Tumor Biol. DOI 10.1007/s13277-015-3669-7

RESEARCH ARTICLE

Down-expression of miR-152 lead to impaired anti-tumor effect of NK via upregulation of HLA-G Xiaokun Bian 1,2 & Yuanquan Si 1,3 & Min Zhang 4 & Ran Wei 1 & Xiaomin Yang 1 & Hao Ren 1 & Guixi Zheng 1 & Chuanxin Wang 1 & Yi Zhang 1

Received: 22 April 2015 / Accepted: 15 June 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015

Abstract It is known that chronic HBV infection (CHB) is the major risk factor for hepatocellular carcinoma (HCC) because CHB could not only cause liver tumorigenesis but also lead to change of local microenviroment and lower immune response to infected and cancerous cells (immune tolerance). Human leucocyte antigen-G (HLA-G) belongs to a nonclassic MHC-I family and was considered to be an immune tolerance molecule, which could bind to immunosuppressive receptors of natural killer cell (NK) and T cells and trigger immunosuppressive signaling. Recently, numerous studies highlighted that microRNAs (miRNAs) were significantly differentially expressed in HCC tumorigenesis, and the expression was tissue-specific, indicating that miRNAs may cause great epigenetic changes in HCC tumorigenesis. In this study, we found that the expression of HLA-G was upregulated by hepatitis B virus (HBV) infection and miR-152; a HLA-Gtargeting miRNA was downregulated by HBV infection. And high expression of HLA-G further suppressed NK

Electronic supplementary material The online version of this article (doi:10.1007/s13277-015-3669-7) contains supplementary material, which is available to authorized users.

against cancer cells, providing a new concept that miR-152 was involved in HBV-induced hepatocellular carcinoma. Keywords HLA-G . HBV . miR-152 . Immune tolerance

Abbreviations HBV Hepatitis B virus CHB Chronic HBV infection HCC Hepatocellular carcinoma HLAHuman leucocyte antigen-G G NK Natural killer cell DC Dendritic cell 3′UTR 3′ Untranslated region HBsAg Hepatitis B surface antigen IHC Immunohistochemical MTT 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide RTReal-time polymerase chain reaction PCR FACS Flow cytometric analysis CTL Cytotoxic T lymphocyte SNPs Single nucleotide polymorphisms

* Yi Zhang [email protected]

Introduction 1

Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan, China

2

Department of Clinical Laboratory, Weifang People’s Hospital, Shandong, China

3

Department of Clinical Laboratory, Provincial Hospital Affiliated to Shandong University, Jinan, China

4

Department of Medicine, Shandong Provincial Chest Hospital, Jinan, China

Human leukocyte antigen (HLA-G) is a non-classical HLA class I molecule, mainly expressed by cytotrophoblasts, that plays a crucial role in fetal-maternal tolerance [1]. Now, HLAG is considered to be an important marker of immune suppression and tolerance. HLA-G could be induced by infection and carcinogenesis factors and further inhibit immune response in many ways, including suppressing cytotoxicity

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and proliferation of CTL and NK, upregulating other inhibitory molecules, and promoting lymphocytes apoptosis [2]. Besides above, it also contributed to the generation of HLAG-positive regulatory T cells and tolerogenic dendritic cell (DC) [3]. So, the regulation of HLA-G expression is a hot spot in the field of immunology. MicroRNAs (miRNAs) are a class of evolutionarily conserved non-coding RNAs and 19 to 25 nucleotides in length, which regulate gene expression by paring with messenger RNA (mRNA) 3′ untranslated region (3′UTR) at partially or fully complementary sites [4]. Deviations from normal miRNA expression have been found to play critical roles in many human diseases, especially in the occurrence and development of tumors. A growing number of both direct and indirect evidence suggest a relationship between differential miRNA expression and infectious diseases. In recent years, besides speeding progress of miRNAs research in basic area, some miRNAs were further developed as potential novel diagnosis and prognosis marker in anti-cancer treatment, showing a promising perspective of miRNA application in oncology and clinical practice [5]. There have been several reports mentioned that some SNPs in HLA-G mRNA may be associated with some miRNAs, which alert HLA-G expression [6]. But how miRNAs regulate HLA-G in infectious disease is still largely unknown. Hepatocellular carcinoma (HCC) is known as one of the most common cancers which lead to about 700,000 deaths per year [7]. HBsAg has been demonstrated to be positive in 80 % of HCC cases, meaning hepatitis B virus (HBV) contribute a lot in the oncogenesis to a certain degree. Progress has been made enormous steps in early diagnosis and treatment of HCC, but due to the advanced tumors often recurring early even after complete surgical operation, the prognosis of HCC patients is still poor [8]. So, it is badly in need of exploiting new therapeutic strategies. In this study, we first investigated the expression of HLA-G and miRNAs in HCC tissues and HBV+ and HBV− hepatoma cell lines and further tried to find out the relationship between HBV infection and HLA-G and miRNA-152 expression in hepatoma cell lines in vitro.

