Human Immunology 77 (2016) 667–673

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Enhancing NK cell cytotoxicity by miR-182 in hepatocellular carcinoma

http://dx.doi.org/10.1016/j.humimm.2016.04.0200198-8859/� 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Abbreviations: B2m, beta-2-microglobulin; cDNA, complementary DNA; DMEM,Dulbecco’s modified eagle’s medium; FBS, fetal bovine serum; GzmB, granzyme B;HCC, hepatocellular carcinoma; HCV, hepatitis C virus; IFN-c, interferon gamma;KIRs, killer immunoglobulin receptors; LDH, lactate dehydrogenase; MHC, majorhistocompatibility complex; miRNAs, microRNAs; NCRs, natural cytotoxicityreceptors; NK-cells, natural killer cells; NKG2A, natural killer cell receptor G2A;NKG2D, natural killer cell receptor G2D; NSCLC, non-small-cell lung carcinoma;PBMCs, peripheral blood mononuclear cells; Prf1, perforin; qRT-PCR, quantitativereal time polymerase chain reaction; RNA, ribonucleic acid; RPMI, Roswell parkmemorial institute medium; TNF-a, tumor necrosis factor alpha; Treg, regulatory Tcells; UTR, untranslated region.⇑ Corresponding author at: AUC Avenue, P.O. Box 74, New Cairo City, 11835 Cairo,

Egypt.E-mail addresses: a.ihab@aucegypt.edu, ahmed.ihab.abdelaziz@gmail.com

(A.I. Abdelaziz).

Mohamed M. Abdelrahman a, Injie O. Fawzy a, Aya A. Bassiouni a, Asmaa I. Gomaa b, Gamal Esmat c,Imam Waked b, Ahmed I. Abdelaziz d,⇑aDepartment of Pharmacology and Toxicology, German University in Cairo, New Cairo City, Main Entrance Al Tagamoa Al Khames, 11835 Cairo, EgyptbDepartment of Hepatology, National Liver Institute, Menoufiya University, Menoufiya, EgyptcDepartment of Endemic Medicine and Hepatology, Faculty of Medicine, Cairo University, Cairo, EgyptdDepartment of Biology, American University in Cairo, New Cairo, Egypt

a r t i c l e i n f o

Article history:Received 23 February 2016Accepted 25 April 2016Available online 2 June 2016

Keywords:HCCMicroRNAsMiR-182Natural killer cellsNKG2ANKG2D

a b s t r a c t

Background and aim: NK-cells are the principle defense line against different malignancies. Their activa-tion status is determined by the balance between activating and inhibitory receptors such as NKG2D andNKG2A, respectively. MicroRNAs are crucial post-transcriptional regulators of gene expression, playingkey roles in modulating NK-cell development and function. The aim of this study is to investigate the roleof miRNAs in regulating the activation and cytotoxic function of NK-cells in HCC.Methods: In silico analysis was performed to predict a potential miRNA that might target NKG2D andNKG2A mRNAs. NK-cells were isolated from HCC patients and healthy controls, after which miRNAand mRNA were quantified. Manipulating miRNA expression was performed followed by investigatingdownstream targets and the cytotoxic activity of NK-cells against Huh-7 cell lines.Results: NK-cells of HCC patients showed miR-182 overexpression compared to controls. NKG2D andNKG2A were upregulated and downregulated, respectively, in HCC NK-cells. Upon forcing miR-182expression in the HCC NK-cells, upregulation of both receptors was observed. Finally, miR-182 wasreported to induce NK-cell cytotoxicity represented in Perforin-1 upregulation and increase in cytolytickilling of co-cultured Huh-7 cells.Conclusion: Our findings suggest that miR-182 may augment NK-cell cytotoxicity against liver cancer viamodulating NKG2D and NKG2A expressions.� 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights

reserved.

