MicroRNA-30c promotes natural killer cell cytotoxicity via up-regulating the expression level of NKG2D

    MicroRNA-30c promotes natural killer cell cytotoxicity via up-regulating the expression level of NKG2D Ying Ma, Jiuyu Gong, Yuan Liu, Wenwei Guo, Boquan Jin, Xiaohong Wang, Lihua Chen PII: DOI: Reference:

S0024-3205(16)30162-X doi: 10.1016/j.lfs.2016.03.012 LFS 14795

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

2 December 2015 28 February 2016 7 March 2016

Please cite this article as: Ma Ying, Gong Jiuyu, Liu Yuan, Guo Wenwei, Jin Boquan, Wang Xiaohong, Chen Lihua, MicroRNA-30c promotes natural killer cell cytotoxicity via up-regulating the expression level of NKG2D, Life Sciences (2016), doi: 10.1016/j.lfs.2016.03.012

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ACCEPTED MANUSCRIPT MicroRNA-30c promotes Natural Killer cell cytotoxicity via up-regulating the

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expression level of NKG2D

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Ying Maa,1, Jiuyu Gonga,b,1, Yuan Liua,c, Wenwei Guoa,c, Boquan Jina, Xiaohong Wangc,*, Lihua Chena,* a

Department of Immunology, the Fourth Military Medical University, Xi’an 710032,

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China

Hospital of Hubei Armed Police Corps, Wuhan, Hubei 430000, China

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Department of Gynecology and Obstetrics, Tangdu Hospital, the Fourth Military

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Medical University, Xi'an 710038, China

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* Corresponding author:

Dr. Lihua Chen, Department of Immunology, the Fourth Military Medical University,

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169 Changle West Road, Xi’an 710032, China. Tel.: +86 029 84774531; fax: +86 029 83253816. E-mail address: [email protected] Dr. Xiaohong Wang, Department of Gynecology and Obstetrics, Tangdu Hospital, the Fourth Military Medical University, Xi'an 710038, China. E-mail address: [email protected]

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These authors contributed equally to this work.

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ACCEPTED MANUSCRIPT Abstract Aims: Natural Killer (NK) cells play critical roles in antitumor immunity. Our

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previous study showed that over-expression of miR-30c-1* enhanced NKL cell

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cytotoxicity through up-regulation of tumor necrosis factor-α via directly targeting transcription factor homeobox containing 1. MiR-30c, the complimentary microRNA of miR-30c-1*, has been found to excert regulatory effect on T cell function. However,

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the effect of miR-30c on NK cells is unknown. Therefore, this study aimed to

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investigate whether miR-30c could play a role to enhance NK cell activation and cytotoxicity.

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Main methods: Chemosynthesis exogenous miR-30c mimics and miR-30c inhibitor

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were transfected into NKL cells and isolated human peripheral blood NK cells,

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respectively. The expression levels of NK group 2, member D (NKG2D), CD107a and FasL on cell surface and cytotoxic ability of miRNAs transfected NKL cells against

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SMMC-7721 cells were evaluated. Key findings: miR-30c could increase the expression of NKG2D and CD107a on NKL cells, and enhance cytotoxic ability of NKL cells to kill SMMC-7721 cells. Moreover, miR-30c could up-regulate the expression of FasL on both NKL cells and human peripheral blood NK cells. However, the peripheral blood NK cells from only four in ten healthy donors appeared high expression levels of NKG2D and CD107a after miR-30c transfection. Significance: miR-30c could promote the cytotoxicity of NKL cells in vitro by up-regulating the expression levels of NKG2D, CD107a and FasL. However, the 3

ACCEPTED MANUSCRIPT effect of miR-30c on ex vivo NK cells from different human individuals is diverse,

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indicating that miR-30c may play complicate and fine adjustment in immune system.

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Keywords: Natural Killer cell; microRNA-30c; NK group 2, member D (NKG2D);

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CD107a; Fas ligand; Cytotoxicity

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ACCEPTED MANUSCRIPT 1. Introduction Natural killer (NK) cells play important roles not only in the innate but also in the

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adaptive immunity to control pathogen infections and malignancies through the

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release of cytolytic granules or the ligation of death-inducing receptors. The important functions of NK cells are controlled and regulated by a wide range of receptors on their surface transmitting signals to NK cells to respond rapidly to the changes of

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environment [1]. Importantly, the activation and cytotoxicity of NK cells were

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determined by the signals delivered through the balance between activating and inhibitory receptors on NK cells [2-4]. Among these receptors, NK group 2, member

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D (NKG2D) is an activating surface molecule that could deliver activating and

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co-stimulatory signals to NK cells and then play an important role in NK

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cell-mediated immune responses.

