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

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.

Figure 1

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).

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.