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. 2014 May 12;11(5):495–502. doi: 10.1038/cmi.2014.30

Ovarian tumor-associated microRNA-20a decreases natural killer cell cytotoxicity by downregulating MICA/B expression

Jingyan Xie 1,4, Mengna Liu 1,4, Yujuan Li 1, Yunzhong Nie 3,3, Qiongyu Mi 1, Shuli Zhao 1,2
PMCID: PMC4197204  PMID: 24813230

Abstract

MicroRNAs (miRNAs) are a class of small non-coding regulatory RNAs, and changes in miRNAs are involved in tumor origin and progression. Studies have shown that miR-20a is overexpressed in human ovarian cancer tissues and that this miRNA enhances long-term cellular proliferation and invasion capabilities. In this study, a positive correlation between serum miR-20a expression and ovarian cancer stage was observed. We found that miR-20a binds directly to the 3′-untranslated region of MICA/B mRNA, resulting in its degradation and reducing its protein levels on the plasma membrane. Reduction of membrane-bound MICA/B proteins, which are ligands of the natural killer group 2 member D (NKG2D) receptor found on natural killer (NK) cells, γδ+ T cells and CD8+ T cells, allows tumor cells to evade immune-mediated killing. Notably, antagonizing miR-20a action enhanced the NKG2D-mediated killing of tumor cells in both in vitro and in vivo models of tumors. Taken together, our data indicate that increased levels of miR-20a in tumor cells may indirectly suppress NK cell cytotoxicity by downregulating MICA/B expression. These data provide a potential link between metastasis capability and immune escape of tumor cells from NK cells.

Keywords: immune escape, MICA/B, miR-20a, NKG2D, ovarian cancer

Introduction

MHC class I chain-related molecules A and B (MICA and MICB, together referred to as MICA/B) are widely expressed on epithelial tumor cells and are recognized by γδ+ T cells, CD8+ T cells and natural killer (NK) cells by the NK group 2 member D (NKG2D) receptor. The NKG2D–MICA/B pathway plays a major role in immune surveillance to identify malignant cells.1,2,3 The MICA/B ligands and their activating receptor NKG2D play an important role in NK, γδ+ and CD8+ T-cell-mediated immune responses to tumors.4,5 These ligands are typically induced by tumor-associated antigen6 and the hypoxic stress commonly associated with cancer.7 However, cancer cells are able to reduce the levels of these surface ligands by inducing their downregulation and/or internalization8 and by causing them to shed their extracellular domains, thus preventing detection by immune cells.9,10 Recently, MICA/B mRNA has been detected in normal healthy human tissues11,12 and in adult and normal embryonic mice.12 This finding indicates that certain post-transcriptional mechanisms may exist to prevent the translation of these mRNAs in healthy individuals, presumably to avoid autoimmunity.

MicroRNAs (miRNAs) are a cluster of small non-coding RNAs that are approximately 22 nucleotides in size. miRNAs post-transcriptionally regulate target mRNA expression by recognizing complementary sites within the 3′-untranslated region (UTR) of the target mRNA, thus stimulating cell-autonomous degradation of the target mRNA.13,14 Increasing evidence indicates that miRNAs are frequently altered in cancers and are therefore believed to play important roles in tumor formation and development. Extensive efforts have been devoted to studying the clinical significance of miRNAs in clinical samples, such as serum, as potential diagnostic biomarkers of cancer. Previous studies have shown that miR-20a is upregulated in severe ovarian carcinomas compared with normal ovarian tissues and that miR-20a expression levels correlate with survival in patients with severe ovarian carcinomas.15,16 These data led us to question whether miR-20a contributes to the post-transcriptional regulation of MICA/B expression and the immune escape of ovarian carcinoma cells.

In this study, we found a positive correlation between the level of serum miR-20a and the stage of ovarian cancer. Next, we studied the effect of miR-20a on the regulation of MICA/B expression using the ovarian cancer cell line SKOV3. We determined that miR-20a inhibited the expression MICA/B by interacting with the 3′-UTR of MICA/B. We further showed that overexpression of miR-20a inhibited MICA/B expression and NK cytotoxicity in vivo. Taken together, the data presented in this study further our understanding of how miR-20a expression might modulate the molecular mechanisms underlying ovarian cancer immune escape.