MicroRNAs (miRNA) are small noncoding RNAs generated by sequential

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processing of primary microRNA transcripts by ribonucleases Drosha and Dicer [5]. MicroRNA-mediated gene regulation represents a fundamental post-transcriptional mechanism with diverse effects on cellular functions such as proliferation, differentiation, and apoptosis [6-8]. Various microRNAs control the gene expression and regulate the effects of lymphocytes which usually related with the onset of immune-mediated diseases, illustrating the importance of microRNA. In fact, microRNAs have been reported to play critical roles in immune system including the early differentiation and maturation of B cells [9-10], the regulation of the lineage induction pathways, the induction,

maintenance and function of regulatory T cells 5

ACCEPTED MANUSCRIPT [11-13], and the regulation of the differentiation of dendritic cells and macrophages via toll-like receptors [14-15].

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It has also been reported that microRNAs have important modulatory functions on

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NK cells. Several microRNAs including let-7c, miR-21, miR-30c, miR-181d and miR-200a* have been proved to be involved in the activation of NK-like (NKL) cells and then to take part in cancer biology, such as the initiation, progress and metastasis

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of the tumor [16-18]. Our previous study found that miR-30c-1* could enhance NKL

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cell cytotoxicity against human hepatoma cells through up-regulation of tumor necrosis factor-α (TNF-) via directly targeting transcription factor homeobox

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containing 1 (HMBOX1) [19]. The microRNA-30c has been proved to participate in T

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cell differentiation and play an important role in adipogenesis, lipid metabolism,

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cardiovascular diseases and cancer [20-21]. Several studies have reported that decreased miR-30c expression was observed in many kinds of cancers, whereas

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overexpression of miR-30c could slow down the progression and metastasis of cancer in both cultured cells and mouse models [22-27]. Thus, miR-30c mimics has been considered to be a potent therapeutic agent for the treatment of cancer and other diseases with low levels of miR-30c [21]. However, whether miR-30c could be involved in the cytotoxicity enhancement of NKL cells and then to take part in cancer biology were largely unknown. Meanwhile, our understanding of the mechanism performed by miRNAs on the function of NK cells remain rather limited. Herein, our study showed that miR-30c may up-regulate the cytotoxicity and killing capacity of NKL cell by increasing the expression level of NKG2D, CD107a and FasL. Moreover, 6

ACCEPTED MANUSCRIPT although miR-30c could increase the cytotoxicity ability of NKL cell in vitro, the effect of miR-30c on peripheral blood NK cells from different person is not exactly

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the same. Thus, our results not only identified miR-30c with the regulation effect on

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the activation and cytotoxicity of NK cells, but also suggested that microRNA

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molecules may play different roles in diverse systems.

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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1 Cell culture

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Cells were cultured following standard procedures. Briefly, α-MEM supplemented

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with 12.5% fetal bovine serum (FBS), 12.5% horse serum, penicillin/streptomycin and 200 U/ml human recombinant interleukin-2 (IL-2) (Peprotech, NJ, USA) was used for NKL cell culture, which was established from human NK cell leukemia.

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Human hepatocellular carcinoma cell line SMMC-7721 cells were maintained in

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RPMI1640 with 10% FBS and penicillin/streptomycin. Human peripheral blood NK cells were isolated by negative immunoselection Dynabeads® Untouched™ Human

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NK Cells (Invitrogen Dynal AS, Oslo, Norway) from peripheral blood mononuclear

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cells (PBMCs) which were isolated from healthy donors obtained from Xijing

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Hospital, Xi’an, China using a standard Ficoll-Hypaque (Sigma-Aldrich, MO, USA) density gradient centrifugation. Human peripheral blood NK cells were cultured in

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RPMI1640 medium supplemented with 10% FBS, 2% human serum and 200 U/ml human recombinant IL-2. All the cells were cultured at 37 °C in 5% CO2 and 100% humidity. The study was approved by the Ethics Committee at the Fourth Military Medical University. The informed consents were obtained from all the donors in this study.

