Long non-coding RNA RUNXOR accelerates MDSC-mediated immunosuppression in lung cancer

One hundred peripheral blood samples which contained lung adenocarcinoma (n = 53), squamous cell lung cancer (n = 24) and small cell lung cancer (n = 23) were collected from lung cancer patients. To separate the cells from the plasma, we centrifuged peripheral blood samples at 20 °C and 2000 rpm for 5 min. Then ACK lysing buffer was used to lyse red blood cells, and the remaining cells were used in the subsequent experiments. Paired peripheral blood samples, pre- and post- surgery, were collected from 40 lung cancer patients. Paired lung cancer tissues and adjacent tissues were collected from 9 patients with lung cancer who underwent primary surgical resection. The study was approved by the respective Ethics Committee of the Affiliated People’s Hospital of Jiangsu University. Written informed consent was obtained from all the subjects in accordance with the Declaration of Helsinki.

Collagenase II (Sigma-Aldrich, St. Louis, MO) was used to digest tumor tissue and adjacent tissue derived from patients with lung cancer that had been cut into small pieces (1–2 mm³) at 37 °C for 2 h on a rotating platform to obtain a single-cell suspension. The cells were collected and stained with human anti-HLA-DR, anti-CD33, anti-CD11b and anti-CD14 mAbs (eBioscience, San Diego, CA) for 30 min. Stained cells were collected and then analyzed via flow cytometry (FACSAria, BD Biosciences).

Density-gradient centrifugation over a Ficoll-Hypaque solution (Haoyang Biological Technology Co., Tianjin, China) was used to isolate human peripheral blood mononuclear cells (PBMCs). Then 40 ng/mL IL-1β (Peprotech, NJ) and 40 ng/mL GM-CSF (Peprotech) were used to stimulate PBMCs from healthy donors for 4 days. At day 4, the cells were collected and isolated by using human anti-CD33 beads (Miltenyi Biotec, Auburn, CA).

To confirm the percentages of CD4+ and CD8+ T cells, 50 ng/ml phorbol myristate acetate (PMA; Sigma-Aldrich, California) and 1 μg/ml ionomycin (Sigma-Aldrich) were used to stimulate PBMCs from lung cancer patients and healthy donors for 2 h and then cells were incubated in the presence of 1 μg/ml brefeldin-A (eBioscience, San Diego) for another 4 h. Post stimulation, cells were then stained with human anti-CD3 and anti-CD8 mAbs (eBioscience), fixed, permeabilized and stained with a human anti-IFN-γ mAb (eBioscience) following the instructions of the Intracellular Staining Kit (Invitrogen, Carlsbad, CA).

Total RNA was extracted from cells with TRIzol reagent (Invitrogen, California) following the manufacturer’s instructions. Random primers and a ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan) were used to synthesize cDNA. Bio-Rad SYBR Green Supermix (Bio-Rad, Hercules) was used to perform quantitative real-time PCR in triplicate. The primer sequences were as follows: human β-actin, sense 5-GAGTGTGGAGACCATCAAGGA-3, antisense 5-TGTATTGCTTTGCGTTGGAC-3; human RUNX1, sense 5-TGATGGCTGGCAATGATGAA-3, antisense 5-TGCGGTGGGTTTGTGAAGAC-3; human 18S, sense 5-CGGACAGGATTGACAGATTG-3, antisense 5- GCCAGAGTCTCGTTCGTTATC-3; human ARG1, sense 5-. CCTTTGCTGACATCCCTAAT-3, antisense 5-GATTCTTCCGTTCTTCTTGACT-3; and human RUNXOR, sense 5-CCTGTTCACGGTCCAAACTGG-3, antisense 5-CGGCAAGATCACAGTCCCTAGC-3. The expression level of each gene was expressed as the ratio to the β-actin transcript level. The data were analyzed with Bio-Rad CFX Manager software.

50 nM RUNXOR siRNA or its negative control (Ribobio Co., Guangzhou, China) was used to transfect MDSCs plated in 48-well plates according to the manufacturer’s protocol.

MDSCs are a population of cells that accumulate during the progression of various cancers. In human, the phenotype of MDSCs is CD11b + CD33 + HLA-DR-CD14-. These cells inhibit the anti-tumor immune response via different mechanisms: MDSCs produce suppressive molecules, such as Arg1, ROS or iNOS, to directly suppress the anti-tumor immune response induced by Th1/CTL cells and promote tumor progression; MDSCs can also promote the production of IL-10 to inhibit the CTL response by inducing Tregs or developing into tumor-associated macrophages (TAMs) [10, 22‐25]. To determine whether the MDSCs proportion changes during lung cancer progression, we detected the percentage of MDSCs in the peripheral blood of healthy controls and lung cancer patients. Compared with the healthy controls, the proportion of MDSCs increased in the peripheral blood of lung cancer patients (P < 0.001, Fig. 1a). We also compared the proportion of MDSCs in the peripheral blood of lung cancer patients with different histological categories. The results indicated that the proportion of MDSCs in various histological categories showed no significant difference (Fig. 1a). At the same time, the proportions of Th1 cells and CTL cells decreased in lung cancer patients (P < 0.01, Fig. 1b). In the subsequent correlation analysis, we found that the proportion of MDSCs was significant negatively correlated with the percentage of Th1 or CTL (Fig. 1c) cells in the peripheral blood of lung cancer patients. These data suggest that MDSCs are a population of cells that inhibit Th1/CTL cells and induce anti-tumor immunity.