Materials and methods

Clinical sample collection and RNA extraction

Serum from 34 ovarian cancer patients, 10 ovarian cysts and 20 age- and gender-matched healthy volunteers were obtained from the Department of Gynecology at the Affiliated Nanjing Hospital of Nanjing Medical University. The serum samples were collected prior to any therapeutic treatment. All studies involving human participants were approved by the Ethics Committee of Nanjing Medical University, and written informed consent was obtained from all donors. The blood samples were centrifuged at 1500g for 10 min at 4 °C to completely remove the remaining cells. The serum samples were collected gently and stored at −80 °C until further use.

Isolation of RNA and quantification of the miR-20a levels in serum and ascites were performed as described previously.17 Total RNA was isolated from each serum sample using Trizol LS Reagent (Invitrogen, California, USA) according to the manufacturer's instructions. Briefly, 200 µl of serum or ascites was mixed with 200 µl of diethylpyrocarbonate-treated water, 200 µL of phenol and 200 µl of chloroform. The mixture was then centrifuged at 12 000g for 15 min at 25 °C, and the upper aqueous layer was collected. Next, 40 µl of 3 M sodium acetate and 800 µl of isopropyl alcohol were added to the aqueous layer. The solution was then incubated at −20 °C for 1.5 h, after which it was centrifuged at 15 000g for 15 min at 4 °C. The RNA pellet was then washed once with 1 ml of 75% ethanol and dried at 37 °C. Finally, the RNA pellet was dissolved in 20 µl of diethylpyrocarbonate-treated H2O and stored at −80 °C until further use.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of miRNAs

The initial miRNA analysis was performed using arrays. Briefly, the sample utilized for the TaqMan Low-Density Array profiling assay (Applied Biosystems, California, USA) consisted of serum pooled from 34 ovarian cancer patients, 10 ovarian cysts or 20 healthy volunteers. The serum miRNA expression was profiled using TaqMan Human miRNA Arrays following the manufacturer's recommended protocol. The expression levels of 667 miRNAs were analyzed together with endogenous control genes and negative controls using this profiling platform, which consists of three 384-well microfluidic cards. First, 20 ng of RNA was reverse transcribed to obtain cDNA with the TaqMan miRNA reverse transcription kit using a GeneAmp PCR System 9700. For real-time PCR analysis, amplification was performed with the TaqMan miRNA multiplex RT assays according to the manufacturer's instructions. The data were analyzed using Sequence Detection Systems Relative Quantification Software. According to the geNorm pairwise variation (V) value, miR16 and miR19b were most similar to the mean of the TaqMan Low-Density Array and were therefore selected as endogenous controls. The miR-20a level in each serum sample was assessed using the One Step PrimeScript miRNA cDNA Synthesis Kit (Dalian, China). Quantitative RT-PCR was performed in duplicate, and the mean was calculated. We utilized the averages of miR16 and miR19b as internal controls for miRNA analysis of serum. The miR-20a expression level in serum was calculated using the following equation:18 ΔCTmiR20a=CTmiR20a−(CtmiR16+CtmiR19b)/2.

Cell lines and transfection

The human ovarian cancer cell lines SKOV3 and ES-2 were obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). The NK-92 cell line (human NK cell line) was provided by Professor Haiming Wei (University of Science and Technology of China) and was originally purchased from the American Type Culture Collection. The SKOV3, ES-2, and K562 cells were grown in RPMI 1640 medium (Gibco BRL, New York, USA) supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin and 100 µg/ml penicillin. NK-92 cells were grown in alpha minimum essential medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 100 U/ml recombinant human IL-2.

The cells were transfected with miR-20a and anti-miR-20a mimics (GenePharma, Shanghai, China); RNA oligos (GenePharma) were utilized as controls. Transient transfection was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. The media were replaced after 24 h of transfection. The cells were harvested in phosphate-buffered saline (PBS) at 96 h post-transfection and then subjected to RT-PCR and flow cytometry (FCM) analysis using an anti-MICA/B-PE antibody (Clone # 6D4; BD PharMingen, San Diego, USA).

DNA constructs and luciferase assay

Luciferase reporters were generated by inserting the wild-type or mutated 3′-UTR of MICA/B at the 3′ end of the open reading frame region in the pGL3 luciferase reporter vector (Promega Corp., Madison, WI, USA).19 The orientation of the inserts was confirmed by sequencing. The SKOV3 and ES-2 cells were transfected with the pGL3 control vector or the PGL3 reconstructed vector containing either the wild type or mutant 3′-UTR in combination with 40 nM miR-20a mimics and 20 ng of the pGL-Renilla luciferase vector as a transfection control. We used 40 nM RNA oligos (Qiagen, Düsseldorf, Germany) as a negative control. The Reporter Assay System Kit (Promega) was used to measure the luciferase activity 48 h after transfection according to the manufacturer's instructions.

