Hepatitis E virus infection upregulates ING5 expression in vitro and in vivo

Abstract

Hepatitis E virus (HEV) is the major pathogen of viral hepatitis. Immunocompromised individuals infected by HEV are prone to chronic hepatitis and increase the risk of hepato-cellular carcinoma (HCC). Inhibitor of growth family member 5 (ING5) is a tumor suppressor that is expressed at low levels in cancer tumors or cells. However, the underlying relationship between ING5 and HEV infection is unclear. In the present study, acute and chronic HEV animal models are used to explore the interaction between ING5 and HEV. Notably, the expression of ING5 is significantly increased in both the livers of acute HEV-infected BALB/c mice and chronic HEV-infected rhesus macaques. In addition, the relationship between HEV infection and ING5 expression is further identified in human hepatoma (HepG-2) cells. In conclusion, HEV infection strongly upregulates ING5 expression both in vivo and in vitro, which has significant implications for further understanding the pathogenic mechanism of HEV infection.

Introduction

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide [1]. HEV infection usually causes acute self-limiting diseases with low mortality in the general population but leads to approximately 25% maternal death in pregnant women [ 2– 4] . Importantly, chronic HEV infection has been reported in immunocompromised patients, such as organ-transplant recipients [ 5, 6] , HIV-infected patients and cancer patients receiving chemotherapy [7]. HEV infection increases the risk of liver decompensation in patients with underlying chronic liver disease, including non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD) and chronic hepatitis C virus (HCV) [ 8– 12] , and aggravates the development of hepato-cellular carcinoma (HCC) in chronic hepatitis B virus (HBV)-infected patients. However, the pathogenesis by which HEV infection promotes the development of HCC is still unclear.

The inhibitor of growth (ING) family (members 1-5) plays important roles in the cell cycle, cell proliferation and growth, apoptosis and senescence [ 13, 14] . The ING family has been identified to be a tumor suppressor gene (TSG) family that interacts with the determinants of chromatin function and gene-specific transcription factors [13]. ING5 encodes a tumor suppressor protein that inhibits cell growth and induces apoptosis [13]. It contains a PHD-type zinc finger and interacts with the tumor suppressors p53 and p300, which are components of the histone acetyltransferase complex, indicating a role in transcriptional regulation [ 15, 16] . ING5 plays a crucial role in chromatin acetylation, oncogenic transformation and cancer development [16]. In cancer cells, ING5 transcript levels are often suppressed; for example, low expression of ING5 was found in esophageal squamous cell carcinoma (ESCC) [17], and suppressed ING5 was reported in HBV-induced HCC [ 18, 19] . The mechanism underlying the suppression of ING5 expression may involve abnormally high methylation level of the ING gene promoter, which are correlated with low transcript levels [13]. Thus, ING5 is considered to be a promising target for cancer treatment.

HEV infection in immunocompromised patients can lead to chronic hepatitis and progression to cirrhosis [ 20, 21] . However, whether ING5 is regulated during HEV infection is still unknown. In the present study, the expression of ING5 in an acute HEV infection animal model and a chronic HEV infection animal model was assessed to explore the potential regulatory relationship between HEV and ING5, providing novel insights into the pathogenic mechanisms of HEV infection.

Materials and Methods

HEV strains and cells

Hepatoma cells (HepG-2, ATCC, Washington, America) were maintained in DMEM (Sevicebio, Wuhan, China) supplemented with 10% FBS (NEWZERUM, Christchurch, New Zealand) at 37°C under 5% CO ₂. HepG-2 cells were infected with HEV (KM01 strain, KJ155502, isolated from an HEV-positive stool sample in Kunming City, genotype 4, 2×10 ⁶ copies/mL) as described in our previous studies [ 22, 23] . The cells were collected at 12, 24, 48 and 72 h post infection (hpi) for viral quantification by quantitative real-time PCR (qRT-PCR) or fixed at 48 and 72 hpi for immunofluorescence assay (IFA). For anti-HEV treatment experiment, HepG-2 cells were infected with HEV for 24 h followed by treatment with IFN-α (500 IU/mL, Kexing, Shenzhen, China) at 72 hpi. Cells were then collected for viral titer quantitation and western blot analysis.

Animal tissues

The animal tissues used in this study were collected from our previous studies [ 24, 25] . In brief, the livers of rhesus macaques with or without HEV infection at 272, 650, and 770 days post infection (dpi) and the livers and ovaries of BALB/c mice with or without HEV infection at 7, 14, 21, 28, and 36 dpi were collected. All animal tissues were preserved in the Laboratory of Viral Infection and Immunology of Medical Faculty at Kunming University of Science and Technology. The animal experimental protocol used in this study was approved by the Animal Protection and Use Committee of Kunming University of Science and Technology.