1. Introduction

Natural killer cells (NK-cells) are large granular lymphocytesfunctioning in the interface between innate and adaptive immu-

nity [1]. As an essential component of the innate immune response,they represent the first line of defense against viral infections andtumors [2]. They perform their effector functions by the directrelease of cytotoxic granules containing perforins and granzymes,and by producing cytokines such as tumor necrosis factor alpha(TNF-a) and interferon gamma (IFN-c) [3]. The NK cell activationand hence its ability to perform its effector functions, isdetermined by a balance between signals from its activating andinhibitory receptors [4]. Inhibitory receptors such as killerimmunoglobulin receptors (KIRs) and natural killer cell receptorG2A (NKG2A) recognize healthy cells by binding to its major histo-compatibility complex (MHC) class I molecules, thus protectingthem from lysis. These inhibitory signals are often attenuated uponbinding to infected and transformed cells due to reduced class IMHC expression, therefore NK cells are activated to kill these targetcells in a process known as ‘‘missing self-recognition” [5,6]. In con-trast, activating receptors such as natural cytotoxicity receptors(NCRs) and natural killer cell receptor G2D (NKG2D) engage with

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specific ligands that are selectively induced on infected and tumorcells in a process called ‘‘induced self-recognition”, and are absentor expressed at low levels on healthy cells [7].

In cancers, there is often an imbalance in these activating andinhibitory signals enabling tumor cells to evade the NK cell medi-ated immune responses [8]. This imbalance can be due to down-regulation of activating receptors on NK cells of cancer patientsor poor expression of activating ligands on tumor cells [9,10]. Onthe other hand, some inhibitory receptors were found to be overex-pressed in NK cells of cancer patients. Several tumors may alsomaintain or increase the expression of MHC molecules to ensureinhibition of NK cells and evasion of killing [11,12]. In hepatocellu-lar carcinoma (HCC), NK cells have shown diminished recognitionand cytotoxicity against the liver tumor cells. Multiple mecha-nisms have been put forward to explicate the decreased NK func-tioning in HCC patients, including the decreased count ofperipheral and tumor-infiltrating NK cells, the reduced capacityto produce IFN-c [13], the downregulation of activating receptorsand their ligands on liver tumor cells, and the upregulation of inhi-bitory receptors and ligands [14–17].

MicroRNAs (miRNAs) are a novel class of small non-codingRNAs. Functioning as key regulators of gene expression on thepost-transcriptional level, miRNAs affect different physiologicaland pathological conditions [18,19]. The role of miRNAs in regulat-ing NK cells has been investigated on different levels. MiR-181 andmiR-150 were reported to regulate the development and matura-tion of NK cells [20,21]. Not only the development but also thefunctionality of NK cells is regulated by miRNAs. MiR-30c-1⁄ wasreported to increase TNF-a production and enhance the NK cellcytotoxicity [22]. Moreover, miR-27a⁄ directly targets the 30

untranslated region (30UTR) of the two cytotoxic proteins perforin(Prf1) and granzyme B (GzmB) affecting the cytolytic activity of NKcells [23]. MiR-155 was found to increase in NK cells upon activa-tion enhancing their IFN-c production [24]. In addition, miR-1245was reported to directly target and suppress the expression of theactivating NKG2D receptor [25].

MiR-182 belongs to the miR-96/182/183 family. It has beenproven in HCC as an oncogenic miRNA, boosting the proliferative,invasive, and metastatic abilities of liver tumor cells, and increas-ing their resistance to chemotherapy [26–28]. Regarding its rolein immunomodulation, miR-182 has been reported to regulatethe important helper T-cell mediated immune responses byinhibiting the proliferation suppressor Foxo1, and hence, allowingthe proliferation and expansion of helper T-lymphocytes [29]. Fur-thermore, miR-182 was found to regulate the immunosuppressiveregulatory T cells (Treg) preserving their stability and suppressorfunction [30].