NK cytotoxicity assay

The NK-92 cell-mediated cytotoxicity against the ovarian cancer cells modified with miR-20a was measured as described previously.20 In brief, the SKOV3 target cells transfected with miR-20a, anti-miR-20a mimics and RNA oligo controls were labeled with 5.0 µM carboxyfluorescein diacetate succinimidyl ester (CFSE) in 5% CO2 for 10 min at 37 °C. The stained cells were washed twice with phosphate-buffered saline and subsequently resuspended in complete RPMI 1640 medium. A target ratio of 20∶1 was used. The stained cells and NK-92 cells were mixed with complete medium and incubated in a 96-well plate (200 µl/well) at 37 °C with 5% CO2 for 4 h. The PI apoptosis kit was used to determine the percentage of stained (apoptotic) cells by flow cytometry.

Lung clearance assay

To evaluate the miR-20a-mediated immune effect in vivo, the in vivo lung clearance assay was used as previously described21 with some modifications. Briefly, 30 male C57B/6 mice (9–10 weeks old) were divided into three groups and were intraperitoneally injected with either an anti-mouse NKG2D monoclonal antibody (mAb) (Clone # C7; Novus, Missouri, USA) at a concentration of 300 µg per mouse or an isotype IgG control. Twenty-four hours later, HeLa cells (negative control cells that are not efficiently killed by mouse NK cells) were labeled with the fluorescent dye Vybrant DiD (Invitrogen), and the various SKOV3 cells (transfected with miR-20a, anti-miR-20a sponge or a control miRNA) were labeled with CFSE (Invitrogen). The stained SKOV3 cells were mixed with the stained HeLa cells of each population at a density of 5×106 cells in 1 ml of phosphate-buffered saline, and a 0.4-ml aliquot of the cells was implanted via the tail vein. The lungs were isolated 5 h later to prepare the single-cell suspensions by employing cell strainers, and fluorescence-activated cell sorting was performed. The mean fluorescence intensity ratio of the stained cells to the HeLa cells was then calculated. The ratio of the SKOV3 cells transfected with control miRNA to the HeLa cells in the group treated with anti-NKG2D IgG was set as 100%.

Statistical analysis

Statistical significance was assessed using one-way analysis of variance and the Bonferroni post-hoc test with SPSS 12.0 software. Survival probabilities (5-year overall survival) were analyzed using the Kaplan–Meier product limit method. The log-rank test was used to analyze the differences between the groups. P values <0.05 were considered statistically significant.

Results

Increased expression of miR-20a in the serum of ovarian cancer patients is correlated with disease progression.

Previous studies employing miRNA microarrays revealed that miR-200a, miR-141, miR-16, miR-200b, miR-200c and miR-20a levels are increased (more than twofold), whereas miR-100 levels are lower in ovarian cancer patients than in with healthy controls.15,16 Among these miRNAs, miR-20a in particular has been reported to regulate T-cell activation genes in both knock-in and knock-down T-cell models.22 To investigate the expression of miR-20a in ovarian cancer patients and to determine the correlation between miR-20a levels and disease severity, we further explored the role of miR-20a in serum specimens from patients with epithelial ovarian cancer.

Serum miR-20a expression was assessed by qRT-PCR in patients with ovarian tumors (n=34), patients with ovarian cysts (n=10) and healthy controls (n=20). As shown in Table 1, the ΔCTmiR20a was significantly lower in the patients than in the normal controls (P<0.05), whereas ΔCTmiR20a in the serum of patients with ovarian cyst disease appeared normal, thus indicating that miR-20a levels were significantly higher in ovarian cancer patients than in both normal controls and ovarian cyst disease controls. To evaluate the association between miR-20a and the clinicopathological features of ovarian cancer, we analyzed miR-20a levels in the sera of patients with ovarian cancers classified by metastasis or the International Federation of Gynecology and Obstetrics (FIGO) disease stage. The miR-20a expression level was significantly increased in the patients with metastasis and stage III–IV disease (P<0.05). There was no correlation between miR-20a expression and other tumor characteristics (Table 1). These results indicate that miR-20a may be associated with the progression of ovarian cancer.

Table 1. Characteristics of study cohort.