2.2 MicroRNA transfection NKL cells or the isolated human peripheral blood NK cells were seeded in 24-well plates at 5×104 cells per well and transfected with chemosynthesis 8

ACCEPTED MANUSCRIPT microRNA mimics and microRNA inhibitors (Genpharma, Suzhou, CN). The sequences

of

the

miR-30c

oligoribonucleotides

were

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follows: miRNA

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5’-CCAGUGUAGGGUAAACACCUCUCUCAGCUUGG-3’.

as

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mimics, miRNA inhibitors, and controls were transiently transfected into cells using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. Each transfection used 50-nM RNA duplexes. The transfection

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efficiency was evaluated by flow cytometric analysis 24 h after fluorescein

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isothiocyanate (FITC) labeled miRNA mimics transfected into the cells. The percentage of FITC positive cell population was considered as the transfection

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efficiency.

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2.3 Flow cytometric analysis

NKL cells and human peripheral blood NK cells were stained with FITC-labeled

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anti-NKG2D monoclonal antibody (mAb) (BD Biosciences, NJ, USA) and phycoerythrin (PE)-Cy5 labeled anti-CD107a mAb (BD Biosciences, NJ, USA) for 30 min on ice. FITC and PE-Cy5 labeled mouse IgG1κ isotype control (BD Biosciences, NJ, USA) were used as negative controls, respectively. For the analysis of the Fas ligand (FasL) expression, the NKL cells or human peripheral blood NK cells transfected with miRNA mimics for 24 h, 48 h or 72 h were stained with PE-labeled anti-FasL mAb (BD Biosciences, NJ, USA) and incubated for 30 min at 4℃. A minimum of 20,000 gated events for each sample were collected on a FACSCalibur and analyzed using CellQuest software. 9

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2.4 Cell-mediated cytotoxicity assay

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For the detection of specific killing activity of NKL cells to human hepatoma cell

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line SMMC-7721 cells, 2×107/ml SMMC-7721 cells were labeled with 10 μM 5, 6-carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes, OR) at 37℃ for 15 min, terminated upon the addition of FBS, washed twice in phosphate buffered

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saline (PBS) and resuspended at 107 cells/ml PBS. Then the labeled SMMC-7721

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cells were plated in 24-well flat-bottom plates at 2×105 cells per well (Costar, US). NKL cells transfected with miRNA control, miR-30c mimics or miR-30c inhibitor

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respectively for 48 h were added into the plates at the ratio of 16:1, 8:1 or 4:1 to target

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cells. The plates were centrifuged at 800 rpm/min for 5 min and incubated at 37 °C in

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5% CO2 and 100% humidity for 4 h. Cells were harvested and washed twice with PBS and stained with propidium iodine (PI) for 15 min at 37 °C. A minimum of

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100,000 gated events for each sample were collected on a FACSCalibur and analyzed using CellQuest software.

2.5 Statistical analyses Each experiment was performed independently at least three times and one representative experiment is presented. All statistical analyses were performed using the SPSS 16.0 (SPSS Inc., Chicago, IL, USA) statistical software package. Graphing were performed using Prism software, version 5.0 (Graphpad; La Jolla, CA). Average values are reported as the mean ± S.D. The significance of difference between two 10

ACCEPTED MANUSCRIPT groups was analyzed statistically by the compared t test with Welch’s correction, or Mann-Whitney U test. A two-tailed P value below 0.05 (P ≤ 0.05) was considered

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statistically significant.

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ACCEPTED MANUSCRIPT 3. Results 3.1 Exogenous miR-30c mimics increased the expression levels of membrane NKG2D

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and CD107a in NKL cells

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Given the importance of NKG2D as an activating receptor expressed on NK cells

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and CD107a as a stable marker for NK cell cytotoxicity, we firstly detected whether miR-30c could regulate NKG2D and CD107a expression levels in vitro. Our database searches of Sanger (http://microrna.sanger.ac.uk) revealed that miR-30c was one of

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the complementary sequences of miR-30c-1* (Fig. 1). Thus, chemosynthesis single

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strand miR-30c mimics were used to explore the function of miRNA in NKL cells and in peripheral blood NK cells. Then the evaluation of the efficiency for miR-30c

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mimics transfection showed that there was similar transfection efficiencies observed

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between NKL cells (34.3%) and peripheral blood NK cells (35.4%) following miR-30c mimics transfection (Fig. 2). After miRNA mimics transfected into NKL

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cells, the expression levels of NKG2D and CD107a on the surface of the cells were analyzed by flow cytometry. Overall, the exogenous miR-30c mimics elevated the