To the best of our knowledge, the role of miRNAs in tuning NKcells in HCC has never been explored. Therefore, this study aimedat identifying the impact of miR-182 on NK cell cytotoxicity inHCC.

2. Materials and methods

2.1. In silico analysis

Several online bioinformatics software such as microrna.org,Diana Lab, and BiBiServ, were used to predict a potential miRNAthat might target both the NKG2D and the NKG2A mRNAs. AmiRNA with good binding scores for both targets on the 30UTRand the 50UTR was selected.

2.2. Subjects and sample collection

A total of 72 non-metastatic hepatitis C virus (HCV)-inducedHCC patients and 12 healthy controls were included in the screen-

ing experiments, while 25 out of the 72 HCC patients wereincluded in the transfection experiments. For all patients, theHCV infection positivity was diagnosed by the presence of anti-HCV antibodies and HCV RNA in the serum. All patients were neg-ative for the hepatitis B surface antigen. Both patients and healthycontrols included in this study gave their written informed con-sent. All experiments were performed in compliance with theguidelines of the Institutional Review Board of Kasr El Aini MedicalSchool in Cairo University and in accordance with the ethical stan-dards of the declaration of Helsinki.

2.3. Peripheral blood mononuclear cells and NK cell isolation

8 ml of peripheral venous blood were withdrawn from patientsand controls. Then peripheral blood mononuclear cells (PBMCs)were isolated from whole blood samples by Ficoll density gradientcentrifugation method using standard procedures (Axis-Shield PoCAS, Norway). 15 � 106 PBMCs were used as a starting count fornegative untouched NK cell selection using MACS human NK cellisolation kit (Miltenyi Biotec, USA) according to the manufacturer’sinstructions.

2.4. NK cells culture and transfection

NK cells were cultured in complete RPMI medium supple-mented with L-glutamine (Lonza, Belgium), containing 5% fetalbovine serum (FBS; Lonza, Belgium) and 1% Penicillin/Strepto-mycin and incubated under normal growth conditions (37 �C, 5%CO2). Cells were seeded at 6 � 104 NK cells/well in 96-well plates.Transfection of NK cells with miR-182 mimics or inhibitors(MSY0000259 and MIN0000259, respectively) (Qiagen, Germany)was performed 24 h after seeding using HiPerfect TransfectionReagent (Qiagen, Germany) according to manufacturer’s instruc-tion. Co-culturing or RNA extraction was performed 48 h post-transfection.

2.5. Total RNA extraction, reverse transcription and quantificationusing quantitative real-time polymerase chain reaction (qRT-PCR)

48 h post-transfection, total RNA was extracted using Biozol(BIOR, China). Later, total cellular RNA was reverse-transcribed intosingle-stranded complementary DNA (cDNA) using the HighCapacity cDNA archive kit (Applied Biosystems, USA) accordingto manufacturer’s instruction. MiRNA was extracted using TaqManmicroRNA Reverse Transcription Kit (Applied Biosystems, USA) andusing specific primers for hsa-miR-182 (Assay ID: 002334) orRNU6B (Assay ID: 001093).

The mRNA and miRNA expression levels were quantified usingStepOne Real-Time PCR instrument (Applied Biosystems, USA).Relative expression was calculated using comparative CT method.Beta-2-microglobulin (B2 m) was used as internal control for cellu-lar genes while RNU6B was used as internal control for miRNA. Theexpression of gene or miRNA in patients was normalized to theaverage expression of those in healthy controls.

2.6. Co-culture of NK cells with Huh-7 cell lines

Huh-7 cells were seeded in a 96-well plate (15,000 cells/well) incomplete DMEM medium and incubated under normal growthconditions (37 �C, 5% CO2) 2 h before co-culturing. Untransfectedor miR-182-mimicked NK cells from HCV-induced HCC patientswere pelleted after 48 h of transfection and then resuspended incomplete RPMI medium, free from any transfection complex. TheseNK cells were then co-cultured with the target Huh-7 cells at dif-ferent effector-to-target ratios (E:T), after removal of the completeDMEM placed before. The co-cultured cells were then incubated

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for 6–8 h at 37 �C and 5% CO2. NK cell cytotoxicity was measuredafter co-culturing.