Material and methods Ethics statement Written informed consent for the use of tissue samples in this study was obtained from every participant. This project was approved by the Clinical Research Ethics Committee of Qilu Hospital of Shandong University.

Cell culture The hepatoma cell lines HepG2 (HBV negative) and HepG2.2.15 (HBV positive) from human species were obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (Gibco-BRL, Carlsbad, CA) with 10 % fetal bovine serum (Gibco-BRL, Carlsbad, CA). Cells were maintained in a humidified 37 °C incubator with an atmosphere of 5 % CO2. Transfection was performed with LipofectamineTM 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Plasmid, miRNA mimics, and transfection pSuper-HBV plasmid (Gift from Dr. He from Health Science Center, University of Texas, San Antonio), double-stranded miR-152/148a/148b mimics (GenePharma, Shanghai, China), or their negative control RNA (non-specific short RNA) was introduced into cells at a final concentration of 100 nM. Transfected cells were harvested at 24, 48, or 72 h. Reverse transcription reaction and quantitative real-time PCR We obtained fresh specimens of tumors and surrounding nontumor hepatic tissues from 14 patients who underwent liver resection for hepatocellular carcinoma between 2011 and 2013 at the Qilu Hospital in Jinan, China. These tissues were used to evaluate the expression of miRNAs, the same as hepatoma cell lines. Total RNA was extracted from hepatic tissues with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the protocols provided by the manufacturer. Reverse transcription was performed using the reverse transcription system kit (Invitrogen, Carlsbad, CA). Real-time polymerase chain reaction (PCR) was performed in triplicate, following a standard SYBR-Green PCR protocol on an ABI StepOne plus Sequence Detection System (Applied Biosystems, Life Technologies). GAPDH was used as an endogenous control for normalization. Western blot analysis Cells were solubilized in lysis buffer (pH 7.5, 50 mM Tris– HCl, 150 mM NaCl, 1 mM EDTA, 0.5 % NP40, 1 mM phenylmethylsulfonyl-fluoride, 1 mM sodium orthovanadate, 15 % glycerol) and incubated on ice for half an hour. After centrifugation, the whole cell supernatant was mixed in Laemmli loading buffer, boiled for 5 min, and then subjected to SDS–PAGE. After electrophoresis, proteins were transferred to nitrocellulose membranes and blocked with 5 % non-fat milk for at least 1 h followed by an overnight blotting with HLA-G antibody (clone4H84, Santa Cruz Biotechnology, Inc.) and β-actin antibody (Cell Signaling

Tumor Biol.