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expression of NKG2D and CD107a on the surface of NKL cells, whereas the inhibitor of miR-30c showed the opposite effects (Fig. 3A). As shown in Fig. 3B and C, miR-30c mimics could significantly up-regulate the expression percentages of both NKG2D and CD107a on the surface of NKL cells at 24 h and 48 h after transfection. Moreover, the mean value of NKG2D expression was obviously up-regulated on NKL cells at 24 h after the transfection of miR-30c mimics, whereas the mean value of NKG2D expression at 48 h and the CD107a expression at both 24 h and 48 h on NKL cells showed no statistical difference between the transfection of miR-30c mimics and transfection of miR-30c inhibitor. However, the up-regulated tendency of mean value of NKG2D and CD107a expression could still be observed in miR-30c mimics 12

ACCEPTED MANUSCRIPT transfection group (Fig. 3D and E). Therefore, we could speculate that miR-30c may have partly prominent regulatory effects on the activation and function of NKL cells,

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which may due to the up-regulation of the expression levels of the important receptor

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NKG2D and cytotoxic molecule CD107a on the surface of NKL cells.

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Fig. 1

Fig. 2

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ACCEPTED MANUSCRIPT

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Fig. 3

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up-regulation of NKG2D

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3.2 Exogenous miR-30c mimics enhanced the cytotoxicity of NKL cell via

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Notably, by transfecting of miR-30c mimics into NKL cells, the expression of CD107a, an effector molecule reflecting the degranulation and cytotoxicity of NK cells, was significantly up-regulated. Therefore, we next set out to determine whether

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miR-30c could have the effects on the cytotoxicity capacity of NKL cells. The CFSE/PI analysis was carried out to investigate the killing capacity of NKL cells transfected with miRNAs mimics to SMMC-7721 cells. This hepatoma cell line SMMC-7721 cells highly express MHC class I-related chain A/B (MICA/B), which is the ligand of NKG2D. It was found that NKL cells transfected with exogenous miR-30c mimics showed higher cytotoxicity percentage against SMMC-7721 cells compared with those transfected with control miRNA mimics at each effect cell to target cell ratio, whereas the inhibitor of miR-30c showed the opposite effects (Fig. 4A). However, when NKG2D blocking antibody was pre-incubated with NKL cells, the enhanced killing activity of miR-30c mimics transfected NKL cells to 14

ACCEPTED MANUSCRIPT SMMC-7721 cells was significantly reversed (Fig. 4B), suggesting that the observed effects may be mediated through a modulation of the receptor NKG2D, and the

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enhanced killing activity of NKL cells was at least in part due to the increased

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expression level of NKG2D after miR-30c mimics transfection.

3.3 Exogenous miR-30c mimics displayed different regulation profiles in peripheral

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blood NK cells from different individuals To better assess the regulatory function of miR-30c on NK cells, we next performed the similar detection on human peripheral blood NK cells ex vivo. The peripheral blood NK cells were isolated from ten healthy individuals numbered from P1 to P10 randomly. Then the chemosynthesis single strand miRNA mimics were transfected into peripheral blood NK cells respectively and the expression levels of NKG2D and CD107a on the surface of NK cells were analyzed. It was found that the expression percentages of NKG2D and CD107a on peripheral blood NK cells from only four of the ten individuals (P1, P4, P9 and P10) were increased significantly after transfection of exogenous miR-30c mimics. However, miR-30c mimics transfection showed no 15

ACCEPTED MANUSCRIPT apparent regulatory effect on the expression of NKG2D and CD107a on peripheral blood NK cells from the other six individuals (P2, P3, P5, P6, P7 and P8) (Fig. 5A and

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B). Take the healthy individuals P1 and P4 as examples, the effects of miR-30c

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mimics on peripheral blood NK cells with the significantly increased expression of

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both NKG2D and CD107a could be observed, whereas the inhibitor of miR-30c showed opposite effect (Fig. 5C), which was the same with the results obtained from the NKL cells. However, compared with the previous in vitro studies, the inconsistent

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effects of miR-30c on the ex vivo peripheral blood NK cells indicated that although

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miR-30c may potentially enhance the activation of NK cells, the individual variability

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was one of the important factors affecting the regulatory function of miR-30c.

