Upregulation of microRNA-328-3p by hepatitis B virus contributes to THLE-2 cell injury by downregulating FOXO4

Background Hepatitis B virus (HBV) remains a major cause of chronic hepatitis and hepatocellular carcinoma, and miRNAs play important roles in HBV pathogenesis. Our previous study has shown that miR-328-3p is upregulated in HBV-infected patients and serves as a potent predictor for the prognosis of HBV-related liver failure. Methods Here, the role of miR-328-3p in modulating cell injury in HBV-infected liver cells THLE-2 was investigated in detail. MiR-328-3p expression was examined using qRT-PCR. The levels of pro-inflammatory cytokines were measured using ELISA. HBV RNA and HBV DNA levels were quantified. The interactions between STAT3 and miR-328-3p promoter as well as miR-328-3p and FOXO4 were analyzed using chromatin immunoprecipitation (CHIP) assay and luciferase reporter assay, respectively. THLE-2 cell injury was evaluated by examining cell viability and apoptosis. Results HBV promoted expression of miR-328-3p through the STAT3 signal pathway and that increasingly expressed miR-328-3p downregulated its target FOXO4, leading to the promotion of cell injury in HBV-infected liver cells THLE-2. Conclusion These data demonstrate that HBV-STAT3-miR-328-3p-FOXO4 regulation pathway may play an important role in the pathogenesis of HBV infection.


Background
Hepatitis B virus (HBV) infection is a major public health problem and affects more than 400 million people worldwide [1,2]. Therefore, a better understanding of the molecular mechanisms underlying the HBV pathogenesis is warranted. A most recent study demonstrated that HBV can regulate apoptosis of liver hepatocellular cells HepG2 via upregulating expression of certain microR-NAs (miRNAs) [3], indicating the potential role of miR-NAs in development of HBV-related liver disease. miRNAs are small noncoding RNAs with 19-23 nucleotide in lengths and play an important regulatory role in various biological processes [4,5]. Recent studies have highlighted the role of miRNAs in HBV pathogenesis [6][7][8]. Our previous study showed that serum miR-146a-5p, miR-122-3p and miR-328-3p levels were upregulated in patients with acute-on-chronic liver failure (ACLF) and chronic hepatitis B (CHB) compared with the chronic asymptomatic HBV carriers (ASC) [9]. Furthermore, the increased levels of these three miRNAs positively correlate with the severity of liver inflammation in ACLF patients and may be useful to predict the severity of HBV-associated ACLF [9]. Among these three miRNAs, miR-146a-5p and miR-328-3p could distinguish ACLF from non-ACLF (ASC and CHB) with high values of specificity and sensitivity [9]. We have also previously demonstrated that miR-146a-5p enhances HBV Page 2 of 15 Fu et al. J Transl Med (2020) 18:143 replication through autophagy to promote aggravation of CHB [10]. MiR-146a has been shown to directly target CXCR4 [11], a gene involved in the advanced liver disease that is associated with hepatitis C virus or HBV [12]. Intriguingly, upregulation of miR-146a induced by HBV X protein (HBx) through NF-κB-mediated enhancement of miR-146a promoter activity contributes to hepatitis development [8]. However, whether HBV can regulate miR-328-3p and the underlying mechanism remain unclear.
Recent studies have suggested that HBV can induce activation of STAT3 (signal transducer and activator of transcription 3) in hepatocytes to foster its own replication but also to prevent apoptosis of infected cells [13,14]. Furthermore, knockout of hepatitis B surface antigen (HBsAg) inhibits STAT3 signaling, while overexpression of HBsAg induces a substantial accumulation of STAT3 phosphorylation [15]. STAT3 has been shown to play important roles in liver inflammatory responses [16]. More importantly, our bioinformatics analysis revealed that STAT3 was identified as a predicted transcription factor for miR-328-3p (LASAGNA-Search 2.0 and Trans-miR v2.0 database). Therefore, we speculated that HBV may promote transcription of miR-328-3p via activating STAT3 signaling.
miRNAs are post-transcriptional regulators that bind to the 3′-untranslated region (3′-UTR) of the target gene messenger RNA [17,18]. Our bioinformatics analysis demonstrated that FOXO4 was a putative target of miR-328-3p by harboring a miR-328-3p binding sequence in the 3′-UTR of its mRNA (Targetscan). FOXO4 has been identified as an endogenous inhibitor of NF-κB [19] which mediates induction of pro-inflammatory cytokines. In addition, we found that FOXO4 expression was down-regulated in HBV-infected human primary hepatocytes by analysis of the GEO database (GSE72068). Therefore, we hypothesized that miR-328-3p may promote hepatocyte injury by down-regulating FOXO4 to promote cellular inflammatory response.
In the current study, we evaluated in detail the roles of miR-328-3p in modulating cell injury in HBV-infected liver cells THLE-2. Furthermore, the putative mechanisms of the HBV-STAT3-miR-328-3p-FOXO4 regulatory axis in this process were also explored.