Technology, New England BioLabs Inc.) at a dilution of 1:1000 at 4 °C, respectively. After washing three times with Tris-buffered saline and Tween 20 (TBST), the membranes were incubated with HRP-conjugated second antibody (Genetech, China). Before detected with an enhanced chemiluminescence system (Millipore ImmobilonTM, USA), membranes were washed with TBST extensively. Flow cytometric analysis Human hepatoma cell lines HepG2 and HepG2.2.15 were harvested and washed twice with 1× PBS, then resuspended in 100 μL PBS, and incubated with antibodies for 45 min at 4 °C. PE-conjugated anti-human HLA-G and mouse IgG2a isotype control PE (R&D systems) were used for phenotype analysis. HLA-G expression was measured using a BD FACS Calibur (BD Biosciences, CA). Cell cytotoxicity assay Human hepatoma cell lines were used as the target cells, including HepG2, HepG2.2.15, or miRNA mimics transfected cells. Target cells were placed in 96-well plates at the concentration of 1×104 cells/well and cultured overnight at 37 °C, 5 % CO2. Then, NKL cells were added as effector cells, and the effector/target (E:T) cell ratio was 10:1, 5:1, and 2.5:1. The cell mixtures were incubated at 37 °C, 5 % CO2 for 6 h, then 20 μL 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT; 5 mg/mL) was added, and cells were incubated for a further 4 h at 37 °C [9]. The absorbance at 570/630 nm in each well was determined with a Microplate Autoreader (Synergy 2, Bio-Rad, USA). Tissues and immunohistochemistry Seven paired human liver tumor tissues and normal adjacent hepatic tissues coming from patients who underwent liver resection for hepatocellular carcinoma were chosen between 2011 and 2013 at Qilu Hospital in Jinan, China. The clinical information of the patients is summarized in Table S1. Immunohistochemical (IHC) staining for HLA-G was performed on formalin-fixed, paraffin-embedded tissue sections of HCC and adjacent nontumorous liver. A mouse monoclonal antibody against HLA-G (clone4H84, Santa Cruz Biotechnology, Inc.) was used at a dilution of 1:100. Immunostaining was operated in IHC system and performed using the HRP detection kit, peroxidase/DAB, Rabbit/ Mouse (ZSGB-BIO, Beijing, China). IHC scoring was performed using modified Histo-score (H-score), which includes semiquantitative assessment of both percentage of positive cells and intensity of staining. The intensity score

was defined as follows: weak (grade 1), moderate (grade 2), or intense (grade 3).

Statistical analysis The data of the expression of miR-152 was done using the GraphPad Prism5 software, single-rank test, and nonfactor test. The other data was determined by the Student’s t test with the SPSS 16.0 software package (SPSS, Chicago). A value of P<0.05 was considered statistically significant.

Results HLA-G expressed highly in HBV+ HCC tissues and cell lines HLA-G was well described as a tolerogenic molecule. It was reported that HLA-G was aberrantly expressed in livers of HCC patients by several studies [10]. As the mechanisms leading to the persistence of hepatitis B virus (HBV) infection are poorly understood and to find out whether HLA-G was involved in HBV persistence in HCC tissues, we firstly evaluated the expression of HLAG in seven pairs of HCC tumor tissues by IHC staining. As shown in Fig. 1a, we did find the expression of HLA-G in HBV+ HCC tissues, but much less in normal adjacent tissues. We then evaluated the HLA-G expression levels in HepG2 cells and HepG2.2.15 cells (a derivative of the human HepG2 cell line that has been stably transformed with a head-to-tail dimer of HBV DNA) in vitro. As shown in Fig. 1b–d, the expression of HLA-G mRNA and protein (including surface and total) was markedly higher in HepG2.2.15 cells than in HepG2 cells. These findings show us that HLA-G was expressed highly in HBV+ HCC tissues and cell lines.

HBV promoted HLA-G expression in HepG2 cells and attenuated NK cytolysis To investigate whether HBV alters HLA-G expression, we evaluated the expression of HLA-G in mRNA and protein levels after the transient transfection of pSuper-HBV into HepG2 cells. As shown in Fig. 2a, b, we found that HLA-G was upregulated in pSuper-HBV-transfected cells in comparison with the pSuper control groups. Then, we found that the cytolytic activity of NKL against HepG2-HBV cells was lower than against HepG2 cells in higher effector-target ratio (Fig. 2c).

Tumor Biol. Fig. 1 HLA-G expressed highly in HBV+ HCC tissues and cell lines. Representative was shown in normal tissues and cancer (HBV-negative and HBV-positive expression) tissues (a). The expression of HLA-G was significantly higher in HBV+ HCC tissues than in HBV− HCC tissues. HepG2 and HepG2.2.15. HCC cells were cultured in 12well plates for 48 h and harvested. The expression of HLA-G was detected by RT-PCR (b), Western blotting (c), and FACS (d). HepG2.2.15 cells expressed more HLA-G than did HepG2 cells. Data shown are means±SD from at least three separate experiments. *P<0.05

