Hepatitis B virus promotes cancer cell migration by downregulating miR-340-5p expression to induce STAT3 overexpression
© The Author(s) 2017
Received: 6 February 2017
Accepted: 6 April 2017
Published: 12 April 2017
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, and infection with hepatitis B virus (HBV) is a leading cause of HCC. Previous studies have demonstrated that expression of the tumor inhibitor miR-340 is significantly downregulated in HCC tissues compared with normal liver tissues. However, the precise biological role of miR-340-5p in HBV–HCC and its molecular mechanism of action remain unknown.
Expression of miR-340-5p was downregulated in HBV-associated HCC liver tissue and HBV-infected cells, facilitating migration of liver cancer cells. Signal transducer and activator of transcription (STAT)3 was found to be a direct functional target of miR-340-5p. The regulation of STAT3 expression by miR-340-5p was assessed using qRT-PCR and western blotting, and the effects of exogenous miR-340-5p and STAT3 on the migration of HBV-infected cells were evaluated in vitro using Transwell® and wound-healing assays. The expression of E-cadherin and vimentin, associated with epithelial–mesenchymal transition, was also assessed using Western blotting after transfection of miR-340-5p mimics and/or STAT3 expression vectors. Overexpression of STAT3 resulted in rescue of HBV effects, decreased E-cadherin expression, increased vimentin expression, and ultimately, enhanced cell migration. Re-introduction of the STAT3 CDS led to marked reversal of the inhibition of cell migration in HBV-infected cells mediated by miR-340-5p.
Hepatitis B virus promotes the migration of liver cancer cells by downregulating miR-340-5p expression to induce STAT3 overexpression. Our results show that STAT3 plays a key role in regulating cell migration in HBV–HCC involving miR-340-5p.
KeywordsmiR-340-5p HBV–HCC STAT3 Cell migration
Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide. Both the incidence and mortality rates of HCC continue to increase. Population-based studies have shown that HCC is the third leading cause of cancer-related deaths worldwide . The development of HCC is believed to be most closely associated with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection . HBV is an enveloped, partially double-stranded DNA virus with a genome size of 3.2 kb. A previous study demonstrated that HBV infection accounts for >60% of all liver cancers in the developing countries but <25% of cases in the developed countries . The binding of the HBV to its receptor leads to the activation of numerous signaling pathways that regulate important biological functions, such as inflammatory and immune responses, as well as tumorigenesis. HCC initiation and progression are currently thought to involve epithelial–mesenchymal transition (EMT) , in which epithelial cells acquire mesenchymal features and increased motility and invasiveness [4, 5]. EMT is characterized by reduced intercellular adhesion, loss of epithelial markers (such as E-cadherin), and acquisition of mesenchymal markers [including vimentin, matrix-degrading proteases [e.g., matrix metalloproteinases (MMPs)], and N-cadherin . The EMT of cancer cells enables them to leave the primary tumor site and migrate to and invade surrounding and distant tissues/organs. However, obtaining a comprehensive understanding of the underlying mechanism by which HBV promotes tumor cell migration requires further elucidation.
EMT involves a number of molecular processes, including the activation of transcription factors, changes in the expression levels of specific cell surface and cytoskeletal proteins, and the production of various extracellular matrix-degrading enzymes. Accumulating evidence suggests that certain microRNAs (miRNAs) play important roles in EMT. miRNAs are endogenous, evolutionarily conserved, small noncoding RNA molecules (18–25 nucleotides in length) that negatively regulate the expression of numerous target genes at the posttranscriptional level through sequence-dependent translational repression and/or target mRNA degradation . Recent studies have revealed that miRNA-340 (miR-340) expression is downregulated in several types of cancer, demonstrating that miR-340 can act as a tumor suppressor and is, thus, involved in cell differentiation, proliferation, apoptosis, and tumor cell migration and invasion [8–11]. In addition, a previous study indicated that miR-340 represses the proliferation, migration, and invasion of HCC cells by targeting the Janus kinase (JAK) via the JAK1/signal transducer and activator of transcription (STAT)3 signaling pathway . However, the underlying mechanism of miR-340 in HBV-associated HCC remains unclear.
