MicroRNA 125a-5p Inhibits Cell Proliferation and Induces Apoptosis in Hepatitis B Virus-Related Hepatocellular Carcinoma by Downregulation of ErbB3.

MicroRNAs, a class of endogenous noncoding RNAs, regulate gene expression at the posttranscriptional level and thus take part in multiple biological processes. An increasing number of miRNAs have been found to be dysregulated in hepatocellular carcinoma (HCC) and are involved in liver tumorigenesis. In this study, miR-125a-5p was found to be obviously downregulated much more in hepatitis B virus (HBV)-related HCC. To investigate the effects of miR-125a-5p, miR-125a-5p was overexpressed in HepG2.2.15 and HepG3X cells. The findings have indicated that overexpression of miR-125a-5p dramatically inhibited cell proliferation and induced cell apoptosis. Furthermore, overexpression of miR-125a-5p could significantly decrease the secretion of HBsAg and HBeAg. In concordance to this, the expression of ErbB3 was upregulated in human HBV-related HCC tissue, HepG2.2.15 cells, and HepG3X cells. miR-125a-5p directly targeted ErbB3 and reduced both mRNA and protein levels of ErbB3, which promoted cell proliferation and suppressed cell apoptosis in HCC cells. Our results provide new insights into the function of miR-125a-5p in HBV-related HCC. It is beneficial to gain insight into the mechanism of HBV infection and pathophysiology of HBV-related HCC.

INTRODUCTION

Hepatocellular carcinoma (HCC) is the most malignant type of liver cancer and is also the third leading cause of cancer-related mortality in the Asian Pacific region1,2. Hepatitis B virus (HBV) belongs to the hepadnavirus family, and its infection is one of the major risk factors for HCC3. In most high-risk HCC regions, HBV is closely related to at least 80% of HCC cases4. However, how HBV contributes to the development of HCC remains unclear. Therefore, insight into the pathogenesis of HBV-related HCC is very meaningful.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have been demonstrated to play important roles in the development and progression of cancer and in the infection of viruses5,6. Some miRNAs have been identified to act as either oncogenes or tumor suppressors in HBV-related HCC. The relative expression levels of miR-122 and miR-22 in HBV-related HCC patients were significantly lower than those in benign liver disease or non-HBV-related HCC patients7. Yen et al. have demonstrated that miR-106b promoted cancer progression in HBV-associated HCC8. These studies of miRNAs provide novel theories for understanding the precise mechanisms of HBV-related HCC and potentially act as effective methods for the diagnosis and treatment of HBV-related HCC.

HER family, also known as EGFR/ErbB family, comprises HER1–4 proteins, and its activation plays critical roles in regulating cell growth and survival9. Under physiological conditions, ligand-mediated endocytic degradation strictly controlled the activation of HER family proteins. Moreover, it has been reported that the expression of HER family proteins is dysregulated, leading to tumorigenesis10. Therefore, high levels of HER family proteins are frequently detected in multiple tumors, including HCC11–17. Notably, overexpression of the ErbB3 protein has been identified as a biomarker in patients with chronic hepatitis and cirrhosis, which is the main cause of most HCC cases17.

So far, the dysregulated level of miR-125a-5p in HBV-related HCC has not been reported. In this study, we evaluated the level of miR-125a-5p in HBV-related HCC tissues and cell lines such as HepG2.2.15 and HepG3X and explored the effects of miR-125a-5p overexpression on proliferation and apoptosis in vitro. We also found that miR-125a-5p could negatively regulate the expression of ErbB3. It was believed that miR-125a-5p could potentially play a therapeutic role in the near future.

MATERIALS AND METHODS

Tissue Samples and Cell Lines

Ten pairs of tumor and adjacent noncancerous tissues were obtained from patients with HBV-associated HCC at The First People’s Hospital of Kunshan (Suzhou, P.R. China). The tissues were snap frozen in liquid nitrogen immediately following resection and stored at −80°C. Informed consent was obtained from all subjects (patient or the patient’s family), and the study was approved by the ethics committee of The First People’s Hospital of Kunshan.