Characteristic   N miR20a (ΔCT)
Healthy   20 3.76±0.98
Ovarian cysts   10 3.47±1.28
Ovarian cancers   34 1.55±1.21*
  CA125 (U/ml)    
   ≤500 14 1.26±0.95*
   >500 20 1.75±0.97*
  Histology    
   Serous 26 1.49±1.03*
   Other 8 1.75±0.91*
  FIGO stage    
   I–II 15 1.92±0.72*
   III–IV 19 1.25±0.99*,#
  Histological grading    
   1, 2 14 1.82±0.89*
   3 20 1.36±1.01*
  Age (years)    
   ≤50 6 1.92±0.84*
   >50 28 1.47±1.07*
  Recurrence (in 24 months)    
   No 11 1.12±0.72*
   Yes 23 1.76±0.93*
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*

Mean P<0.05 compared with healthy groups.

#

Mean P<0.05 compared with the same characteristic group.

miR-20a expression and ovarian cancer prognosis

To determine whether miR-20a is associated with the prognosis of ovarian cancer patients, the median value (ΔCT=1.55) of the relative serum miR-20a expression of all patients was used to define the high- and low-expression groups (n=15 and 19, respectively). Patients in the high-expression group were more likely to have a shorter overall survival (P=0.048), compared with patients with low miR-20a expression (Figure 1). This suggests that miR-20a may play an important role in ovarian tumorigenesis and anti-tumor immunity.

Figure 1.

Figure 1

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Survival curves of ovarian cancer patients according to miR-20a expression status. Patients with high miR-20a expression (n=15) had significantly poorer prognoses than those with low miR-20a expression (n=19) (P=0.048; log-rank test).

miR-20a downregulates MICA/B expression

To investigate whether miR-20a was involved in immune regulation by targeting the MICA/B mRNA, we used the TargetScan algorithm,23 which identified potential binding sites for miR-20a in the 3′-UTR of the MICA/B ligands.

To experimentally confirm whether miR-20a regulates MICA/B protein expression in cancer tissue, a functional study was performed in SKOV3 and ES-2 ovarian carcinoma cells in vitro. miR-20a mimics and anti-miR-20a oligonucleotides were transfected into the cells, and miR-20a, MICA/B mRNA and MICA/B protein levels on the surface of the cells were analyzed by qRT-PCR and FCM (Figure 2).

Figure 2.

Figure 2

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miR-20a specifically downregulates MICA/B expression in SKOV3 ovarian carcinoma cells. (a) Polymerase chain reaction analysis of miR-20a levels in SKOV3 cells transfected with miR-20a (left) or an oligonucleotide inhibitor of miR-20a (right). (b) The mRNA levels of MICA/B in the transfected SKOV3 cells were analyzed by real-time PCR. The data are shown as the means±s.e.m. of three independent experiments. (c) Expression of MICA/B on the surface of transfected SKOV3 cells was detected via flow cytometry. The data are shown as the means±s.d. of three independent experiments. *P<0.05 and ***P<0.01 versus the control. PCR, polymerase chain reaction.

The results showed that MICA/B mRNA (Figure 2a and b) and protein (Figure 2c) were significantly reduced in the miR-20a-transfected cells in vitro. Moreover, downregulation of miR-20a by transfection of the cells with anti-miR-20a (three- to four-fold) led to increased expression of MICA/B mRNA and protein. These data demonstrate that miR-20a regulates MICA/B expression in ovarian carcinoma cells.

miR-20a directly binds to the MICA/B 3′-UTR

To test whether miR-20a directly targets MICA/B, we generated four firefly luciferase reporter vectors: two containing the wild-type MICA and MICB 3′-UTRs and the other two containing the MICA and MICB 3′-UTRs with a mutated miR-20a binding site (predicted by the TargetScan algorithm; Figure 3a and b). These vectors were transfected into SKOV3 cells that were also transfected with miR-20a (40 and 80 nM) or control (non-targeting) RNA oligos.

Figure 3.

Figure 3

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miR-20a directly binds the MICA/B 3′-UTR. (a) Schematic representation of the MICA/B 3′-UTR and the predicted binding sites of miR-20a (seed position bases 207–213 in the 3′-UTR of MICA and seed position bases 50–56 in the 3′-UTR of MICB). (b) Alignment of miR-20a and the mutated 3′-UTR of MICA/B. (c) Relative luciferase activity after transfection of the indicated reporter plasmids (MICA/B 3′-UTR or the mutant MICA/B mut 3′-UTR) into SKOV3 cells expressing 40 nm hsa-miR-10b, 80 nm hsa-miR-10b or the control miRNA. Firefly luciferase activity was normalized to Renilla luciferase activity and subsequently normalized to the average activity of the control reporter. The data are shown as the means±s.d. of three independent experiments. *P<0.05 and ***P<0.01 versus the control. miRNA, microRNA; UTR, untranslated region.