Human samples
Patients with acute-on-chronic liver failure (ACLF, n = 25, 19 male, mean age: 46.7 ± 10.1 years), chronic hepatitis B (CHB, n = 25, 17 male, mean age: 41.3 ± 11.5 years), chronic asymptomatic HBV carriers (ASC, n = 25, 18 female, mean age: 40.5 ± 10.8 years), and healthy volunteers as normal controls (NC, n = 25, 14 male, mean age: 40.5 ± 11.3 years) were enrolled from the Xiangya Hospital, from October 2016 to September 2017. ASC, CHB, and ACLF were diagnosed according to the Guideline of Prevention and Treatment for Chronic Hepatitis B (2015 Update) [20]. The diagnosis of ACLF was based on the following criteria: Acute hepatic insult manifested as jaundice (serum bilirubin ≥ 5 mg/dL or 85 µmol/L) and coagulopathy [international normalized ratio (INR) > 1.5 or prothrombin activity < 40%], complicated within 4 weeks by ascites and/or encephalopathy in a patient with CHB. CHB is defined as chronic HBV infection with clinical evidences of liver diseases such as biochemical, virological and histological features together with exclusion of other causes. Patients with CHB can be divided into HBeAg-positive and HBeAg negative. ASC is defined as HBsAg-positive, anti-HBe-positive with persistent normal serum alanine aminotransferase (ALT) and HBV-DNA < 2000 IU/mL, and no evidence of liver injury [21]. Patients exhibiting the following were excluded from the study: pregnancy, drug-induced liver injury, alcoholic liver disease, acute fatty liver, autoimmune liver disease, hepatolenticular degeneration, and liver failure after transplantation. The whole blood samples were collected from individuals and immediately centrifuged at 3000 rpm for 10 min at 4 °C. The resulting supernatant was serum that was stored at − 80 °C for subsequent experiments. All the experimental procedures were approved by the Ethics Committee of the Xiangya Hospital. Written informed consent was obtained from each participant.

Quantification of HBV RNA and HBV DNA
Quantification of HBV RNA in THLE-2 cells was performed as previously described [24]. HBV RNA was isolated using the EasyPure Viral RNA Kit (TransGen Biotech, Beijing, China) and reverse transcribed using RevertAid First Strand DNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). The levels of HBV RNA were detected by quantitative real-time PCR with a SYBR Green or TaqMan probe method using LightCycler 480 II Real-time PCR Detection System (Roche, Mannheim, Germany).
The level of HBV DNA was quantified by artus HBV PCR Kits CE (QIAGEN) according to the manufacturer's instructions.
Levels of HBsAg and HBeAg in THLE-2 cell supernatants were measured using their commercial ELISA kits (LifeSpan BioSciences) following the manufacturers' protocols.