We used real-time fluorescence quantitative PCR (qRT-PCR) to detect the expression of lncRNA RUNXOR in the peripheral blood of lung cancer patients. We found that compared with healthy controls, the RUNXOR level increased in the peripheral blood of lung cancer patients (P < 0.001, Fig. 2a). In addition, by analyzing the RUNXOR level in different types of lung cancers, we found that RUNXOR was differently expressed between squamous cell lung cancer and lung adenocarcinoma, which indicated that RUNXOR might be used to distinguish lung cancer types (P < 0.05, Fig. 2b). Interestingly, we found that RUNXOR expression was significantly downregulated in the blood of lung cancer patients who have underwent surgery (P < 0.05, Fig. 2c). Additionally, the data in Table 1 showed that the RUNXOR level was remarkably correlated with smoking history (P = 0.039), TNM stage (P < 0.0001), histological tumor type (P = 0.016) and lymph node metastasis (P = 0.0028) in lung cancer. These results show that the lncRNA RUNXOR level is closely related with lung cancer.

Previous work has revealed that RUNXOR is significantly upregulated in AML which is characterized by the proliferation of immature myeloid cells [21]. As described above, RUNXOR is associated with lung cancer. Based on these findings, we hypothesized that RUNXOR might be involved in MDSC-induced immunosuppression in lung cancer. According to the correlation analysis, RUNXOR expression was positively correlated with the proportion of MDSCs and Arg1 level (Fig. 3a), which is the main suppressive molecule of MDSCs, in the peripheral blood of lung cancer patients. Meanwhile, RUNXOR expression was negatively correlated with the percentage of Th1 and CTL (Fig. 3b) cells in the peripheral blood of lung cancer patients. To further determine whether RUNXOR was expressed by MDSCs, we isolated CD11b + CD33 + HLA-DR-CD14- MDSCs from tumor and adjacent tissues of lung cancer patients and detected the expression of RUNXOR by using qRT-PCR. Compared with cells of the same phenotype from adjacent tissue, RUNXOR expression was increased in MDSCs from tumor tissue (P < 0.05, Fig. 3c). We also used a specific siRNA to inhibit the expression of RUNXOR and then detected the Arg1 level in MDSCs from tumor tissue. The production of Arg1 by MDSCs from tumor tissue was significantly downregulated (P < 0.01, Fig. 3d). We also used PBMCs from normal donors to induce MDSCs with GM-CSF + IL-1β and detected the expression of RUNXOR in the induced CD33+ MDSCs. We found that the RUNXOR level was increased in the induced MDSCs compared with PBMCs without stimulation (P < 0.001, Fig. 3e). Arg1 expression was clearly decreased in induced CD33+ MDSCs after RUNXOR knockdown (P < 0.01, Fig. 3f). To further verify whether RUNXOR could regulate the development of MDSCs from progenitor cells in the bone marrow, we detected the proportion of induced CD33+ cells in PBMCs post transfection with siRUNXOR, and found that the percentage of MDSCs decreased after knockdown of RUNXOR (P = 0.065, Fig. 3g). These data show that the expression of RUNXOR is positively correlated with MDSC-induced immunosuppression in lung cancer.

RUNX1 is a critical molecule in the development of myeloid cells [26‐28]. In our previous study, we confirmed that miR-9 regulates the immunosuppression and maturation of MDSCs by targeting RUNX1. In addition, RUNX1 is reported to be a target gene of RUNXOR, and RUNXOR is involved in the epigenetic regulation of RUNX1. RUNXOR interacts with the promoters and enhancers of RUNX1 gene via its 3′-terminal fragment. RUNXOR also participates in the formation of an intrachromosomal loop and then interacts with the H3-K27 methylase EZH2 and RUNX1 protein, which are known to regulate the gene function of RUNX1 [12, 21]. Here, we detected the expression of RUNX1 in the peripheral blood of lung cancer patients and found that compared with healthy controls, the RUNX1 level was decreased in lung cancer patients (P < 0.001, Fig. 4a). And RUNX1 level was the highest in the peripheral blood of patients with lung adenocarcinoma (Fig. 4b). In addition, RUNX1 expression in both the MDSCs from the tumor tissue of lung cancer patients and MDSCs induced with GM-CSF and IL-1β was decreased compared with cells of the same phenotype from adjacent tissue and PBMCs, respectively (P < 0.05, Fig. 4c). Meanwhile, we also showed that RUNX1 expression was negatively correlated with the proportion of MDSCs (Fig. 4d) in the peripheral blood of lung cancer patients. These results demonstrate that RUNX1 is negatively correlated with the immunosuppression of MDSCs.

To confirm whether RUNXOR regulates the expression of RUNX1 in MDSCs, we firstly analyzed the correlation between RUNXOR and RUNX1 and found that RUNXOR expression was negatively correlated with RUNX1 expression (Fig. 5a). In addition, the expression of RUNX1 increased after surgery in the peripheral blood of lung cancer patients, while the expression of RUNXOR decreased after surgery in the peripheral blood of lung cancer patients (P < 0.001, Fig. 5b). We next used a specific siRNA to interfere with RUNXOR expression and detected the RUNX1 expression in both MDSCs from the tumor tissue of lung cancer patients and MDSCs induced from healthy donor PBMCs with GM-CSF + IL-1β. After knockdown of RUNXOR, the expression of RUNX1 was upregulated in isolated and induced MDSCs (Fig. 5c). Thus, RUNXOR knockdown can increase the expression of RUNX1 in MDSCs.