All the cells lines used in this study were routinely used and preserved in our laboratory, including the human hepatoma cell lines HepG2, HepG2.2.15, HepG3, and HepG3X. The HBx-overexpressed stable clone Hep3BX was established from Hep3B and selected with G418 antibiotic agent. The HepG2.2.15 cells were established by transfecting the HBV genome into HepG2 cells and selecting with G418. All cells were cultured in Dulbecco’s modified Eagle’s medium/F12 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin G, and 100 mg/ml streptomycin sulfate, at 37°C in a humidified incubator under an atmosphere of 5% CO2 in air.

Transient Transfection

The miR-125a-5p mimic, miR-negative control of mimic (miR-NC), pcDNA-ErbB3, and pcDNA3.1 vectors were synthesized and purified by GenePharma (Shanghai, P.R. China). HepG2.2.15 and HepG3X cells were transfected with 50 nM miR-125a-5p mimic and miR-NC using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocols. Total RNA and protein were collected 48 h after transfection.

RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Total RNA of tissues and cells was isolated by TRIzol reagent (Invitrogen) following the manufacturer’s protocol. The mRNA expression levels were detected by qRT-PCR. The complementary DNA (cDNA) was obtained by reverse transcription utilizing the miScript II RT Kit (TAKARA, Kusatsu, Japan). The SYBR Green PCR Kits (TAKARA) were employed to conduct quantitative PCR. Primers were synthetized by RiboBio (Guangzhou, P.R. China). The following primers were used: ErbB3, 5′-GGTGATGGGGAACCTTGAGAT-3′ (forward) and 5′-CTGTCACTTCTCGAATCCACTG-3′ (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-ACAACTTTGGTATCGTGGAAGG-3′ (forward) and 5′-GCCATCACGCCACAGTTTC-3′ (reverse). Each sample was assessed in triplicate. The expression levels of mRNAs were normalized to levels of GAPDH, which acted as a housekeeping gene.

For the detection of human miRNAs, the TaqMan MicroRNA Reverse Transcription Kit and TaqMan miRNA assay (Qiagen, Hilden, Germany) were used to perform reverse transcription and PCR according to the manufacturer’s instructions. Reverse transcription was performed using the miScript II RT Kit (TAKARA), which was followed by qRT-PCR utilizing the miScript SYBR Green PCR Kit (TAKARA) and hsa-miR-125a-5p and hsa-miR-4319 specific primers that were purchased from RiboBio. The levels of miRNAs were normalized to levels of U6 small nuclear RNA (snRNA).

Cell Viability

For estimating cell viability, the cell counting kit-8 (CCK-8; Thermo Fisher Scientific, Carlsbad, CA, USA) assay was performed. HepG2.2.15 and HepG3X cells were seeded in 96-well culture plates at 2 × 104 cells per well. After 24 h, the cells were transfected with miR-125a-5p mimic or miR-NC for 48 h. Then 210 μl of CCK-8 reagents was separately added to each well before and after transfection. The cell viability rates were shown by optical density (OD) value at 450 nm via a microplate reader (Bio-Rad, Hercules, CA, USA).

Cell Proliferation Assay

To explore the effect of miR-125a-5p on the proliferation of HepG2.2.15 and HepG3X cells, 2 × 104 cells were seeded in 96-well plates and allowed to grow overnight in complete medium. The medium was removed, and then cells were transfected with miR-125a-5p mimic or miR-NC for 48 h at 37°C. Cell Proliferation ELISA-BrdU (colorimetric) Kit (Roche, Inc., Basel, Switzerland) was used to detect the cell proliferation according to the manufacturer’s protocols.

Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Analysis

Flow cytometry (FCM) was performed using an Annexin-V-FITC Apoptosis Detection Kit (Nanjing KeyGEN Biotech. Co., Ltd., Nanjing, P.R. China). HepG2.2.15 and HepG3x cells were transfected with miR-125a-5p mimic for 48 h. After transfection, cells were harvested and washed twice in cold phosphate-buffered saline (PBS) and double stained with annexin V-FITC and PI for 30 min in the dark. After that, all samples were quantitatively analyzed at 488-nm emission and 570-nm excitation by a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

Detection of Apoptosis

According to a previous study18, apoptosis was determined by using the Cell Death ELISA Detection Kit (Roche), which measures cytoplasmic DNA–histone complexes generated during apoptotic DNA fragmentation. Cell apoptosis detection was performed under the manufacturer’s instructions and monitored spectrometrically at 405 nm.

Caspase 3 Activity Assay

Caspase 3 fluorescence assay kit (Nanjing KeyGEN Biotech) was used to detect caspase activity according to a previous study18. Briefly, after treatment cells were lysed in the lysis buffer and centrifuged at 10,000 × g for 1 min, and then the supernatants were collected. Equal amounts of protein samples were reacted with the synthetic fluorescent substrates at 37°C for 1.5 h, and the reactions were read at 405 nm in a microplate reader (Bio-Rad). Fold increases in caspase 3 activity were determined with values obtained from the treatment samples divided by those from the controls.

Western Blot Analysis

After transfection with miR-125a-5p mimic and miR-NC for 48 h, HepG2.2.15 and HepG3x cells were washed twice in cold PBS, and then lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, P.R. China) with protease inhibitor cocktail (Merk, Darmstadt, Germany) to extract protein. The protein concentration of cell lysates was quantified by BCA Kit (Beyotime Institute of Biotechnology), and 50 μg of each of the proteins was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Boston, MA, USA). The membranes were blocked in 5% skimmed milk diluted in TBST at room temperature for 2 h and incubated overnight at 4°C with primary antibody against human ErbB3 protein (No. 32121; 1:1,000; Abcam, Cambridge, MA, USA). The membranes were then incubated with goat anti-rabbit (No. 14708) IgG conjugated to horseradish peroxidase secondary antibody (1:1,000; Cell Signaling Technology Inc., MA, USA) for 2 h. The proteins were visualized using ECL Plus reagents (Amersham Biosciences Corp., Piscataway, NJ, USA). The density of the bands was measured using the ImageJ software (USA), and values were normalized to the densitometric values of α-tubulin (T5168; 1:1,000; Sigma-Aldrich, St. Louis, MO, USA) in each sample.

Detection of HBV DNA Expression

HBV DNA expression was detected according to a previously published study19. Briefly, HepG2.2.15 and HepG3X cells were seeded in six-well plates at 1 × 105 cells per well. After 24 h, the cells were transfected with miR-125a-5p mimic or miR-NC (50 nM) using Lipofectamine 3000 reagent (Invitrogen) following the manufacturer’s protocol. After transfection for 48 h, HBV DNA in culture supernatants was collected and then extracted by standard methods. The HBV DNA was quantified using HBV PCR Fluorescence Quantitative Detection Kit (Bioer, Hangzhou, P.R. China) according to the manufacturer’s protocol. The average threshold cycle values were used to determine the concentration of HBV DNA. The inhibitory rate was calculated according to the formula: inhibitory rate (%) = (Ccontrol − Ctested)/Ccontrol × 100%.

Detection of HBsAg and HBeAg

HBsAg and HBeAg were detected according to a previously published study19. After transfection for 48 h, the concentrations of HBV surface antigen (HBsAg) and HBe antigen (HBeAg) in culture supernatants were collected and measured by an HBsAg and HBeAg enzyme diagnostic kit (Autobio, Zhengzhou, P.R. China) according to the manufacturer’s instructions. The OD values were read at 450 nm using a microplate reader (Thermo Fisher Scientific). Inhibitory rates were calculated according to the formula: inhibitory rate (%) = (Ccontrol − Ctested)/Ccontrol × 100%.