2.7. NK cell-mediated cytotoxicity assay

The cytolytic activity of the NK cells from HCV-induced HCCpatients was assessed in vitro against target Huh-7 cells usingthe Lactate Dehydrogenase Activity Assay kit (MAK066-1K1-Sigma-Aldrich) according to the manufacturer’s instructions. Uponoptimization the effector-to-target ratio (5:1) was chosen, where15 � 103 Huh-7 cells were co-cultured with 75 � 103 NK cells. Inbrief, the assay was terminated after co-culturing by removingthe media and centrifugation of adherent Huh-7 cells in freshmedia, then the supernatant was collected for lactate dehydroge-nase (LDH) activity measurement. The LDH activity was calculatedusing the following equation provided by the manufacturer:

LDH activ ity¼Amount ðnmoleÞ of NADH generated � Sample Dilution FactorReaction Time � sample volume ðmLÞ added to well

The target maximum LDH activity was determined from non-co-cultured wells containing target Huh-7 cells of equal count(15 � 103). All the sample LDH activity and maximum LDH activitymeasurements were performed in triplicates.

Finally the percentage of cytotoxicity was calculated using thefollowing equation:

% Cytotoxicity¼ Target maximum LDH activity�Sample LDH activityTarget maximum LDH activity �100

2.8. Statistical analysis

The relative quantification of the results obtained by qRT-PCRwas performed using 2–DDCT. MiRNA and mRNA expression werecompared using the Student’s unpaired t-test and correlation anal-ysis was carried out using Spearman analysis. Results were consid-ered statistically significant at p < 0.05. Analysis was performedusing the GraphPad Prism 5.00 software.

3. Results

3.1. In silico prediction

In silico analysis using different online software revealed thatmiR-182 may target the mRNAs of NKG2D and NKG2A, and haveputative binding sites on the 30UTR and the 50UTR of both mRNAs(Tables 1 and 2).

3.2. Screening of miR-182 expression in NK cells of HCC patients

Given its prediction to be a potential regulator of both NKG2Dand NKG2A using bioinformatic analysis, miR-182 expressionwas analyzed in NK cells of HCC patients as well as healthy con-trols, where it was found to be significantly upregulated in NK cellsof HCC patients (p = 0.0146) (Fig. 1a).

3.3. Screening of NKG2D and NKG2A expressions in NK cells of HCCpatients

NKG2D and NKG2A baseline expression levels were quantifiedin the NK cells of HCC patients as well as healthy controls. A signif-icant upregulation of NKG2DmRNAwas observed in the NK cells ofHCC patients compared to healthy controls (p = 0.0217) (Fig. 1b).On the other hand, a significant downregulation of NKG2A mRNAwas observed in the NK cells of HCC patients compared to healthycontrols (p = 0.0153) (Fig. 1c).

Upon correlating miR-182 expression to NKG2D and NKG2AmRNA expression, a significant direct correlation was revealedbetween miR-182 and NKG2D expression (p = 0.0104, r = 0.6848)(Fig. 2A) and between miR-182 and NKG2A expression(p = 0.0013, r = 0.7832) (Fig. 2B).