A significant decrease in the luciferase activity of MICA and MICB was observed in the presence of miR-20a, whereas the mutations in the MICA and MICB 3′-UTRs abolished this effect (Figure 3c). This result indicates that miR-20a directly targets MICA/B at the predicted binding site.

Downregulation of MICA/B by miR-20a reduces NKG2D-mediated killing

Next, we investigated whether miR-20a-mediated downregulation of MICA/B affects biological functions in vitro. SKOV3 target cells were transfected with miR-20a, anti-miR-20a mimics or control RNA oligos. Next, we pre-labeled the transfected SKOV3 cells with CFSE and evaluated the role of miR-20a in NK cytotoxicity assays, which were performed by coculturing SKOV3 cells with NK cells. As shown in Figure 4, transfection with miR-20a resulted in a moderate decrease in the cytolytic activity of the NK92 cells at various effector-to-target ratios. In addition, anti-miR-20a reversed these inhibitory effects. Taken together, these results indicate that the miR-20a-mediated decrease in MICA/B expression reduced killing by NK cells.

Figure 4.

Figure 4

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The downregulation of MICA/B by miR-20a results in reduced NKG2D-mediated killing. (a) A dual parameter dot plot shows living SKOV3 target cells (top left quadrant, Q1) labeled with CFSE versus dead target cells (top right quadrant, Q2) labeled with CFSE and PI. The cells were transfected with miR-Ctrl, miR-20a, anti-miR-Ctrl or anti-miR-20a. A plot that is representative of three independent experiments is shown. (b) The right histogram represents the statistical data shown in the left plot (Q2/(Q2+Q1)×100%). The experiment was performed with an effector-to-target cell ratio of 20∶1 as described in the section on ‘Materials and methods'. The data are reported as the mean±s.d. of three independent experiments. *P<0.05 and ***P<0.01 versus control. CFSE, carboxyfluorescein diacetate succinimidyl ester; NKG2D, natural killer group 2 member D; PI, propidium iodide.

miR-20a mediated immune evasion in vivo

Despite the fact that human MICA/B are different from those of the mouse, immunocompetent mice can eliminate human malignant cells via the NKG2D–MICA/B pathway.21 Mice treated with a blocking anti-NKG2D mAb (to deplete NKG2D+ immune cells) were used to investigate miR-20a-mediated immune effects in vivo. Five hours after injection with different mixtures of cells (SKVO3 cells labeled with CFSE and HeLa cells labeled with DiD), signal-cell suspensions of the lungs were prepared, and the significance of miR-20a targeting of MICA/B in vivo was examined by fluorescence-activated cell sorting. As shown in Figure 5a, the SKOV3 cells (CFSE+) and HeLa cells (Vybrant DiD+) could be successfully distinguished by FCM. The ratio of SKOV3 to HeLa cells in Figure 5b suggests that there was a significant enrichment of miR-20a-overexpressing SKOV3 cells in the lungs of the injected mice as compared with cells expressing control miRNA. The anti-miR-20a sponge resulted in significantly lower tumor cell survival in the lungs because it increased MICA/B expression. The anti-NKG2D mAb abolished the observed differences in the blockage of NKG2D recognition (Figure 5b). Thus, the miR-20a-mediated reduction of MICA/B protects tumor cells from the immunologic cytotoxicity of NK cells in vivo.

Figure 5.

Figure 5

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miR-20a-mediated immune evasion in vivo. (a) FACS analysis for the cells labeled with Vybrant DID (HeLa cells, lower right) and CFSE (SKOV3 cells transfected with miR-Ctrl, miR-20a or an inhibitor of miR-20a, upper left) in single-cell suspensions from lungs of the isotype IgG group. (b) The SKOV3 to HeLa tumor cell ratio in the lungs was calculated 5 h post injection in the isotype IgG group and the anti-NKG2D IgG group. HeLa cells are not efficiently killed by mouse NK cells and were therefore used as an internal control. The ratio reflects the survival of SKOV3 cells transfected with various constructs. The anti-NKG2D mAb abolished the observed differences. The data are reported as the mean±s.d. *P<0.05 versus the control. CFSE, carboxyfluorescein diacetate succinimidyl ester; FACS, fluorescence-activated cell sorting; mAb, monoclonal antibody; NK, natural killer; NKG2D, natural killer group 2 member D.