Fig. 5

3.4 Exogenous miR-30c mimics induces up-regulation of FasL expression on both NKL cells and human peripheral blood NK cells The mechanisms of NK cell-mediated cytotoxicity and apoptosis include not only the cytolytic mediators, but also Fas-FasL interaction. FasL is a member of the tumor 16

ACCEPTED MANUSCRIPT necrosis factor family and expressed on the membrane of activated NK cells, being involved in the apoptosis of the target cells. Therefore, we speculated that the

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expression level of FasL on NK cells may be also regulated by miR-30c. To confirm

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this hypothesis, we measured the expression levels of FasL at different time points following miR-30c mimics transfection into NKL cells and human peripheral blood NK cells, respectively. The results showed that FasL exhibited increased percentages

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of expression on the surface of NKL cells or peripheral blood NK cells after

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transfecting miR-30c mimics (Fig. 6A). Compared with the control miRNA mimics, transfection of miR-30c mimics led to a significant elevation of FasL expression

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percentages on both NKL cells and peripheral blood NK cells at all the three time

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points 24 h, 48 h and 72 h (Fig. 6B and C). Meanwhile, the mean values of FasL

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expression were significantly up-regulated at 72 h on both NKL cells and peripheral blood NK cells after miR-30c mimics transfection (Fig. 6B and C). Therefore, the

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results from our study indicated that FasL could be regulated by miR-30c to promote the function of NK cells. The up-regulation of FasL expression may be one of the mechanisms involved in the enhancement of NK cell activation induced by miR-30c.

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Fig. 6

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ACCEPTED MANUSCRIPT 4. Discussion miRNAs have been proved to exert pleiotropic functions in the modulation of

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immune system. Several miRNAs with known function take part in the development

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and activation of many immune cell-type [28]. In this study, another regulatory

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miR-30c promoting NKL cell cytotoxicity was identified. We showed that exogenous overexpression of miR-30c enhanced NKL cell cytotoxicity against hepatoma cell line SMMC-7721 cells through up-regulation of NKG2D, CD107a and FasL expression

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on NKL cells. Furthermore, we also found that miR-30c played different roles in ex

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vivo peripheral blood NK cells from different persons, suggesting that there might be flexible and complexity for miR-30c to exert the regulatory function.

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Previous studies have shown that cytokines such as interferon (IFN)-, IL-2, IL-12

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and IL-15 [29-31] and transcription factors such as activator protein 1 (AP-1) [32-33], E4BP4 (also known as nuclear factor, interleukin 3 regulated (NFIL3)) [34-35] and

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HMBOX1 [36], may contribute to the regulation of signal transduction in NK cells. Notably, different microRNAs may play diverse effects on the function of NK cells.

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Several studies have reported the down-regulating effects on the activation of NK cells by microRNA molecules. MiR-150 repressed NK cell lytic activity by targeting perforin-1 [37]. The human cytomegalovirus derived miR-UL112 could attenuate NK cell activity by inhibiting type I interferon secretion [38]. MiR-23a decreased granzyme B activity leading to impaired NK cell cytotoxicity [39]. Downregulation of miR-302c and miR-520c enhanced the susceptibility of tumour cells to NK cell-mediated cytotoxicity [40]. MiR-146a was proved to be involved in hepatocellular carcinoma cells-induced NK cell dysfunction [41]. Ovarian tumor-associated miR-20a decreased NK cell cytotoxicity by downregulating MICA/B expression [42]. The overexpression of miR-583 had an inhibitory effect on 19

ACCEPTED MANUSCRIPT NK cell differentiation by silencing IL-2Rγ [43]. MiR-10b inhibited the expression of MICB, a stress-induced ligand of the activating NK cell receptor NKG2D [44].

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TGF-β–inducible miR-183 silenced tumor-associated NK cells [45]. In contract, some

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microRNAs may contribute to the differentiation and function of NK cells. MiR-181a

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and miR-181b are critical for human NK cell development [46]. The miR-155 is important for the enhanced NK cell development, homeostasis, and activation [47-49], and could regulate IFN-γ production in NK cells after chronic hepatitis C virus

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infection [50].

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Our previous study found that miR-30c-1* may promote the function of NKL cells against hepatoma cells [19]. Interestingly, miR-30c was one of the complementary

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sequences of miR-30c-1*. Thus, there may be certain relationship of the functions

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between these two miRNA molecules. MiR-30c has been found to be highly

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conserved among vertebrates and is mostly expressed in the heart, skeletal muscle, and kidneys [51]. Several studies have demonstrated a key role of miR-30c in cancer

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pathogenesis and found that the expression of miR-30c was reduced in many kinds of carcinomas [21,52]. Moreover, it is speculated that the tumor suppressor miR-30c may have diagnostic and therapeutic potential. Therefore, in this study, we tried to figure out whether miR-30c could play an effect on the activation and cytotoxicity of NK cells as one of the mechanisms to control tumor progression.