Dual-Luciferase Reporter Assay

The 3′-untranslated region (3′-UTR) of the ErbB3 gene containing the target sequence of miR-125a-5p was cloned by PCR. The 3′-UTR was cloned into the luciferase reporter pGL3-control vector (Promega, Madison, WI, USA) to construct the pGL3-ErbB3 vector.

HepG2.2.15 and HepG3X cells were seeded in 24-well plates and incubated for 1 day before transfection. Overnight, cells were cotransfected with pGL3-ErbB3/miR-125a-5p mimic and pGL3-ErbB3/miR-NC. The pRL-TK plasmid was the cotransfection (Promega) plasmid used as an internal control. Forty-eight hours after transfection, both firefly and Renilla luciferase activities were quantified using the Dual-Luciferase Reporter Assay system (Promega) according to the manufacturer’s instructions. All experiments were performed in triplicate.

Statistical Analysis

All statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Data from each group were expressed as mean ± standard error of the mean (SEM) and statistically analyzed by Student’s t-test. Differences were considered statistically significant at a value of p < 0.05.

RESULTS

The Expression of ErbB3 Was Upregulated and the Level of miR-125a-5p Was Downregulated in HBV-HCC Tissues and Cells

To study the expression of ErbB3 in HBV-HCC, we detected ErbB3 in 10 HBV-HCC tissues and 10 normal liver tissues by using qRT-PCR. Strikingly, the mRNA and protein expressions of ErbB3 were significantly increased in HBV-HCC tissues compared to those in the adjacent normal tissues (p < 0.05) (Fig. 1A). To confirm the increased tendency of ErbB3 in cell lines, we then determined the expression of ErbB3 in HepG2.2.15, HepG3X, HepG2, and HepG3 by qRT-PCR and Western blot, respectively. The results showed that ErbB3 was also obviously higher in HepG2.2.15 and HepG3X than in HepG2 and HepG3, respectively (p < 0.01) (Fig. 1B). Subsequently, the online database (TargetScan 6.2; www.targetscan.org) predicted that miR-125a-5p and miR-4319 could directly target ErbB3. Our results showed that the level of miR-125a-5p was significantly reduced in HBV-HCC tissues and cells, but the miR-4319 level was not changed (Fig. 1C and D). Together, the data suggested that the decrease of miR-125a-5p expression was closely related to HBV-HCC.

Figure 1

The levels of ErbB3 and miR-125a-5p in hepatitis B virus (HBV) tissues and cells. (A) The mRNA and protein expressions of ErbB3 in normal liver tissues (n = 10) and HBV-HCC tissues (n = 10) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. (B) The mRNA and protein expressions of ErbB3 in HepG2, HepG2.2.15, HepG3, and HepG3X cells. (C) The levels of miR-125a-5p and miR-4319 in normal liver tissues (n = 10) and HBV-HCC tissues (n = 10) were detected by qRT-PCR. (D) The levels of miR-125a-5p and miR-4319 in HepG2, HepG2.2.15, HepG3, and HepG3X cells. The data shown are the means ± standard error of the mean (SEM), n = 4. ***p < 0.001 versus normal liver tissues; ##p < 0.01, ###p < 0.001 versus HepG2 or HepG3.

miR-125a-5p Inhibited the Secretion of HBV but not the Replication of HVB-DNA

After transfection with miR-125a-5p mimic, the qRT-PCR analysis showed that the level of miR-125a-5p was significantly upregulated in the miR-125a-5p mimic group compared to the miR-NC group in HepG2.2.15 and HepG3X cells (p < 0.01) (Fig. 2A). To explore the effect of miR-125a-5p on HBV at the DNA level, qRT-PCR was performed after transfection with miR-125a-5p mimic. Our results showed that the number of copies of HBV DNA was not significantly changed in the miR-125a-5p mimic group compared with the miR-NC group (Fig. 2B).