3.4. Impact of miR-182 on NKG2D and NKG2A mRNA expression in NKcells of HCC patients

To further characterize the impact of miR-182 on NKG2D andNKG2A mRNA expression, NK cells from HCC patients were eitherkept untransfected or transfected with miR-182 mimics or antago-mirs, and the change in gene expression was determined by realtime qRT-PCR. Transfection efficiency was checked after eachexperiment, where NK cells transfected with miR-182 mimicsshowed approximately a 1300-fold increase in the expression ofmiR-182 compared to untransfected NK cells (p < 0.0005) (Fig. 3a).NKG2D and NKG2A expression was then assessed in untransfected,miR-182-mimicked and miR-182-antagonized NK cells of HCCpatients. It was found that upon forcing the expression of miR-182, a significant increase was observed in the expression of bothNKG2D mRNA (p = 0.0459) (Fig. 3b) and NKG2A mRNA (p = 0.0230)(Fig. 3c) compared to untransfected NK cells. However, anti-miR-182 did not yield significant differences in the expression of nei-ther NKG2D mRNA (p = 0.2525) (Fig. 3b) nor NKG2A mRNA(p = 0.2797) (Fig. 3c) compared to untransfected NK cells.

3.5. Impact of miR-182 on perforin mRNA expression in NK cells of HCCpatients

To evaluate whether miR-182 affects the cytotoxicity of NKcells, mRNA expression of the pore-forming protein, Prf1, wasassessed upon forcing miR-182 expression in the NK cells of HCCpatients. Prf1 mRNA was found to be significantly elevated inmiR-182 mimicked cells compared to untransfected cells(p = 0.0194) (Fig. 4a).

3.6. Impact of miR-182 on NK cell cytotoxicity upon co-culturing withtarget tumor cells

Finally, it was crucial to investigate the overall impact of miR-182 manipulation on the NK cell cytotoxicity against their targettumor cells. NK cells were co-cultured for 6–8 h with Huh-7 cellline at E:T of 5:1. Following, the LDH activity of the remainingadherent Huh-7 cells was measured as an indicator of target cellviability, and the percentage of NK cell cytotoxicity was calculated.MiR-182-mimicked NK cells showed 36% increase in the cytolyticactivity compared to untransfected NK cells (p = 0.0175) (Fig. 4b).

4. Discussion

HCC is a major health burden worldwide. With a yearly globalincidence far exceeding a half million new cases, HCC is regardedas one of the most rapidly spreading cancers in the world makingup 5.5% of all diagnosed cancers. Regarding mortality, HCC is con-sidered the second most lethal type of cancer accounting for 9.1%of the total cancer deaths. In Egypt, HCC was reported to be theworst type of cancer in males and the second worst in females interms of incidence and mortality [31]. Unfortunately, conventionalHCC treatment strategies are far from satisfactory, with few cura-tive therapeutic modalities available for a limited number of HCCpatients with specific criteria [32,33]. In view of these facts, devel-opment of new alternative and innovative therapeutic modalitiesis crucial, especially for advanced HCC patients. Immunotherapyhas recently emerged as a potential therapeutic approach, aiming

Table 1MiR-182 binding to NKG2D untranslated region.

Different bioinformatics tools were used to identifyNKG2D as a putative target for miR-182. For microRNA.org, the mirSVR score was used and no cutoff value present but thescores are interpreted as the more negative score the better the binding. For DIANA lab, the miTG score was used and the score is interpreted as the higher the value thehigher the probability of targeting. For BiBiServ, minimum free energy is used as indicator to the stability of the microRNA-mRNA binding.

Table 2MiR-182 binding to NKG2A untranslated region.

Different bioinformatics tools were used to identifyNKG2A as a putative target for miR-182. For microRNA.org, the mirSVR score was used and no cutoff value present but thescores are interpreted as the more negative score the better the binding. For DIANA lab, the miTG score was used and the score is interpreted as the higher the value thehigher the probability of targeting. For BiBiServ, minimum free energy is used as indicator to the stability of the microRNA-mRNA binding.

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to provide an efficient and selective targeting of tumor cells byboosting our tumor-specific immune responses.