Intriguingly, we

found that the exogenous miR-30c enhanced the cytotoxic ability of NKL cells through up-regulation of the expression of NKG2D, CD107a and FasL on the surface of NKL cells, which was similar with the effects of miR-30c-1* on NK cells, although the mechanisms may be not exactly the same. 20

ACCEPTED MANUSCRIPT Notably, miRNAs exert their function by regulating the expression of their downstream target genes. Based on the bioinformatics method and experimental proof,

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our previous study has further confirmed that the inhibitory transcription factor

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HMBOX1, which has been reported to be related with the suppression to the activating of NK cells, was the direct target gene of miR-30c-1* in NKL cell activation [19]. Overall, several studies revealed that miR-30c might be involved in

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tumor suppression in different ways by targeting different genes and then regulate key

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steps in the metastatic process of cancers. For example, miR-30c has been validated to negatively regulate cancer metastasis with early response protein 2 as a direct target

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gene in SMMC-7721 and HepG2 cells [52]. Moreover, in lung cancer, the increased

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miR-30c expression targeting tumor-suppressor genes was proved to be associated

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with acquired TRAIL resistance [22]. MiR-30c was also shown to be dramatically down-regulated in colon cancer tissues and inhibit cancer cell growth, migration and

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invasion in vitro via targeting ADAM19 [23]. In non-small cell lung cancer, miR-30c could inhibit cancer cell proliferation by targeting Rab18 [24]. Furthermore, miR-30c may directly inhibit target gene MTA-1 expression and function as a tumor suppressor via the miR-30c-MTA-1 signalling pathway in endometrial cancer [25]. In acute myeloid leukemia, miR-30c was downregulated with NOTCH1 as the experimentally validated target [26]. Many other studies of breast cancer showed that miR-30c could significantly suppress the ability of breast cancer cell line MCF-7/ADR to resist doxorubicin by regulating anti-apoptotic gene YWHAZ as a target, and miR-30c might suppress breast cancer cell growth potentially through 21

ACCEPTED MANUSCRIPT inhibition of Kirsten rat sarcoma viral oncogene (KRAS) signaling [27,53]. Thus, although we have provided evidence that overexpression of miR-30c might enhance

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NKL cell activity to kill hepatoma cell line SMMC-7721 cells through up-regulation

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of NKG2D, CD107a and FasL expression on NKL cells, the mechanism for miR-30c to promote NK cell cytotoxicity and its downstream target genes were still the important issues for us to be determined in the next step.

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It is generally accepted that different receptors on the surface of NK cell not only

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could define the NK subsets with different functional properties, but also could be served as the target genes for miRNAs to regulate. The interaction of specific NK

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cell-activating receptors with their cognate ligands on target cells was required for NK

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cell-lysis. NKG2D, a C-lectin type activating receptor on NK cells, could deliver

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activating and co-stimulatory signals resulting in cytotoxicity and release of cytokines. Ligands binding to NKG2D include RAE1, H60 in the mouse and MICA/B and

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ULBP1 in human, which could always be induced expression on the cells undergone “stress” such as virus infections and tumorigenesis [54]. Furthermore, NKG2D was considered as molecular target for NK cell-mediated control and immunotherapy in lymphomas [55]. The expression level of NKG2D on NK cells is affected by some activating molecules like IL-2 and poly I:C. Importantly, several miRNAs have been proved to regulate the expression of NKG2D [56]. In our study, we verified the up-regulation of NKG2D expression on NKL cells following the exogenous miR-30c mimics transfection, which may be one of the mechanisms for the improved cytotoxicity of NKL cell to kill the hepatoma cell line SMMC-7721 cells. Importantly, 22

ACCEPTED MANUSCRIPT once triggered to kill, the NK cells lyse a target always through two independent lytic mechanisms: the degranulation of perforin/granzyme and the death receptor pathway.