RNA isolation and qRT-PCR

Total RNA was extracted from tissue by using TRIzol reagent (15596-026, Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. Reverse transcription was performed using M-MLV reverse transcriptase (2641A, Takara, Tokyo, Japan). RNA expression levels were quantified by TB Green-based qRT-PCR (RR430A, Takara) with the primers of ING5 sense, 5′-CTTCATCCA AAACGATGATGC-3′ and ING5 antisense, 5′-CGTTCCAAAAAATA CTTTATT-3′. HEV RNA was quantified as described in a previous study [26]. The qRT-PCR was performed on a Bio-Rad CFX96TM Real-Time PCR System. GAPDH served as a loading control. The relative gene expression was calculated by the 2 ^–ΔΔCt method.

Western blot analysis

Cells and tissues were harvested and lysed with RIPA buffer (BD0031, Bioworld, Bloomington, USA). An equivalent amount of total protein was separated through 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane. Nonspecific binding sites were blocked with 5% skim milk, and the membrane was incubated overnight with primary antibodies, including anti-ING5 (10665-1-AP, Proteintech, Boston, USA, 1:1000 dilution) and anti-GAPDH (ab9485, Abcam, Cambridge, UK, 1:5000 dilution). HRP‐conjugated IgG (ab205718, Abcam, 1:5000 dilution) was used as the secondary antibody. The bands were visualized using an Immobilon ECL Kit (WBKLS0500, Milillpore, Billerica, USA) and exposed to X‐ray films. GAPDH served as the loading control.

Immunohistochemistry (IHC)

Tissue samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. The specimens were cut into 4-μm serial sections. Standard hematoxylin staining was performed, and the tissues were examined under a microscope. The tissues were deparaffinized, hydrated, heated in a water bath for antigen retrieval, and blocked with 3% hydrogen peroxide for 15 min. The tissue sections were incubated with primary antibodies, including anti-ING5 (10665-1-AP, Proteintech, 1:100 dilution) and anti-HEV (ab233244, Abcam, 1:200 dilution), overnight at 4°C. The sections were washed with PBS three times and incubated with HRP-labeled secondary antibodies (ab205718, Abcam) at 37°C for 1 h. Chromogenic detection was performed by using DAB (ab64238, Abcam) followed by hematoxylin counterstaining. Finally, sections were viewed under a microscope (E200, Nikon, Tokyo, Japan).

Immunofluorescence assay (IFA)

At 48 or 72 hpi, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10 min, and washed again with PBS. The cells were treated with primary antibodies, including anti-ING5 (10665-1-AP, Proteintech, 1:100 dilution) and anti-HEV ORF2 (ab233244, Abcam, 1:200 dilution), and then incubated with TRITC- and FITC-labeled secondary antibodies (AS026 and AS011, ABclonal, Boston, USA) at 37°C for 1 h. The nuclei were stained with DAPI (C1006, Beyotime, Shanghai, China). Finally, cells were observed under a confocal fluorescence microscope (Ts2, Nikon, Tokyo, Japan).

Statistical analysis

GraphPad Prism 9 and Image-Pro-Plus 6.0 were used for statistical analysis. Normally distributed data are expressed as the mean± standard deviation, and Student’s t test was used for comparison between two groups. The non-normal distribution measurement data are presented by the median (quartile), and the Mann-Whitney U test was used to compare the difference between two groups. P< 0.05 was considered to indicate statistically significant difference.

Results

Acute HEV infection significantly upregulates ING5 expression in vivo

HEV infects BALB/c mice and induces acute infection symptoms, as confirmed in our previous study [25]; for instance, acute hepatitis E and the viral genome are detectable for approximately 4 weeks. Accordingly, using this HEV infection model, we found that ING5 protein expression was significantly increased upon HEV infection for 7, 14, 21, 28, or 36 days after evaluating the liver tissues by IHC ( Figure 1A). The HEV and ING5 protein expression levels were quantified by grayscale analysis ( Figure 1B). Interestingly, the expression of ING5 and HEV tended to be similar, indicating a reciprocal interaction between ING5 expression and HEV infection. For example, the ING5 protein level was stably upregulated throughout the 3 weeks but was only slightly downregulated at 28 dpi. A similar trend was observed for HEV replication. However, no positive signal of the HEV ORF2 were detected at 36 dpi, indicating that the viruses were cleared and that ING5 expression was consistently decreased. Subsequently, the expressions of HEV RNA and ING5 at the mRNA and protein levels in the liver were quantified by qRT-PCR and western blot analysis respectively. Notably, the expression levels of the HEV RNA and ING5 mRNA and protein levels in the liver still increased at 36 dpi even after the virus was cleared ( Figure 1C,D).

Figure 1 .

