEPATOMA CELLS IN VITRO AND IN VIVO

We then evaluated the significance of MSL2 in hepatocarcinogenesis mediated by HBV. Assays for 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide, 5‐ethynyl‐2′‐deoxyuridine, and colony formation revealed that the proliferation ability of HepG2.2.15 cells was reduced significantly by treatment with MSL2 siRNAs#1 (100 nM) or MSL2 siRNAs#2 (100 nM) (Fig. 8A; Supporting Fig. S8A,B). Moreover, we observed that the tumorigenicity was remarkably suppressed in nude mice injected with HepG2.2.15 cells pretreated with MSL2 siRNAs#2 (100 nM) in vivo (Fig. 8B). The silence efficiency of MSL2 by si‐MSL2 was confirmed by way of western blot analysis in the tumor tissues from the nude mice (Fig. 8C). In addition, immunohistochemistry assays revealed that the expression levels of Ki‐67 were significantly decreased in the si‐MSL2–transfected group (Supporting Fig. S8C), suggesting that MSL2 is able to enhance the growth of hepatoma cells expressing HBV in vitro and in vivo.

hep29316-fig-0008
Figure 8:
MSL2 enhances the growth of hepatoma cells in vitro and in vivo. (A, D) The effect of MSL2 siRNAs (si‐MSL2#1 or si‐MSL2#2, 100 nM) on cell proliferation was determined by way of MTT assays in HepG2.2.15/HepG2‐X cells. (B, E) Left: Photographs of dissected tumors from nude mice. Right: Growth curve and average weight of the tumors transplanted with HepG2.2.15 cells or HepG2‐X cells pretreated with si‐MSL2#2 (100 nM) or control siRNAs (si‐Ctrl, 100 nM) in nude mice. (C, F) Protein expression levels of MSL2 or HBx were examined using western blot analysis in the tumor tissues from nude mice, respectively. Error bars represent the mean ± standard deviation (n = 3). Statistically significant differences are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001 (Student t test).

We further evaluated the role of MSL2 in the promotion of hepatoma cells mediated by HBx in vitro and in vivo. We found that the treatment with MSL2 siRNAs#1 (100 nM) or MSL2 siRNAs#2 (100 nM) significantly reduced the proliferation ability of HepG2‐X cells (Fig. 8D; Supporting Fig. S8D,E). Moreover, the tumorigenicity was remarkably decreased in nude mice injected with HepG2‐X cells pretreated with MSL2 siRNAs#2 (100 nM) (Fig. 8E). The silence efficiency of MSL2 by si‐MSL2 and the expression levels of HBx were also confirmed by way of western blot analysis (Fig. 8F). Immunohistochemistry assays showed that the Ki‐67 levels were reduced in the si‐MSL2–transfected group (Supporting Fig. S8F), suggesting that HBx promotes the growth of hepatoma cells through MSL2.

Next, we considered whether MSL2 could function as an oncogene to promote hepatocarcinogenesis in HBV‐free hepatoma cells. As expected, we observed that MSL2 was able to enhance the growth of HepG2 cells in vitro and in vivo (Supporting Fig. S8G‐I, S8J‐N), suggesting that MSL2 is a potential oncogene in hepatocarcinogenesis. Taken together, we conclude that MSL2 enhances the growth of hepatoma cells in vitro and in vivo.

Discussion

Chronic hepatitis B virus infection is a leading cause of cirrhosis and liver cancer.33 Most researchers have focused on the mechanism by which HBV leads to hepatocarcinogenesis; however, the influence of host liver cancer on HBV replication has been ignored. In the present study, we investigated the mechanism by which the key factors in host liver cells affect HBV cccDNA.

To better understand the mechanism of HBV cccDNA mediated by host factors, we examined the expression levels of epigenetic genes in the liver tissues of HBV‐Tg mice, from which we selected MSL2 for further experiments. Moreover, the results were validated in clinical HBV‐related HCC tissues that MSL2 was positively expressed with HBV production. It suggests that MSL2 may serve as a crucial mediator of HBV–host interaction. Microarray assays revealed the profiling of MSL2 responding genes that mainly played function in some biological processes involving cell activation and response to virus. We then validated that MSL2 was required for HBV replication and the activation of HBV cccDNA in hepatoma cells. Accordingly, we identified that MSL2 promoted HBV replication through degradation of APOBEC3B by ubiquitylation. Thus, our findings provide insight into the mechanism by which MSL2 modulates HBV cccDNA.

Interestingly, we demonstrated that HBV was able to modulate MSL2 in hepatoma cells. We observed that the expression levels of MSL2 were affected by HBx, rather than HBc or HBs. HBx‐Tg mice have been created in a variety of genetic backgrounds, with HBx expression driven by viral or cellular promoters.34 The expression levels of MSL2 were up‐regulated in the liver tissue of HBx‐Tg mice. Mechanically, we identified that HBx increased MSL2 at the transcriptional level by enhancing the binding property of FoxA1 to the promoter region of MSL2. We identified that HBx up‐regulates MSL2 by activating YAP/FoxA1 signaling. As a member of the MSL complex, MSL2 is required for the increased acetylation of histone H4 along the male X chromosome.35 As MSL2 underwent sex‐specific regulation in Drosophila,36 we first checked whether the expression of MSL2 had sex difference in HBV‐Tg mice and human HCC samples. The results revealed that MSL2 was not significantly highly expressed in males (Supporting Fig. S1E,F; Supporting Table S3). However, the role of MSL2 in hepatocarcinogenesis is not well documented. In this study, we found that MSL2 contributed to hepatocarcinogenesis in vitro and in vivo. Of note, we speculated that MSL2 in the HBV‐positive system enhanced the promotion of HCC depending on many aspects: on the one hand, MSL2 built a viral cccDNA persistence reservoir by decreasing APOBEC3B to accelerate HBV life cycle; on the other hand, MSL2 stimulated the oncogenic signaling, such as MAPK, TNF‐α, and so on, to promote the HCC together.

In conclusion, we present a model of MSL2 up‐regulation in the liver tissues of HBV‐Tg mice, clinical HCC patients and hepatoma cells (Supporting Fig. S9). MSL2 contributes to the activation of HBV cccDNA by inducing degradation of APOBEC3B through ubiquitylation. Moreover, HBx is responsible for the up‐regulation of MSL2 by activating YAP/FoxA1 signaling, forming a positive feedback loop of HBx/MSL2/cccDNA/HBV. Functionally, MSL2 promotes the growth of hepatoma cells in vitro and in vivo.

Author names in bold designate shared co‐first authorship.

Acknowledgment

We thank Lingyi Chen (Nankai University, Tianjin, China) for providing the vector of pX330‐U6‐Chimeric‐BB‐CBh‐hSpCas9.

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