This study aimed at investigating the role of miRNAs in regulat-ing the activation and cytotoxic function of NK cells in HCC. NKG2Dand NKG2A are powerful activating and inhibitory receptors,respectively, belonging to the same family of NK cell receptors.MiR-182 was found to have an immunomodulatory effect by regu-lating helper and regulatory T-cells [29,30]. It was also reported asa known oncomir in HCC [26–28]. MiR-182 was predicted to haveputative binding sites on the 30UTR and the 50UTR of both NKG2Dand NKG2A mRNAs (Tables 1 and 2). MiR-182 expression wasinvestigated in this study in the NK cells of HCC patients andhealthy controls, where it was found to be significantly upregu-lated in HCC patients (Fig. 1a). This is the first reported expressionprofiling of miR-182 in NK cells of HCC patients.

NKG2D and NKG2A expression levels were upregulated anddownregulated, respectively, in the NK cells of HCC patients com-pared to healthy controls (Fig. 1b and c). These findings contradicta previous study that showed underexpression of NKG2D in NKcells of HBV-induced HCC patients [14]. In another study, NKG2Dexpression was also reported to be downregulated in other typesof cancers [11]. Furthermore, the observed downregulation of

NKG2A in this study contradicts a previous study in which NKG2Aexpression was found to be upregulated in NK cells of advancedHCC patients [17]. This discrepancy could be due to the differencein the etiological factors leading to HCC because all the patients inthis study were HCV-induced HCC patients. This can also be relatedto the fact that in this work all the patients were non-metastaticHCC patients of early stages. The previous justification goes in linewith a study by Jinushi et al. [15] in which an increased NKG2Dexpression was revealed in early stages of HCC (stage I and II),where soluble MICA (sMICA) levels were undetected. Yet inadvanced stages (stage III and IV), NKG2D expression levels weredownregulated and the NK cell cytotoxicity was diminished dueto significantly high sMICA levels. Moreover, the reported upregu-lation of NKG2D and downregulation of NKG2A in this work couldbe explained as a compensatory mechanism by which the NK cellstry to act against the liver tumor cells in early stages of HCC. Inadvanced stages of HCC this relative expression of NKG2D andNKG2A was found to be reversed and hence halting the cytotoxicactivity of NK cells [15,17]. The previous explanation impeccablycorrelates to the findings of a recent study about the differentialexpression of NKG2D and NKG2A in cytotoxic T-cells from earlyand late stages of non-small-cell lung carcinoma (NSCLC); whereas

Fig. 1. Screening of miR-182 and its possible targets NKG2D and NKG2A. (a) miR-182 expression in NK cells of HCC patients. MiR-182 expression was found to be upregulatedin the NK cells of HCC patients compared to healthy controls (p = 0.0146). (b) NKG2D expression in NK cells of HCC patients. NKG2D mRNA expression was upregulated in theNK cells of HCC patients compared to healthy controls (p = 0.0217). (c) NKG2A expression in NK cells of HCC patients. NKG2D mRNA expression was found to bedownregulated in the NK cells of HCC patients compared to healthy controls (p = 0.0153).

Fig. 2. Correlation of miR-182 expression to NKG2D and NKG2A mRNA expression. (a) miR-182 correlation with NKG2D. A direct correlation was found between miR-182and NKG2D mRNA expression (p = 0.0104, r = 0.6848). (b) miR-182 correlation with NKG2A. NKG2A mRNA is directly correlated with miR-182 expression (p = 0.0013,r = 0.7832).

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high levels of the activating receptor and low levels of the inhibi-tory receptor were reported in early stages, in late stages theexpressions of both receptors were reversed [34].

Upon correlating miR-182 expression to NKG2D and to NKG2Aexpressions in NK cells of HCC patients, it was found that as thelevels of miR-182 increase, the levels of both NKG2D and NKG2AmRNAs increase accordingly (Fig. 2a and b). Furthermore, forcingthe expression of miR-182 significantly increased NKG2D andNKG2A mRNA expression compared to untransfected NK cells(Fig. 3b and c). It is worth mentioning that this study is amongthe first of its kind to manipulate the expression of miRNAs in pri-mary NK cells isolated from HCC patients.