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Of note, NK cells exhibit obvious CD107 expression immediately following a target

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cell interaction. In fact, as shown to be required for perforin trafficking and granule movement [57], CD107a (LAMP-1) has been considered as a marker for NK cell cytotoxicity, which is not only stable but also may amplify over time during NK cell

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degranulation [58]. Moreover, the CD107a degranulation assay which could detect the

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expression of CD107a on NK cell surface is now also used to evaluate cytotoxicity capacity of NK cells [59]. In the present study, exogenous miR-30c mimics could

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significantly elevate the expression levels of membrane CD107a on NKL cells,

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suggesting that miR-30c may have regulatory effects on the activation and

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degranulation of NK cells. Our study also gives us a hint that miR-30c may take part in the apoptosis effect of NKL cells by up-regulating the expression of FasL, which is

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a member of tumor necrosis factor family proteins and triggers cell death depending on Fas/FasL pathway. FasL has been proved to be served as the target for many miRNAs [60-61]. The fact that NKL cells and human peripheral blood NK cells transfected with exogenous miR-30c mimics expressed higher percentage of FasL than control miRNA comfirmed that miR-30c could promote the function of NK cells through up-modulate the expression of FasL. The temporal and spatial regulation which determined by the flexible and various expression pattern of miRNAs, is one of the interesting characteristics of miRNAs. A miRNA could regulate expression of a series of target genes, whereas the expression 23

ACCEPTED MANUSCRIPT of one miRNA should be affected by a group of transcription factors. The complicated regulating network determines the difficulty in researches on miRNAs’ function. Our

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finding that miR-30c could increase the NKG2D and CD107a expression on

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peripheral blood NK cells from only four in ten healthy individuals, instead of all of the ten persons indicated that there may be other factors influencing the effect of miR-30c when the detection performed ex vivo. One of the possible explanations

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about the perplexing outcome that miR-30c failed to play the uniform effects on

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different individuals might be restricted to certain NK subgroups of the population. It is known that NK cell can be divided into different subsets based on CD56 and CD16

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expression, including CD56brightCD16−, CD56brightCD16+ and CD56dimCD16++ NK

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cells [62]. The CD56dimCD16++ NK cell subset, known to be the mature NK cells, is

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mainly responsible for NK cell cytotoxicity and degranulation [63]. Moreover, acquisition of the inhibitory receptor killer Ig-like receptors (KIRs) has been

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demonstrated as a sign of NK cell differentiation and maturation [64-65]. The experienced cytotoxic CD56dimCD16++KIR+ NK cells would express high level of CD107a and NKG2D. Based on these, we speculated that miR-30c may regulate the expression of NKG2D and CD107a more in activated mature NK cells rather than in immature ones. Since the percentages of the three NK cell subsets may be inconsistent in different individuals, the mature cytotoxic CD56dim phenotype might be the mainly NK cell subset in the four persons with significant up-regulated NKG2D and CD107a expression after miR-30c mimics transfection, whereas the immature non-cytotoxic CD56bright subset may be the primary NK cell subset in other six individuals. In fact, 24

ACCEPTED MANUSCRIPT the effects of miRNA in different system may be controlled by various mechanisms. Although several miRNAs are enriched in lymphocytes, no miRNA is known to be

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exclusively expressed in the immune system. Therefore, to explore the exact

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functional characterization of each miRNA may be a very challenging work in the

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future [28].

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ACCEPTED MANUSCRIPT 5. Conclusion MiR-30c as one of the important regulatory miRNAs may be critical for normal

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function of NK cell. Our study proved that miR-30c could enhance the cytotoxicity of

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NKL cells against hepatoma cell line SMMC-7721 cells in vitro through up-regulation

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of the NKG2D, CD107a and FasL expression on the NKL cells, whereas miR-30c have a inconsistent improved effect on the function of the ex vivo peripheral blood NK cells. These findings about the enhancement effects of miR-30c on the

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cytotoxicity of NKL cells may be a remarkable development in NK cell-based

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therapies. However, the exact regulation mechanisms of miR-30c on NK cells have not yet been clarified. Further research will undoubtedly provide more accurate

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targets for miR-30c in regulating the function of NK cells as well as NK cell-mediated

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diseases.

Conflict of interest statement

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The authors declare that they have no conflict of interest.

Acknowledgements Dr. Lihua Chen and Dr. Xiaohong Wang are supported by grant no. 91442108 and 81370710 respectively, financed by the National Natural Science Foundation of China.