Figure 2

miR-125a-5p inhibited the secretion of HBV and not the replication of HBV-DNA. HepG2.2.15 and HepG3X cells were transfected with miR-125a-5p mimic or miR-negative control of mimic (miR-NC) for 48 h. (A) The level of miR-125a-5p in HepG2.2.15 and HepG3X cells was determined by qRT-PCR. (B) The copies of HBV DNA were not significantly reduced in HepG2.2.15 and HepG3X cells transfected with miR-125a-5p mimic compared with cells transfected with miR-NC. Transfection with miR-125a-5p mimic had a significant inhibitory effect on (C) HBsAg and (D) HBeAg levels compared with cells transfected with miR-NC. All data are presented as mean ± SEM, n = 6. ##p < 0.01, ###p < 0.001 versus miR-NC.

To further study the effect of miR-125a-5p overexpression on the expression of HBV, the concentrations of HBsAg and HBeAg were determined after transfection with miR-125a-5p mimic. As shown in Figure 2C and D, the inhibitory ratio of HBsAg and HBeAg was 53.67%, 45.33%, 50.00%, and 52.00% in the miR-125a-5p mimic group compared to the miR-NC group, respectively (HBsAg: p < 0.01; HBeAg: p < 0.01, respectively). The above results showed that miR-125a-5p mimic could suppress the expression of HBV gene but did not affect HBV replication at the DNA level.

miR-125a-5p Inhibited Proliferation and Induced Apoptosis

To preliminarily determine the biological effects of miR-125a-5p on cell viability, proliferation, and apoptosis, we performed CCK-8, bromodeoxyuridine (BrdU)-enzyme-linked immunosorbent assay (ELISA), and FCM assays after transfection with miR-125a-5p mimic in both HepG2.2.15 and HepG3X cells. The CCK-8 assay revealed that overexpression of miR-125a-5p significantly inhibited the viability of the HepG2.2.15 and HepG3X cells (Fig. 3A). Next, introduction of miR-125a-5p remarkably suppressed cell proliferation in both cell lines (Fig. 3B). For further study, the FCM and ELISA assays revealed that miR-125a-5p dramatically induced cell apoptosis of HepG2.2.15 and HepG3X cells (Fig. 3C and D). Finally, to confirm the above apoptosis results, we detected the activity of caspase 3. After transfection with miR-125a-5p mimic, the activity of caspase 3 was significantly increased (Fig. 3E).

Figure 3

Effects of miR-125a-5p on cell viability, proliferation, and apoptosis in HepG2.2.15 and HepG3X cells. HepG2.2.15 and HepG3X cells were transfected with miR-125a-5p mimic or miR-NC for 48 h. (A) Cell viability was assessed by cell counting kit-8 (CCK-8) assay. (B) Cell proliferation was assessed by bromodeoxyuridine (BrdU)-enzyme-linked immunosorbent assay (ELISA) assay. Cell apoptosis was measured by flow cytometric analysis of cells labeled with (C) annexin V/propidium iodide (PI) double staining and (D) nucleosomal degradation by using Roche’s Cell Death ELISA Detection Kit, respectively. (E) The activities of caspase 3 were determined by caspase 3 activity detection assay. All data are presented as mean ± SEM, n = 6. ##p < 0.01, ###p < 0.001 versus miR-NC.

ErbB3 Was Identified as one of miR-125a-5p Direct Targets

To identify putative targets of miR-125a-5p, the online database TargetScan 6.2 was used in this study. ErbB3 was simultaneously predicted to have a complementary site at the 3′-UTR with miR-125a-5p and be preliminarily recognized as a putative target of miR-125a-5p. The prediction results are listed in Figure 4A.