Acute HEV infection increases ING5 expression in the livers and ovaries of BALB/c mice

(A) BALB/c mice were infected with HEV for different durations. IHC showed that HEV ORF2 was expressed at higher levels in infected, but not in control, animal liver tissue, and ING5 was expressed at higher levels in the infected group. Scale bar: 50 μm. (B) The positive signals of HEV particles and the ING5 protein in liver tissue were measured at 7, 14, 21, 28, or 36 dpi ( n=3). (C) HEV RNA and ING5 mRNA levels in liver tissues were quantified by qRT-PCR ( n=3). (D) ING5 protein levels in liver lysates were determined by western blot analysis ( n=3). (E) IHC was used to analyze HEV infection and ING5 expression in the ovary tissues. Scale bar: 50 μm. (F) The specific levels of HEV infection and ING5 expression in the ovaries were quantified and analyzed ( n=3). (G) HEV RNA and ING5 mRNA levels in ovary tissues were quantified by qRT-PCR ( n=3). (H) ING5 protein levels in ovary lysates were determined by western blot analysis ( n=3). Data are expressed as the mean±SD. ns, no statistical significance, * P<0.05; ** P<0.01; *** P<0.001.

HEV has multiple extra-hepatic replication sites, including the placenta, uterus, and ovary [ 25, 27, 28] . Accordingly, we further profiled the dynamics of ING5 protein expression in ovary tissues upon HEV infection. Increased ING5 and HEV levels were observed over time ( Figure 1E,F). As expected, a similar trend in ING5 activation and HEV replication was observed in liver and ovary tissues. The expressions of HEV RNA and ING5 in ovarian tissues further confirmed the potential relationship between HEV infection and ING5 activation ( Figure 1G,H). All these results showed a dynamic coordination of ING5 expression and HEV infection in the BALB/c murine model.

Chronic HEV infection potently increases ING5 expression in vivo

Chronic HEV infection has been identified in various patients, including recipients of solid-organ transplantation, those receiving immunosuppressive therapies, those with HIV coinfection, and those with liver disease. Persistent HEV infection has a high probability of causing liver fibrosis and rapidly progressing to cirrhosis. Thus, the potential interaction between ING5 regulation and HEV infection in chronic HEV-infected rhesus macaques was further investigated. Notably, the ING5 protein expression level was stably upregulated throughout the chronic HEV infection period (up to 770 dpi) ( Figure 2A). Subsequently, the level of ING5 protein was calculated ( Figure 2B). The increased HEV RNA and ING5 mRNA and protein expression levels further confirmed the potential cooperation between HEV infection and ING5 activation in the HEV chronic infected animal model ( Figure 2C,D), which is consistent with the findings in HEV-infected BALB/c mice. Notably, HEV infection activated the expression of ING5 in both acute and chronic infections.

Figure 2 .

ING5 expression in the liver tissue of chronic HEV-infected rhesus macaques

(A) IHC showing HEV ORF2 and ING5 protein expression in the liver tissues of chronic HEV-infected rhesus macaques at 272, 650, or 770 dpi. Scale bar: 20 μm. (B) Quantification of the positive signal intensity of the HEV ORF2 and ING5 proteins in liver tissues ( n=3). (C) HEV RNA and ING5 mRNA levels of liver tissues quantified by qRT-PCR ( n=3). (D) ING5 protein levels in liver lysates were determined by western blot analysis ( n=3). Data are presented as the mean±SD. * P<0.05; ** P<0.01; *** P<0.001.

Acute HEV infection robustly activates ING5 expression in vitro

Given the interaction between upregulated ING5 expression and HEV infection in BALB/c mice and rhesus macaques, we subsequently investigated the expression of ING5 in HEV-infected HepG-2 cells. First, HEV RNA and protein were detected by qRT-PCR and IFA to confirm viral replication after inoculation with the infectious virus. HEV RNA and antigens were detected at 12–72 hpi ( Figure 3A,B). Second, ING5 mRNA and protein expression levels were quantified by qRT-PCR, IFA, and western blot analysis at the same time points, and a similar trend was observed ( Figure 3A–C). In conclusion, HEV replication positively regulated ING5 expression.

Figure 3 .

HEV-infected HepG-2 cells increases ING5 expression

(A) HEV RNA and ING5 mRNA levels were quantified by qRT-PCR ( n=3) in HEV-infected HepG-2 cells. (B) HepG-2 cells were infected with HEV for 48 and 72 h. Co-localization of ING5 (green) and HEV ORF2 (red) was examined under a confocal microscope. (C) HepG-2 cells were infected with HEV for 12, 24, 48, or 72 h. ING5 protein levels in the lysates were determined by western blot analysis ( n=3). The nuclei were stained with DAPI (blue). Scale bar: 20 μm; 40×oil immersion objective. Data are presented as the mean±SD. *** P<0.001.