Due to the increase in the expression of both the activatingNKG2D and the inhibitory NKG2A receptors, the net effect ofmiR-182 on the NK cell activation status and cytotoxicity remainedambiguous. Therefore, the impact of miR-182 on Prf1 expressionwas analyzed. Perforins are cytolytic pore forming proteins thatcreate pores in the plasma membrane of the NK target cellsenabling the entrance of granzymes which will later induce apop-tosis in these target cells [35]. Forcing the expression of miR-182resulted in an increased Prf1 mRNA expression in NK cells ofHCC patients (Fig. 4a). Despite using several software, we couldnot identify any in silico predicted binding between miR-182 andPrf1, which might suggest that the mechanism by which

Fig. 3. Impact of miR-182 on NKG2D and NKG2A mRNA expression. (a) Confirmation of transfection. A greater than 1300 fold increase in the expression levels of miR-182was observed in miR-182-mimicked NK cells compared to untransfected NK cells (p < 0.0005). (b) Impact of miR-182 on NKG2D expression. A significant upregulation in theNKG2D mRNA expression was found in miR-182-mimicked NK cells compared to untransfected NK cells (p = 0.0459). MiR-182-antagonized NK cells showed similar NKG2Dexpression pattern when compared to untransfected NK cells (p = 0.2525). (c) Impact of miR-182 on NKG2A expression. A significant upregulation in the NKG2A mRNAexpression was found in miR-182-mimicked NK cells compared to untransfected NK cells (p = 0.0459). MiR-182-antagonized NK cells showed similar NKG2A expressionpattern when compared to untransfected NK cells (p = 0.2797).

Fig. 4. Impact of miR-182 manipulation on cytotoxic effect of NK cells on target cells. (a) Impact of miR-182 on Prf1 expression. A significant upregulation in the expression ofPrf1 mRNA was observed in miR-182-mimicked NK cells compared to untransfected NK cells (p = 0. 0194). (b) Impact of miR-182 on the NK cell cytotoxicity against Huh-7cells in E:T (5:1). After forcing the expression of miR-182 in NK cells of HCC patients, NK cells were co-cultured with Huh-7 cell line. After 6–8 h, the LDH activity of remainingviable Huh-7 cells was measured and % NK cytotoxicity was calculated. The cytotoxic activity was increased in miR-182-mimicked NK cells compared to untransfected NKcells as shown by 36% increase in the cytotoxicity of co-cultured Huh-7 cells (p = 0.0175).

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miR-182 affects Prf1 could be indirect, perhaps through modulat-ing the expression of the NKG2D and NKG2A. It was also crucialto investigate the overall impact of miR-182 manipulation on theNK cell cytotoxicity against their target tumor cells upon

co-culturing. Forcing miR-182 expression in NK cells of HCCpatients increased their cytolytic activity against Huh-7 cancer celllines by 36% compared to untransfected NK cells (Fig. 4b). Theabovementioned increase in the cytotoxicity of NK cells suggests

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that after forcing the expression of miR-182 the activation signalsof the NKG2D overrode the inhibitory signals of the NKG2A. Thisgoes in line with a previous report which revealed NKG2D to havea powerful role in the activation of NK cells, and reported theinduction of its cytotoxicity even in the presence of inhibitory sig-nals [36].

In conclusion, forcing the expression of miR-182 increases theexpression of two crucial NK cell receptors and ultimatelyenhances the NK cell activation and cell lytic activity in liver tumorcells. This finding opens up a new approach for developing a ther-apeutic strategy whereby the manipulation of miRNAs in NK cells -may be an effective approach to activate NK cell-mediatedclearance of tumor cells and may enhance NK cell-basedimmunotherapy against various cancers.

Declaration of interest

The authors declare that they have no conflict of interest.

Acknowledgement

The author would like to acknowledge the rest of the MolecularPathology Research Group (MPRG) members for their valuablesupport and guidance in this study.

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