Discussion

The activating receptor NKG2D and its ligands play important roles in NK, γδ+ and CD8+ T cell-mediated immune responses to tumors.14,17 However, tumors have developed various mechanisms to escape immune cell–mediated elimination, thus allowing tumor relapse.24 Recent studies have found that tumor cells abnormally express a number of miRNAs, and this abnormal miRNA expression profile significantly contributes to tumor immune escape.25 Previous studies have reported that miR-20a is not only related to tumor development26,27 but also associated with invasive activity in ovarian carcinoma cells.28 In this study, we investigated the role of miR-20a and its post-transcriptional regulation of MICA/B in ovarian carcinoma.

Although previous studies have revealed that miR-20a is highly expressed in ovarian cancer, these studies mainly focused on miR-20a expression in tissues. Our study confirms that miR-20a expression is not only significantly higher in ovarian cancer patients than in normal controls but also significantly associated with metastasis and poor prognosis of ovarian cancer. miRNAs in serum, which are mostly derived from cells with a damaged plasma membrane,29 circulate in the blood in a cell-free and relatively stable form30 and are regarded as a promising new class of biomarkers.31 However, further clinical studies are warranted to fully evaluate the diagnostic potential of serum miR-20a in ovarian cancer in specific prospective studies. In this study, we report that miR-20a expression is significantly enhanced in ovarian cancer patients with CA-125 ≤500 U/ml but not in those with CA-125 >500 U/ml, which may be caused by individual variations. However, further clinical studies are warranted to fully evaluate the relationship of CA-125 and serum miR-20a in ovarian cancer.

Because miR-20a levels are correlated with disease metastasis, we hypothesized that this association might be due to the miR-20a-mediated downregulation of MICA/B mRNA. To test this possibility, SKOV3 and ES-2 cells were transfected with miR-20a to determine the effect of miR-20a expression on MICA/B levels. RT-PCR and FCM analyses indicated that miR-20a reduced the levels of MICA/B mRNA and protein in transfected cells. Next, using an in vitro model, we determined that miR-20a targets the 3′-UTR of MICA/B, resulting in diminished NKG2D recognition and subsequently reduced K562 cell killing by NK cells. Finally, we found that miR-20a-mediated downregulation of MICA/B reduces NK cell elimination in vivo by diminishing NKG2D recognition.

Given that MICA/B is not expressed in mice, it is difficult to assess miR-20a-mediated immune evasion in vivo. Recent work by the Pinchas group21 showed that mouse NK cells could significantly kill human tumor cells via the NKG2D–MICA/B pathway in vivo. Here, we showed that transfection with miR-20a resulted in the downregulation of MICA/B on the surface of tumor cells, thus enhancing the survival of tumor cells in vivo. Moreover, we showed that a competitive inhibitor of miR-20a (sponge-miR-20a) significantly cleared tumor cells in vivo. Furthermore, the survival difference observed in various tumor cells in the mouse lung could be eliminated by blocking anti-NKG2D mAb.

In conclusion, we determined that miR-20a targets MICA/B to avoid NKG2D-mediated immune attack and that the upregulation of miR-20a in ovarian cancer reduces MICA/B expression through interaction with its 3′-UTR. This desensitizes tumor cells expressing low levels of MICA/B to NK/T-cell killing. Taken together, these data indicate a new role for miR-20a in tumor development and provide novel insights into MICA/B modulation during cancer immune surveillance.

Contributions

Shuli Zhao designed the study, performed experiments, analyzed data and wrote the manuscript. Mengna Liu and Jingyan Xie performed experiments, assisted with the study design, analyzed the data and commented on the manuscript. Yujuan Li, Yunzhong Nie and Qiongyu Mi performed the experiments.

Acknowledgments

This work was supported by the Medical Science and Technology Development Foundation of Nanjing (Department of Health, grant numbers YKK12076 and QRX11243) and the National Natural Science Foundation of China (grant number 81201598). We thank Dr Hui Wang and Professor Yayi Hou for their helpful comments on the manuscript.

The authors do not report any conflict of interest regarding this work.