The expression of miR-152 was lower in HBV+ HCC and hepatoma cells Firstly, we assessed the aberrant expression of miR-152 in HCC tissues by quantitative reverse transcription PCR. We compared the miRNA profile of HBV+ HCC tumor tissues with HBV− tumor tissues and found that miR-152 Fig. 2 HBV promoted HLA-G expression in HepG2 cells and attenuated NK cytolysis. 3×105 HepG2 cells were transfected with HBV genome using a plasmid vector (pSuper-HBV) and cultured in a six-well plate for 48 h, while empty vector transfected cells were used as control. The expression of HLAG was detected using RT-PCR (a) and Western blotting (b). The cytolytic activity of NKL against HepG2-HBV cells was lower than against HepG2 cells in high effector-target ratio (c). Data shown are means±SD from at least three separate experiments. *P<0.05

was significantly downregulated in HBV+ HCC tissues (Fig. 3a). Next, we determined whether miR-152 was expressed differently in human HCC cells. The expression of miR-152 was markedly lower in the HepG2.2.15 cells versus HepG2 cells (Fig. 3b). In order to verify whether this downregulation was correlated with the HBV expression, HepG2 cells were

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HBV plasmid transfected cells in comparison with control plasmid transfected cells (Fig. 3c). The expression of HLA-G was downregulated by MiR-152 in hepatoma cells Since it has been demonstrated that in human placental choriocarcinoma line, JEG-3, miR-152 targeted HLA-G mRNA directly [11], we then evaluated that if miR-152/148a/148b would downregulate HLA-G in hepatocyte-derived cells, we transfected miR-152/148a/148b mimics or negative control into HepG2.2.15 cells. After another 48 or 72 h, we measured the mRNA and protein expression levels of HLA-G, respectively. The results of our experiments showed that a reduction of HLA-G expression at protein level (Fig. 4a) but not mRNA level (Fig. 4b) was lead by the enforced miR-152 expression in comparison with the control group in HepG2.2.15 cells. miR-152 overexpression enhanced NK cytolysis against hepatoma cells HLA-G was considered to inhibit different kinds of immune cells directly, such as NK [12]. We firstly used NKL cells as effector cells to evaluate the cytolytic activity against HBV+/− HCC cells in vitro. As shown in Fig. 5a, b, the cytolytic activity of NKL cells against HepG2.2.15 and HepG2-HBV cells was lower than HepG2 cells in high effector-target ratio. Next, we investigated whether the aberrant expression of miR152 could functionally influence anti-HBV immune response in HBV+ HCC cells or not. The miR-152/148a/148b mimics were transfected into HepG2.2.15 cells, meanwhile the miRNA negative control served as negative control. In comparison with negative control transfected group, we observed an improvement of 62 to 89 % in cell lysis for the HepG2.2.15 cells introduced with miR-152 mimics (Fig. 5c).

Fig. 3 The expression of miR-152/148a/148b was lower in HBV+ HCC and hepatoma cells. This assay was performed in seven HBV− HCC tissues and seven HBV+ HCC tissues. Total cellular RNA was isolated using TRIzol Reagent, and the expressions of miR-152/148a/148b were detected by qPCR. The expression of miR-152 was downregulated in HBV+ HCC tissues against HBV− HCC tissues (*P<0.05), while no different expression of miR-148a/148b was detected (a). 5×105 HepG2 and HepG2.2.15 cells were harvested, and the total cellular RNA was isolated using TRIzol Reagent; mRNA levels of HLA-G-associated genes such as miR-148a, miR-148b, and miR-152 were detected by qPCR (b). 3×105 HepG2 cells were transfected with HBV genome using a plasmid vector and cultured in a six-well plate for 48 h. The expression levels of miR-148a, miR-148b, and miR-152 were detected by qPCR (c). Data shown are means±SD from at least three separate experiments. *P<0.05

transiently transfected with pSuper-HBV and empty vector. Forty-eight hours later, miR-152 was downregulated in

Discussion HLA-G belongs to a non-classic MHC-I family and was considered to be an immune inhibitory molecule, which could bind to immunosuppressive receptors and induce immunosuppressive signaling [13]. Preliminary studies found that HLAG was highly expressed in various kinds of tumors, such as gastric carcinoma and lung cancer cells, which lead to tumor immune escape. But epigenetic alteration of HLA-G gene expression remains unclear until now [14]. An increasing number of evidence has showed that viral genes played critical roles in the process of tumor formation, but the concrete mechanisms involved are still poorly understood; we need to do more to explore these. miRNAs are an endogenous, small, non-coding RNAs which can bind to specific target complementary mRNA and