Chromatin immunoprecipitation (CHIP)
CHIP assay was performed to analyze the interaction between STAT3 and miR-328-3p promoter using Simple ChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology). Briefly, cells were cross-linked with 1% formaldehyde, harvested, and then incubated on ice for 10 min in lysis buffer. The lysates were sonicated to shear DNA. Subsequently, the sheared chromatin was incubated with anti-STAT3 or normal serum IgG (Cell Signaling Technology) overnight at 4 °C. Then, protein G beads were added to the mixtures for 2 h of incubation. The antibody-bound protein/DNA complexes were washed and eluted from the beads. After reversing crosslinks, DNA was purified and subjected to qRT-PCR analysis using promoter-specific primers (miR-328-3p promoter-F: TGT CAA GGT TCA GCG ATG CT; miR-328-3p promoter-R: CCT TCT TCC TGC AGT CCC TG). An aliquot of chromatin that was not incubated with an antibody was used as the input control sample.

Cell proliferation assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to analyze the cell growth at different time points in THLE-2 cells. After different treatment, cells were plated into 96-well plates at a density of 1500-2000 cells/well for 24 h of incubation. Then MTT (Sigma; 20 μL; 5 mg/mL) was added into each well. After 4 h of incubation, the supernatant was discarded carefully and DMSO (Sigma; 150 μL) was added to dissolve the formazan product for 10 min. The optical density (OD) at 490 nm was measured by a microplate reader (Multiskan Mk3, Thermo Labsystems, Finland).

Cell apoptosis assay
An annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) cell apoptosis kit was used to qualify the cell apoptosis in THLE-2 cells. After the designated treatment, cells were harvested, washed twice with PBS, and re-suspended in the staining buffer provided in the kit. After this, Annexin V-FITC (5 µL) and PI (5 µL) were mixed with the cells for 10 min at room temperature in the dark. Finally, the apoptotic rates were detected using BD FACSAria flow cytometry (BD Biosciences, San Jose, CA, USA).

Statistical analysis
All statistical analyses were performed using SPSS version 16.0 (SPSS, Inc., Chicago, USA). The unpaired Student's t-test and one-way analysis of variance (ANOVA) were used to analyze differences between groups. Values are presented as the mean ± standard deviation (SD) from three independent experiments. P < 0.05 was considered to indicate a statistically significant difference.

FOXO4 suppresses HBV activity and attenuates cell injury under LPS and HBV stimulation
To explore the effect of FOXO4 expression on cell injury, we overexpressed and silenced FOXO4 in THLE-2 cells, followed by LPS and HBV stimulation. Successful overexpression and knockdown were confirmed by qRT-PCR (Additional file 2: Figure  S2A). FOXO4 overexpression inhibited cell proliferation (Fig. 6c), promoted cell apoptosis ( Fig. 6d and Additional file 2: Figure S2B), and decreased proinflammatory cytokines (Fig. 6e), under stimulation with LPS and HBV. In contrast, FOXO4 knockdown exerted the opposite effect ( Fig. 6c-e). These data suggest that FOXO4 attenuates cell injury. We next explored the effect of FOXO4 on HBV activity in THLE-2 cells. FOXO4 overexpression significantly abrogated the pHBV1.3-induced elevation in both RNA and DNA levels of HBV (Fig. 7a), and levels of HBsAg and HBeAg (Fig. 7b), and pro-inflammatory cytokines (Fig. 7c). Overall, these data indicate that FOXO4 overexpression suppresses HBV activity.