STAT3 is a latent transcription factor that is activated in response to cytokine-induced stimuli. After cytokine binding, the receptors are quickly phosphorylated by JAK kinases. STAT3 is then phosphorylated by JAK kinase, after which STAT3 dimerizes and is translocated into the nucleus, where it regulates the expression of target genes . STAT3 is constitutively activated in various cancers, including HCC [14, 15]. Activated STAT3 contributes to oncogenesis by promoting cell proliferation, preventing apoptosis, and impairing host anti-tumor immune responses [16, 17]. Knockdown of the STAT3 protein via STAT3 antisense oligonucleotide was shown to markedly inhibit the proliferation and tumorigenic growth of an HCC cell line transplanted into nude mice . Constitutive activation of STAT3 plays a pivotal role in the development of many types of human tumors , and increased levels of STAT3 protein are found in many human tumors, including HCC [14, 16, 20]. An increase in the level of un-phosphorylated STAT3 contributes greatly to the development of cancers by driving EMT. However, the role and mechanism of the upregulation of total STAT3 in tumorigenesis and cancer progression remain unclear. Our study shows that HBV regulates the STAT3 signaling pathway via miR-340-5p to facilitate cancer cell migration. In the present study, we sought to gain insights into the regulation of miR-340-5p in HBV-associated HCC. Our findings indicate that miR-340-5p directly regulates STAT3 in HBV-mediated promotion of hepatoma cell migration. Our data, thus, provide new insights into the mechanism through which HBV promotes hepatoma cell migration.
Clinical HCC specimens were obtained from Zhongnan Hospital of Wuhan University. The specimens were verified and classified according to the World Health Organization Classification of Tumors guidelines by two experienced clinical pathologists. Clinical samples were collected from patients after obtaining informed consent in accordance with a protocol approved by the Ethics Committee of Wuhan University (Wuhan, China).
Reagents and cell culture
HepG2, HepG2.2.15, HuH7, and HEK293T cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco/Life Technologies, US) supplemented with 10% fetal bovine serum (FBS; Gibco/Life Technologies) as a complete growth medium and maintained at 37 °C with 5% CO2 in a humidified chamber. The miR-340-5p mimics, inhibitors, corresponding controls, and siSTAT3 RNA sequence were designed and synthesized by RiboBio (Guangzhou, China). Cell transfection and co-transfection were performed using Lipofectamine 2000 when the cells reached 70% confluence. Transfection efficiency was verified by quantitative real-time PCR. After 48 h, the cells were harvested for further analysis.
RNA extraction and quantitative real-time PCR
Primers used in this study
Primer sequences (5′–3′) forward (F), reverse (R)
GenBank accession number
F: GCGCGCGCGGACTAGTT CCGAT AATAGACTTATTTTCCT
R: CGACGCGTAATAT AACCACCTTATAGGTAGGTAAGC
F: GCGCGCGCGGACTAGTT CCGAT AATAGACTTATTTTCCT
R: CGACGCGTAATAT AACCACCTTATAGGTAGGTAAGC
Luciferase reporter assay
The putative binding sites of the 3′-untranslated region (UTR) of the human genes for miR-340-5p targeting were predicted using the TargetScan Human computational methods (http://www.targetscan.org/). The partial 3′-UTR fragment of STAT3, the wild STAT3 including predicted binding sites, and the mutant STAT3 inserts with an opposite mutation in the miRNA seed sequence binding sites, were cloned into the pMIR-REPORT™ miRNA Expression Reporter Vector (Applied Biosystems). All primers were designed as shown in Table 1. HEK293T cells were seeded in triplicate at a density of 2 × 105 cells per 24-well plate. Luciferase activity was monitored using the Luciferase Assay Kit (Promega) and normalized to Renilla luciferase activity, as previously described .
Cells were transfected for 24 h in 12-well plates, and upon reaching ~90% confluency, the cell monolayer was scratched with a 10-μL sterile pipette tip and then gently washed three times with phosphate-buffered saline (PBS), after which culture medium containing 2.5% FBS was added. Images of the cells along the scrape line were captured under a microscope at 0, 24, and 48 h. The wound-healing capacity was determined by measuring the change in the size of the scraped area, resulting from cell migration, using ImageJ software.
Cell migration assays
Cell migration assays were performed using Transwell chambers measuring 6.5 mm in diameter (8-μm pore size, Corning). After transfection for 24 h, a total of 5 × 104 transfected cells in FBS-free medium were seeded in the upper chamber of an uncoated Transwell chamber for the migration assay. Medium, containing 10% FBS, was then added to the lower chamber. After 24 h, cells that did not migrate were removed using a cotton swab. Cells that migrated to the lower chamber were fixed in 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) for 15 min, rinsed with PBS, and then counted under a microscope. Five random views were used to count the cells.
Western blotting analysis
Total cells or tissues were extracted using cell lysis buffer followed by immunoblotting with anti-STAT3 (Proteintech, Wuhan, China), anti-E-cadherin, anti-vimentin and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p-STAT3 and anti-GAPDH (Cell Signaling Technology) antibodies, as described previously .