References

  1. Wu J, Groh V, Spies T. T cell antigen receptor engagement and specificity in the recognition of stress-inducible MHC class I-related chains by human epithelial gamma delta T cells. J Immunol. 2002;169:1236–1240. doi: 10.4049/jimmunol.169.3.1236. [DOI] [PubMed] [Google Scholar]
  2. Oppenheim DE, Roberts SJ, Clarke SL, Filler R, Lewis JM, Tigelaar RE, et al. Sustained localized expression of ligand for the activating NKG2D receptor impairs natural cytotoxicity in vivo and reduces tumor immunosurveillance. Nat Immunol. 2005;6:928–937. doi: 10.1038/ni1239. [DOI] [PubMed] [Google Scholar]
  3. Eleme K, Taner SB, Onfelt B, Collinson LM, McCann FE, Chalupny NJ, et al. Cell surface organization of stress-inducible proteins ULBP and MICA that stimulate human NK cells and T cells via NKG2D. J Exp Med. 2004;199:1005–1010. doi: 10.1084/jem.20032194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Andresen L, Hansen KA, Jensen H, Pedersen SF, Stougaard P, Hansen HR, et al. Propionic acid secreted from propionibacteria induces NKG2D ligand expression on human-activated t lymphocytes and cancer cells. J Immunol. 2009;183:897–906. doi: 10.4049/jimmunol.0803014. [DOI] [PubMed] [Google Scholar]
  5. Li K, Mandai M, Hamanishi J, Matsumura N, Suzuki A, Yagi H, et al. Clinical significance of the NKG2D ligands, MICA/B and ULBP2 in ovarian cancer: high expression of ULBP2 is an indicator of poor prognosis. Cancer Immunol Immunother. 2009;58:641–652. doi: 10.1007/s00262-008-0585-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Zhao S, Wang H, Nie Y, Mi Q, Chen X, Hou Y. Midkine upregulates MICA/B expression in human gastric cancer cells and decreases natural killer cell cytotoxicity. Cancer Immunol Immunother. 2012;61:1745–1753. doi: 10.1007/s00262-012-1235-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hedlund M, Nagaeva O, Kargl D, Baranov V, Mincheva-Nilsson L. Thermal- and oxidative stress causes enhanced release of NKG2D ligand-bearing immunosuppressive exosomes in leukemia/lymphoma T and B cells. PLoS ONE. 2011;6:e16899. doi: 10.1371/journal.pone.0016899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheng M, Chen Y, Xiao W, Sun R, Tian Z. NK cell-based immunotherapy for malignant diseases. Cell Mol Immunol. 2013;10:230–252. doi: 10.1038/cmi.2013.10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kaiser BK, Yim D, Chow IT, Gonzalez S, Dai Z, Mann HH, et al. Disulphide-isomerase-enabled shedding of tumor-associated NKG2D ligands. Nature. 2007;447:482–486. doi: 10.1038/nature05768. [DOI] [PubMed] [Google Scholar]
  10. Zhang C, Zhang J, Wei H, Tian Z. Imbalance of NKG2D and its inhibitory counterparts: how does tumor escape from innate immunity. Int Immunopharmacol. 2005;5:1099–1111. doi: 10.1016/j.intimp.2005.03.003. [DOI] [PubMed] [Google Scholar]
  11. Perera L, Shao L, Patel A, Evans K, Meresse B, Blumberg R, et al. Expression of nonclassical class I molecules by intestinal epithelial cells. Inflamm Bowel Dis. 2007;13:298–307. doi: 10.1002/ibd.20026. [DOI] [PubMed] [Google Scholar]
  12. Champsaur M, Lanier LL. Effect of NKG2D ligand expression on host immune responses. Immunol Rev. 2010;235:267–285. doi: 10.1111/j.0105-2896.2010.00893.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–297. doi: 10.1016/s0092-8674(04)00045-5. [DOI] [PubMed] [Google Scholar]
  14. Lai EC. Micro RNAs are complementary to 3′UTR sequence motifs that mediate negative post-transcriptional regulation. Nat Genet. 2002;30:363–364. doi: 10.1038/ng865. [DOI] [PubMed] [Google Scholar]
  15. Nam EJ, Yoon H, Kim SW, Kim H, Kim YT, Kim JH, et al. MicroRNA expression profiles in serous ovarian carcinoma. Clin Cancer Res. 2008;14:2690–2695. doi: 10.1158/1078-0432.CCR-07-1731. [DOI] [PubMed] [Google Scholar]