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ACCEPTED MANUSCRIPT Figure Legends Fig. 1. The sequences of the miR-30c oligoribonucleotides.

method

used

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database

searches

of

Sanger

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bioinformatics

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The sequences of the miR-30c are complimentary microRNA of miR-30c-1*. The

(http://microrna.sanger.ac.uk/).

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cells and peripheral blood NK cells.

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Fig. 2. The transfection efficiency for the transfection of miR-30c mimics into NKL

(A) NKL cells and (B) peripheral blood NK (PNK) cells were transfected with

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miR-30c mimics labeled with FITC respectively for 24 h. The transfection efficiency

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was determined as the percentages of FITC positive cell population.

Fig. 3. The detection of NKG2D and CD107a expression on NKL cells after

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transfection of microRNA mimics. (A) The expression of membrane NKG2D and CD107a on NKL were analyzed by flow cytometry 48 hours after transfected with miR-30c mimics (left panel) or miR-30c inhibitor (right panel) (black line) and control miRNA (Gray shaded histograms). Solid histograms represent background staining with control mAb. (B) and (C) The comparison of expression percentages of membrane (B) NKG2D and (C) CD107a on NKL cells after transfected with control miRNA, miR-30c mimics or miR-30c inhibitor at 24 hours and 48 hours, respectively. (D) and (E) The comparison of mean value of membrane (D) NKG2D and (E) CD107a on NKL 36

ACCEPTED MANUSCRIPT cells after transfected with control miRNA, miR-30c mimics or miR-30c inhibitor at 24 hours and 48 hours, respectively. Data are present in means ± on. Three

**

P < 0.01, significant differences between groups

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statistical evaluation. *P < 0.05,

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independent experiments performed in triplicates. Mann-Whitney U test was used for

and control.

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SMMC-7721 cells by CFSE/PI staining.

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Fig. 4. Cytotoxicity assay for NKL cells transfected with microRNA mimics against

(A) Specific killing activity of NKL cells after transfecting miRNA mimics for 48

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hours was analyzed with CFSE/PI assay. NKL cells were incubated with target cell

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SMMC-7721 for 4 hours at the indicated effector:target (E:T) ratios. (B) Specific

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killing activity of NKL cells transfected miRNA mimics for 48 hours and preincubated with anti-NKG2D mAb was analyzed with CFSE/PI assay. Bulk NK

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cells were preincubated either with anti-NKG2D mAb (white) or with isotype-match control mAb (black). Labeled SMMC-7721 cells were then added and incubated for 4 hours at the indicated effector:target (E:T) ratios. Data are present in means±SD. Three independent experiments performed in triplicates. Mann-Whitney U test was used for statistical evaluation. *P < 0.05,

**

P < 0.01, significant differences between

(A) miR-30c mimics and control miRNA or (B) miR-30c mimics with control mAb and miR-30c mimics with anti-NKG2D mAb. mAb, monoclonal antibody.

Fig. 5. Influences of miR-30c on NKG2D and CD107a expression in human 37

ACCEPTED MANUSCRIPT peripheral blood NK cells. (A) and (B) Expression of membrane (A) NKG2D and (B) CD107a on peripheral

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blood NK cells from ten individuals (P1 to P10) after transfection with control

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miRNA, miR-30c mimics or miR-30c inhibitor were detected with flow cytometry, respectively. (C) The examples of histogram of NKG2D and CD107a expression on human peripheral blood NK cells from P1 and P4 transfected with miR-30c mimics

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(left panel) or miR-30c inhibitor (right panel) (black line) and control miRNA (Gray

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shaded histograms). Solid histograms represent background staining with control

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mAb.

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Fig. 6. MiR-30c mimics up-regulate the expression levels of FasL on NKL cells and

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human peripheral blood NK cells.

(A) The representative flow cytometry results of FasL expression on NKL cells

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transfected with microRNA mimics after 24 hours, 48 hours and 72 hours, respectively. Red line histograms represent background staining with control mAb. Green line histograms represent anti-FasL mAb with control miRNA. Blue line histograms represent anti-FasL mAb with miR-30c mimics. (B) and (C) The statistical results of the expression percentages (left panel) and mean value (right panel) of FasL on (B) NKL cells and (C) human peripheral blood NK cells (PNK) transfected with microRNA mimics after 24 hours, 48 hours and 72 hours, respectively. Data are present in means±SD. Three independent experiments performed in triplicates. Mann-Whitney U test was used for statistical evaluation. *P < 0.05, **P < 0.01, 38

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significant differences between groups and control. mAb, monoclonal antibody.

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