All data are expressed as the mean ± standard deviation (SD) (n = 3). Statistical analyses were performed using SPSS software, version 16.0 (SPSS, Chicago, IL, USA). Differences between two groups were analyzed using the unpaired Student’s t test, and differences between multiple groups were evaluated using one-way analysis of variance. P < 0.05 was considered indicative of statistical significance.
HBV attenuates the expression of miR-340-5p both in vivo and in vitro
miR-340-5p inhibits the migration of hepatoma cells
miR-340-5p downregulates STAT3 expression by directly targeting the STAT3 3′-UTR
miR-340-5p regulates the migration of Huh7 cells via targeting of STAT3
HBV promotes cell migration via downregulation of miR-340-5p expression to induce STAT3 expression
Molecular mechanism through which HBV induces the migration of HuH7 cells
Hepatocellular carcinoma is the most common type of primary liver cancer and is the third leading cause of cancer-related deaths . HBV is one of the most important causes of HCC. Recent studies have shown that miRNAs play a fundamental role in HCC, thereby opening an avenue for novel investigations of the molecular mechanisms of HBV-HCC pathogenesis. A number of studies have shown that miR-340 acts as a tumor suppressor in several cancers, including neurofibromatosis type 1, breast cancer, and HCC [10, 11, 23, 24]. Consistent with previous studies, our data indicate that miR-340-5p expression is downregulated in HCC tissues, and importantly, the HCC tissues that we used in the present study were derived from an HBV-infected patient. Our results suggest that a decline in miR-340-5p level is closely linked to HBV-associated liver carcinogenesis. Through in vitro experiments, we verified that the downregulation of miR-340-5p expression is mediated by HBV. And, the significant effects of HBV on miR-340-5p in HBV-associated liver cancer require further investigation.
Metastasis of tumors represents the most life-threatening stage for patients with cancer. Metastasis involves a complex cascade of events leading to detachment of cancer cells from the original tumor because of altered adhesion properties and establishment of new, distant tumors. Other cellular changes promote migration, local invasion, proteolysis, angiogenesis, and survival of the tumor cells in the circulatory system. Of these events, the migration of cancer cells into surrounding tissues is a crucial early step. miRNAs are an important group of regulators of the EMT process. The overexpression of miR-340 suppresses cell migration, invasion, and metastasis in these cancers . It has been reported that miR-340 suppresses the proliferation of, and invasion by, HCC cells via the regulation of JAK1 . JAK1 activates STAT proteins in response to various cytokines and growth factors. The JAK1/STAT3 pathway, which is generally regarded as one of the most active signaling pathways, plays an important role in the process of malignant transformation. Recent studies have also shown that miR-340 can function as a tumor metastasis suppressor, inhibiting the metastasis of multiple malignant human tumors, such as melanoma , breast cancer , colorectal cancer , and liver cancer. Our results showed that overexpression of miR-340 inhibits the migration of HCC cells. We also demonstrated that STAT3 is a direct target of miR-340. In our study, miR-340-5p inhibited cell migration induced by HBV. Additional analysis confirmed that STAT3 rescues the miR-340-5p-mediated inhibition of HBV-induced migration of HCC cells, which is an important step in hepatocarcinogenesis. Our findings, showing that increased STAT3 expression, resulting from downregulation of the migration inhibitor miR-340-5p, thus provide a theoretical basis for the development of novel clinical treatments of HBV-associated liver cancer.
HBV promotes the migration of liver cancer cells by downregulating miR-340-5p expression to induce STAT3 overexpression. Our results show that STAT3 plays a key role in regulating cell migration in HBV-HCC involving miR-340-5p.
hepatitis B virus
signal transducer and activator of transcription
Conceived, designed and supervised the project: GS, DG. Performed the experiments: QX, SW, JW, XZ, JC, MW, HG, CF, LC, GS. Analyzed data: GS, LC, DG and QX. Wrote the paper: GS, DG, QX. All authors read and approved the final manuscript.
We thank Ph.D Dan Liu gave our work scientific suggestions.
The authors declare that they have no competing interests.
Availability of data and materials
The data supporting the conclusions of this article is available to all interested researchers upon request.
Consent for publication
All the co-authors Consented the work is published in the Cell & Bioscience.
Ethics approval and consent to participate
This study was approved by the Research Ethics Committee of Wuhan University (Wuhan, China).
This work was supported by the National Natural Science Foundation of China (Nos. 31370187, 81572447 and 31570762). The funders had no role in the design of the study, data collection and interpretation, or writing the manuscript.
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