Inhibition of HEV infection suppresses ING5 activation

Based on the results showing that HEV infection activated ING5 expression in both in vivo and in vitro models, the degree of HEV infection was positively correlated with ING5 expression. To further confirm this interaction, we inhibited HEV infection by using a classical inhibitor interferon α (IFN-α) to analyze its effect on ING5 expression. The mRNA and protein expression of ING5 upon HEV inhibition was assessed by qRT-PCR and western blot analysis. The results showed that HEV was significantly suppressed by IFN-α, and the expression of ING5 was decreased accordingly ( Figure 4A,B), consistent with the reduction in the acute and chronic animal models. Overall, these results confirmed that the expression of ING5 was influenced by HEV infection.

Figure 4 .

Inhibition of HEV replication abolishes HEV-induced ING5 expression

HepG-2 cells were infected with HEV for 24 h and then treated with IFN-α (500 IU/mL) for 72 h. (A) HEV RNA and ING5 mRNA levels were quantified by qRT-PCR ( n=3). The uninfected group was set as the negative control. (B) ING5 protein level was determined by western blot analysis ( n=3). The uninfected group was set as the negative control (set as 1). Data are presented as the mean±SD. ** P<0.01; *** P<0.001.

Discussion

HEV infection is thought to be an acute infection, but chronic HEV infection is widely reported in immunocompromised patients with rapid progression to liver cirrhosis [21]. However, the pathogenesis of HEV-induced liver cirrhosis is unexplored. In the present study, significantly activated ING5 was observed in both acute HEV-infected BALB/c mice and chronic HEV-infected rhesus macaques. Although suppressed ING5 expression was found in HBV-induced HCC, increased ING5 expression was detected in HEV-infected human HCC cell lines (HepG-2), which indicated that there is an interaction between HEV infection and ING5 expression.

HEV infection causes a self-limiting disease, but acute liver failure or fulminant hepatic failure occurs in HEV-infected pregnant women [ 3, 4] . HEV infection is associated with chronic hepatitis and liver cirrhosis, and increases the risk of HCC [29]. However, the relationship between HEV and HCC has rarely been reported, and the role of HEV in the development of HCC is unclear. HEV infection activates severe inflammatory responses in the liver [25], and long-term persistent inflammatory stimuli aggravate liver damage, which may contribute to liver fibrosis, cirrhosis and ultimately HCC if no effective treatment is available. Furthermore, whether there is an association between increased apoptosis in the liver of HEV-infected animals/patients and the upregulation of ING5 related to HEV infection needs to be explored.

ING5 is a newly identified TSG. ING5 has been reported to inhibit the ability of lung cancer cells to invade normal tumor-adjacent tissues and distant metastasis through the AKT pathway [30]. ING5 overexpression in colorectal cancer cells suppresses cell proliferation, migration, and invasion, and promotes cell apoptosis, which can be reversed by ING5 knockdown [31]. Similarly, a negative correlation was found between the expression of ING5 and cell proliferation/migration in ovarian cancer cells [32]. ING5 inhibits the migration and invasion of esophageal cancer cells by downregulating the IL-6/CXCL12 signaling pathway [30]. In addition, HBV infection suppresses ING5 to promote the proliferation of HCC cells [18]. ING5 is a potential molecular marker for carcinogenesis and subsequent progression and a target for gene therapy. However, ING5 expression was significantly increased in an HEV-infected acute BALB/c murine model and a chronic rhesus macaque model, as well as in human hepatoma cells (HepG-2). An increase in ING5 caused by HEV infection may play a critical role in the development of HCC, and more attention should be given to this increase. For example, the acetylated proteins in the liver should be further identified, and their functions should be explored, which will improve the understanding of HEV pathogenesis.

In summary, HEV infection activates ING5 expression both in vivo (in BALB/c mice and rhesus macaques) and in vitro (in HepG-2 cells). Our results demonstrate that HEV infection positively regulates ING5 expression, and more attention should be given to exploring the potential regulatory mechanism, which will provide new insight into the pathogenesis of HEV infection.

COMPETING INTERESTS

The authors declare that they have no conflict of interest.

Funding Statement

This work was supported by the grants from the National Natural Science Foundation of China (Nos. 82260396 and 82302509), the Natural Science Foundation of Yunnan Province (No. 202401AS070057), the Yunnan Provincial Key Laboratory of Clinical Virology (No. 2023A4010403-01), the Program for Innovative Research Team (in Science and Technology) of the University of Yunnan Province (2020), the Program for Cultivating Reserve Talents in Medical Disciplines from the Health Committee of Yunnan Province (No. H-2019043), and the Technical Innovation Talents of Yunnan Province (No. 202205AD160008).