Figure 4

ErbB3 was a direct target of miR-125a-5p. (A) Schematic representation of ErbB3 3′-untranslated regions (3′-UTRs) showing putative miRNA target site. (B) The mRNA expression of ErbB3 was determined by qRT-PCR in HepG2.2.15 and HepG3X cells transfected with miR-125a-5p mimic or miR-NC. (C) The protein expression of ErbB3 was determined by Western blot in HepG2.2.15 and HepG3X cells transfected with miR-125a-5p mimic or miR-NC. α-Tubulin was detected as a loading control. (D) The analysis of the relative luciferase activities of ErbB3-wild type (WT) or ErbB3-mutant (MUT) in HepG2.2.15 and HepG3X cells. All data are presented as mean ± SEM, n = 6. ##p < 0.01 versus miR-NC.

Based on the decreased level of miR-125a-5p and increased expression of ErbB3 in HBV tissues and HBV-HCC cells, we carried out qRT-PCR and Western blotting to determine the effect of miR-125a-5p overexpression on the target ErbB3. The qRT-PCR analysis showed that the expression of ErbB3 was obviously decreased after overexpression of miR-125a-5p at the mRNA level (p < 0.01) (Fig. 4B). The Western blotting analysis revealed that the expression of ErbB3 was also lowered at the protein levels in HepG2.2.15 and HepG3X cells transfected with miR-125a-5p mimic compared to the miR-NC group (Fig. 4C). To further demonstrate the interaction of miR-125a-5p and the 3′-UTR region of ErbB3, we performed dual-luciferase reporter assays. After cotransfection of miR-125a-5p mimic and wild-type (WT) pGL3-ErbB3, the luciferase activity was dramatically reduced compared to the miR-NC group (p < 0.01) (Fig. 4D). Altogether, these results demonstrated that ErbB3 was indeed a direct downstream target of miR-125a-5p in HBV-HCC.

Introduction of ErbB3 Reversed the Effects of miR-125a-5p Mimic on Proliferation and Apoptosis of HepG2.2.15 and HepG3X Cells

To determine whether miR-125a-5p overexpression protected HCC cells from HBV-induced apoptosis in an ErbB3-dependent manner, we cotransfected HepG2.2.15 and HepG3X cells with miR-125a-5p mimic and pcDNA-ErbB3. We found that the expression of ErbB3 was dramatically increased after transfection with miR-125a-5p mimic and pcDNA-ErbB3, compared with miR-125a-5p mimic and pcDNA3.1, in HepG2.2.15 and HepG3X cells (data not shown). Analysis by CCK-8 and BrdU-ELISA assays indicated that upregulation of ErbB3 in cells transfected with the miR-125a-5p mimic increased the viabilities and proliferation of HepG2.2.15 and HepG3X cells transfected with miR-125a-5p mimic only (Fig. 5A and B). Moreover, the results also showed that increased ErbB3 expression could reverse the proapoptotic effect of miR-125a-5p mimic on HepG2.2.15 and HepG3X cells (Fig. 5C–E). Our results clearly demonstrated that overexpression of miR-125a-5p inhibited proliferation and induced apoptosis of HepG2.2.15 and HepG3X cells by downregulation of ErbB3 and that knockdown of ErbB3 was essential for the proapoptotic effect of miR-125a-5p overexpression on HepG2.2.15 and HepG3X cells.

Figure 5

The effects of ErbB3 overexpression on cell proliferation and apoptosis in HepG2.2.15 and HepG3X cells transfected with miR-125a-5p mimic. HepG2.2.15 and HepG3X cells were transfected with miR-125a-5p mimic and pcDNA-ErbB3 or pcDNA3.1. (A) Cell viability was assessed by CCK-8 assay. (B) Cell proliferation was assessed by BrdU-ELISA assay. Cell apoptosis was measured by flow cytometric analysis of cells labeled with (C) annexin V/PI double staining and (D) nucleosomal degradation by using Roche’s Cell Death ELISA Detection Kit, respectively. (E) The activities of caspase 3 were determined by caspase 3 activity detection assay. All data are presented as mean ± SEM, n = 6. #p < 0.05, ##p < 0.01 versus miR-125a-5p mimic + pcDNA3.1.