  16. Wyman SK, Parkin RK, Mitchell PS, Fritz BR, O'Briant K, Godwin AK, et al. Repertoire of microRNAs in epithelial ovarian cancer as determined by next generation sequencing of small RNA cDNA libraries. PLoS ONE. 2009;4:e5311. doi: 10.1371/journal.pone.0005311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Li C, Fang Z, Jiang T, Zhang Q, Liu C, Zhang C, et al. Serum microRNAs profile from genome-wide serves as a fingerprint for diagnosis of acute myocardial infarction and angina pectoris. BMC Med Genomics. 2013;6:16. doi: 10.1186/1755-8794-6-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Zeng X, Xiang J, Wu M, Xiong W, Tang H, Deng M, et al. Circulating miR-17, miR-20a, miR-29c, and miR-223 combined as non-invasive biomarkers in nasopharyngeal carcinoma. PLoS ONE. 2012;7:e46367. doi: 10.1371/journal.pone.0046367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Liao Y, Du X, Lönnerdal B. miR-214 regulates lactoferrin expression and pro-apoptotic function in mammary epithelial cells. J Nutr. 2010;140:1552–1556. doi: 10.3945/jn.110.124289. [DOI] [PubMed] [Google Scholar]
  20. Li J, Cui L, He W. Distinct pattern of human Vdelta1 gammadelta T cells recognizing MICA. Cell Mol Immunol. 2005;2:253–258. [PubMed] [Google Scholar]
  21. Tsukerman P, Stern-Ginossar N, Gur C, Glasner A, Nachmani D, Bauman Y, et al. MiR-10b downregulates the stress-induced cell surface molecule MICB, a critical ligand for cancer cell recognition by natural killer cells. Cancer Res. 2012;72:5463–5472. doi: 10.1158/0008-5472.CAN-11-2671. [DOI] [PubMed] [Google Scholar]
  22. Cox MB, Cairns MJ, Gandhi KS, Carroll AP, Moscovis S, Stewart GJ, et al. MicroRNAs miR-17 and miR-20a inhibit T cell activation genes and are under-expressed in MS whole blood. PloS ONE. 2010;5:e12132. doi: 10.1371/journal.pone.0012132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell. 2005;120:15–20. doi: 10.1016/j.cell.2004.12.035. [DOI] [PubMed] [Google Scholar]
  24. Schreiber RD, Old LJ, Smyth MJ. Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion. Science. 2011;331:1565–1570. doi: 10.1126/science.1203486. [DOI] [PubMed] [Google Scholar]
  25. Matejuk A, Collet G, Nadim M, Grillon C, Kieda C. MicroRNAs and tumor vasculature normalization: impact on anti-tumor immune response. Arch Immunol Ther Exp. 2013;61:285–299. doi: 10.1007/s00005-013-0231-4. [DOI] [PubMed] [Google Scholar]
  26. Li X, Zhang Z, Yu M, Li L, Du G, Xiao W, et al. Involvement of miR-20a in promoting gastric cancer progression by targeting early growth response 2 (EGR2) Int J Mol Sci. 2013;14:16226–16239. doi: 10.3390/ijms140816226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wu Q, Yang Z, Wang F, Hu S, Yang L, Shi Y, et al. MiR-19b/20a/92a regulates the self-renewal and proliferation of gastric cancer stem cells. J Cell Sci. 2013;126:4220–4229. doi: 10.1242/jcs.127944. [DOI] [PubMed] [Google Scholar]
  28. Fan X, Liu Y, Jiang J, Ma Z, Wu H, Liu T, et al. miR-20a promotes proliferation and invasion by targeting APP in human ovarian cancer cells. Acta Biochim Biophys Sin. 2010;42:318–324. doi: 10.1093/abbs/gmq026. [DOI] [PubMed] [Google Scholar]
  29. Wang K, Zhang S, Weber J, Baxter D, Galas DJ. Export of microRNAs and microRNA-protective protein by mammalian cells. Nucl Acids Res. 2010;38:7248–7259. doi: 10.1093/nar/gkq601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, et al. Circulating microRNAs as stable blood based markers for cancer detection. Proc Natl Acad Sci USA. 2008;105:10513–10518. doi: 10.1073/pnas.0804549105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Chen X, Ba Y, Ma L, Cai X, Yin Y, Wang K, et al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 2008;18:997–1006. doi: 10.1038/cr.2008.282. [DOI] [PubMed] [Google Scholar]

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