Discussion
The results described above in this study support a HBV-STAT3-miR-328-3p-FOXO4 regulatory cascade in regulating HBV-infected THLE-2 cell injury: (i) HBV increases miR-328-3p expression through a STAT3mediated activation of miR-328-3p transcription and (ii) HBV-induced miR-328-3p downregulates FOXO4 by targeting its 3′-UTR. The resulting reduced FOXO4 would lead to activation of the NF-κB pathway and cell inflammation and injury. Therefore, we propose that the HBV-STAT3-miR-328-3p-FOXO4 regulation pathway may underlie the pathogenesis related to chronic HBV infection. To our knowledge, the molecular details of STAT3-dependent activation of miR-328-3p by HBV and downregulation of FOXO4 by miR-328-3p in THLE-2 cells are reported here for the first time.
The pathogenesis of HBV-induced hepatitis is complicated. Previous studies have indicated that expression of serum miR-328 is associated with several diseases such as cancer [25,26], atherosclerosis [27], and immunityrelated diseases [28]. Importantly, downregulated miR-328 had an inhibitory effect on cell invasion and growth in hepatocellular carcinoma [29]. To date, multiple targets of miR-328 have been identified [27,29]. In HBVrelated studies, miR-328-3p has been identified by us as a potent predictor for the prognosis of HBV-related ACLF [9]. The results presented here showed that serum miR-328-3p expression was significantly upregulated in both CHB and ACLF patients compared with the normal control subjects. Our in vitro assay revealed that in human liver cell line THLE-2, HBV/HBc/HBx increased miR-328-3p expression. Importantly, miR-328-3p inhibitor significantly suppressed HBV activity and attenuated the HBV-mediated cell injury, whereas miR-328-3p mimic exerted the opposite effect. Therefore, our studies indicate that miR-328-3p plays an important role in the pathogenesis of HBV infection and serve as a potential therapeutic target for HBV infection.
STAT3 has been shown to play important roles in liver inflammatory responses [16]. Previous studies showed that HBV activates STAT3 signaling in hepatocytes to foster its own replication but also to prevent apoptosis of infected cells [14]. Furthermore, knockout of HbsAg inhibits STAT3 signaling, while overexpression of HBsAg induces accumulation of STAT3 phosphorylation [15]. Consistent with these observations, our results showed that HBV/HBc/HBx increased miR-328-3p expression and STAT3 phosphorylation. Furthermore, our bioinformatics analysis (LASAGNA-Search 2.0 and TransmiR v2.0 database) and CHIP assay confirmed the direct binding of STAT3 to the miR-328-3p promoter. Further functional assay revealed that STAT3 activated miR-328-3p transcription and mediated the HBV/HBc/ HBx-induced upregulation of miR-328-3p. Therefore, these findings suggest the potential role of the STAT3-miR-328-3p axis in regulating cell injury in HBV-infected THLE-2 cells. FOXO4 is a member of the Forkhead (Fox) transcription factor O family and was initially identified as a tumor suppressor which limits cell proliferation and induces apoptosis [30]. Interestingly, HBV/HBc/HBx increased miR-328-3p expression but decreased FOXO4 expression. Furthermore, miR-328-3p targeted FOXO4 and decreased FOXO4 protein expression. Moreover, FOXO4 overexpression significantly suppressed HBV activity and attenuated the HBV-mediated cell injury, whereas FOXO4 silencing caused the opposite effect. Importantly, FOXO4 overexpression abrogated the miR-328-3p mimic-mediated cell injury under LPS and HBV stimulation. The results presented here also demonstrated miR-328 mimic decreased IκB-α protein expression and increased p65 phosphorylation, indicating the activation of the NF-κB pathway. In contrast, miR-328 inhibitor suppressed NF-κB pathway. Researches have shown that FOXO4-knock out (KO) mice exhibits upregulated inflammatory cytokines in colons [19], and mechanistically, FOXO4 interacts with NF-κB and inhibits its DNA binding and transcriptional activity [19]. They also proposed that inflammatory signals activate NF-κB and c Levels of TNF-α, IL-6, IL-8, IL-12, and IL-18 were measured by ELISA. Values are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01 vs. LPS + HBV + Vector + mimic NC. # P < 0.05, ## P < 0.01 vs. LPS + HBV + Vector + miR-328-3p mimic