DISCUSSION

HCC is one of the leading causes of cancer deaths worldwide20. In the past decade, although the diagnosis and treatment are greatly improved, the outcome of patients with HCC is still very poor. Therefore, there is an urgent need to obtain a profound understanding of the mechanisms underlying its pathogenesis to treat this intractable disease. Since most HCCs are generated from chronic hepatitis, particularly with cirrhosis, it is of significance to explore the relationship between HBV infection and formation of HCC21. HBV, a widespread pathogen, is the most significant etiological factor of HCC. Many dysregulated miRNAs have been reported after infection of HBV. miRNAs, including miR-106b8, miR-2122, miR-22123, and miR-58124, were reported to be upregulated after HBV infection. In this study, we determined that the level of miR-125a-5p was dramatically downregulated in HBV-HCC tissues and cell lines such as HepG2.2.15 and HepG3X compared with their control. These results suggested that the decreased level of miR-125a-5p was closely associated with HBV, and miR-125a-5p could serve as an effective tool for diagnosing HBV-related HCC.

Generally, many reports have demonstrated that miRNAs could affect viral replication either by directly interacting with the HBV genome or by indirectly regulating transcription factors. miR-199a-3p and miR-210 were upregulated and found to possess binding sites in the S and pre-S regions25, resulting in repressing HBV replication. In addition, miRNAs could indirectly regulate HBV replication. For example, miR-1 promotes HBV replication by targeting histone deacetylase 4 (HDAC4) and increasing the expression of farnesoid X receptor (FXR)19. However, in this study, we found that miR-125a-5p did not obviously affect the replication of the viral gene. Therefore, we speculated that miR-125a-5p may indirectly affect the viral gene expression by regulating transcription factors. Moreover, the expressions of HBsAg and HBeAg were dramatically inhibited after transfection with miR-125a-5p mimic. The mechanism causing these similar changes needs further study. These results would be very helpful in understanding the mechanism of HBV infection.

miRNAs act as effectors of disease progression and play various important roles in pathways related to HBV-HCC. As regulators at the posttranscriptional level, miRNAs perform their function mainly by targeting mRNAs at their 3′-UTR, leading to repression of their translation. In this study, ErbB3 was identified as one of the miR-125a-5p targets. ErbB3, which belongs to the human epidermal growth factor receptor (EGFR/ERBB) family, is responsible for delivering extracellular signaling and transmitting the downstream intracellular signaling cascades to regulate cell proliferation, migration, and differentiation26. Moreover, ErbB3 is overexpressed and abnormally activated in many human cancers, such as breast, lung, prostate, and liver cancers, emphasizing the critical role of ErbB3 in oncogenic signaling in human cancers27–31. A previous study showed that the level of ErbB3 was significantly increased in HBx-expressed HCC tissues, which illustrated the importance of ErbB3 in the development of HCC32. In this study, the expression of ErbB3 was higher in HBV tissues, HepG2.2.15 cells, and HepG3X cells compared with the corresponding controls. After overexpression of miR-125a-5p, ErbB3 expression was significantly decreased at the mRNA and protein levels. Therefore, we speculated that miR-125a-5p inhibited proliferation and induced apoptosis potentially by regulating ErbB3 signaling. However, the mechanisms need further study. More studies are required to further elucidate the mechanisms of miR-125a-5p in HBV-HCC. Especially, we need to further examine in a model in vivo.

Our results showed that miR-125a-5p was downregulated in HBV-HCC tissues and cells. miR-125a-5p could inhibit the secretion of HBV proteins indirectly, but not inhibit HBV replication at the DNA level. Moreover, the upregulation of miR-125a-5p after HBV infection leads to an increased expression of one target, ErbB3, which might play important roles in chronic HBV infection and HCC development. In conclusion, our study provided a novel miRNA which is beneficial to gain insight into the mechanism and pathophysiology of HBV-related HCC.