Tumor Biol. Fig. 4 The expression of HLA-G was downregulated by miR-152/ 148a/148b in hepatoma cells. 3× 105 HepG2.2.15 cells were transfected with microRNA mimics using Lipofactamine™ 2000 and cultured in a six-well plate for 48 h. The expression of HLA-G was detected using Western blotting (a) and RT-PCR (b). Data shown are means±SD from at least three separate experiments. *P<0.05

Fig. 5 miR-152/148a/148b overexpression enhanced NK cytolysis against hepatoma cells. NKL cells were cocultured in graded E:T ratios with HepG2 and HepG2.2.15 cells for 6 h, and cellular cytotoxicity was tested by MTT assay. The cytolytic activity of NKL against HepG2.2.15 and HepG2-HBV cells was lower than HepG2 cells in high effectortarget ratio (a, b). The cytolytic activity of NKL against HepG2.2.15 cells, which were transfected with miR-148a, miR148b, and miR-152 mimics, was higher than against HepG2.2.15 cells in high effector-target ratio (c). Data shown are means±SD from at least three separate experiments. *P<0.05

Tumor Biol.

inhibit translation [15]. MiRNAs play important roles in various biological processes, including cellular death, proliferation, and differentiation, and also involve in various diseases such as cancer. So far, none of HBV-encoded miRNAs has been validated in a laboratory and recorded by miRBase, but many evidence have indicated that miRNAs had important roles in HBV infection and HBV-associated HCC. HBV infection altered the expression profiles of human cellular miRNAs directly or indirectly, to facilitate its replication and transcription, or maintain latency [16]. And in return, some miRNAs, such as miR-125 [17] and miR-199 [18], directly targeted HBV transcripts and change its life cycle. Some miRNAs, like miR-373 [19] and let-7 [20], targeted host genes that regulated HBV infection to participate in HBV pathogenesis indirectly. Taken together, miRNAs are novel angle of view to understand the interaction between HBV and human hepatocytes. What deserves to be mentioned particularly is miRNAs are also involved in anti-HBV immune response and HBV-induced immune evasion. miR-146a [21, 22], miR-155 [23], and miR-181a [24] have been proven to affect immune genes to support viral evasion or promote viral clearance. In this study, we focused on a known immune inhibitory molecular, HLA-G, which could present immunomodulatory functions in many ways. In the context of hepatocellular carcinoma, HLA-G regulates anti-cancer immune response widely by influencing DC, CD8+ T cell, and NK [25]. Even sHLAG levels in serum could be used as a useful diagnostic marker for HBV and HCV-related HCC [26]. But whether and how HBV regulate HLA-G in hepatocytes and the detailed role of HLA-G in HBV infection and prognosis is still largely unknown [27]. Our data showed that HBV infection could cause immune tolerance of HCC partly by a microRNA-dependent manner, including miR-148a, miR-148b, and miR-152. Also, HLA-G was one target of these miRNAs, which could be downregulated by these miRNAs, and further attenuates NK-mediated cytotoxicity towards tumor cells (as shown in Fig. 6). So targeting miR-152 might reverse highly expressed HLA-G and recover normal anti-tumor immune response

during HBV infection and HBV+ HCC. Since virus, not limited to HBV, may establish chronic infection and carcinomatous change by alter cellular miRNAs pattern, certain miRNAs which bind to immune genes may be promising targets for treating viral diseases, including HBV-related HCC. Taken together, this understanding between miRNAs and HBV interaction need further investigation, and HLA-G and miRNAs might be new potentially biomarkers for diagnosis and therapy of HBV infection and HBV-positive HCC. A c k n o w l e d g m e n t s We t h a n k P r o f . Z h a n g ( I n s t i t u t e o f Immunopharmacology and Immunotherapy, School of Pharmaceutical Sciences, Shandong University, China) for providing NKL cell lines and Dr. He (Health Science Center, University of Texas, San Antonio) for providing pSuper-HBV plasmid. This study was supported in part by grants from the Shandong Province Science Foundation for Key Programs (2008GG30002017 and 2010GSF10274), the University Innovation Program from Jinan, Shandong Province (201004050), and National Key Clinical Medical Specialties foundation. Conflicts of interest None

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12. Fig. 6 Working model

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