miR-219a-5p enhances the radiosensitivity of non-small cell lung cancer cells through targeting CD164

Lung cancer is the most frequently diagnosed malignant cancer globally and one of the leading causes of cancer-associated mortality [1]. According to the statistics, the incidence of lung cancer ranks only second to prostate cancer in all malignant tumors in men and only less than breast cancer in women [2]. Lung cancer mainly consists of small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC), and NSCLC accounts for 70–85% of the total cases of lung cancer [3]. Radiotherapy combined with surgery and chemotherapy is an effective treatment for malignant tumors, including lung cancer [4].

Radiotherapy mainly indicates ionizing radiation which can induce damage to cellular DNA and subsequently result in cell death via direct or indirect mechanisms [5]. The efficacy of radiotherapy relies on the sensitivity of tumor cells to radiation. Thus, radioresistance usually occurs when tumor cells become insensitive to radiation, leading to the recovery of damaged cells [6]. In particular, radioresistance frequently develops in NSCLC in the middle and later stages of radiotherapy [7]. Thereof, it is of importance to identify new mechanisms of radioresistance in NSCLC.

In recent years, emerging studies indicate that dysregulation of microRNAs (miRNAs) is associated with radioresistance of many types of cancers [8–11]. It is demonstrated that miRNAs could not only regulate key biochemical processes that are critical for tumorigenesis [12] but also respond to radiation and regulate DNA damage response [13]. miR-219 is down-regulated in NSCLC and could inhibit tumor cell growth and metastasis [14]. In the present study, we aimed to investigate whether the dysregulation of miR-219a-5p is involved in radioresistance of NSCLC. Using tumor cell and xenograft animal models, we explored the mechanism of miR-219a-5p-induced regulation of radiosensitivity in NSCLC.

Fresh NSCLC tissue specimens and blood samples were collected from 30 patients, who received surgical treatment at the Anyang Tumor Hospital between April 2016 and June 2017. None of the patients had received prior neoadjuvant treatment. The patients at stage II or III underwent curative resection, followed by radiotherapy (total cumulative dose, 60 Gy with photon beams of cobalt 60 and weekly dose was 10 Gy). The clinicopathological features are shown in Table 1. Radiotherapy sensitivity was mainly assessed according to the overall survival rate and local or distant recurrence rate. Cancer growth in the same lung or in the mediastinum was defined as local recurrence. Distant recurrence was defined as a new peripheral contralateral lesion. Based on these criteria, the patients were divided into radiotherapy-sensitive (12, 40%) and -resistant (18, 60%) groups. Thirteen non-tumor lung tissues and blood samples were collected from non-tumor patient with thoracic trauma. Tissue samples were immediately frozen in liquid nitrogen and stored at −80°C. Serum samples were extracted from whole blood through centrifugation at 2800×g for 10 min and stored at −80°C. Written informed consent was obtained from all patients. The protocol was approved by the Ethics Committee of Anyang Tumor Hospital.

NSCLC cell lines, A549, H23, H358, H520, H460, H841 and H1299, were cultured in RPMI 1640 (Life Technologies, Carlsbad, CA, U.S.A.) supplemented with heat-inactivated 10% FBS (Life Technologies, Carlsbad, CA, U.S.A.), 1% Penicillin/Streptomycin (P/S, Life Technologies, Carlsbad, CA, U.S.A.), and cultured at 5% CO₂ and 37°C.

For the induction of irradiation, a Gammacell® 40 Exactor (Atomic Energy of Canada Limited, Chalk River, ON, Canada) was employed. ¹³⁷Cs γ-ray irradiation was delivered at a dose rate of 1 Gy/min. The cells were exposed to 0–10 Gy irradiation for the observational experiments. Then, 5 Gy irradiation was used for the subsequent experiments. A549 and H358 cells were exposed to sequentially increasing doses of irradiation to generate radioresistant NSCLC cell lines. The established radioresistant cell lines were defined as A549-(radioresistant) RR and H358-RR cells. The parental radiosensitive cell lines were defined as A549-(radiosensitive) RS and H358-RS cells.

To evaluate the function of miR-219-5p, cells were transfected with miR-219-5p inhibitor, miR-219-5p mimics or miR-NC (negative control) (Thermo Fisher Scientific, Inc., U.S.A.). To evaluate the function of CD164, cells were transfected with pcDNA3.1 (Invitrogen; Thermo Fisher Scientific, Inc., U.S.A.)-CD164, shCD164 or NC (Santa Cruz, CA, U.S.A.). The transfection was performed using Lipofectamine® 2000 and Opti-MEM I reduced serum medium (Invitrogen; Thermo Fisher Scientific, Inc., U.S.A.), as per the manufacturer’s instructions. Forty-eight hours post transfection, cells were used for further examination.

HEK293 cells were co-transfected with 100 nM miR-219-5p inhibitor, miR-219-5p mimics or miR-NC, 250 ng pGL3 reporter vector carrying CD164-3′UTR with WT or mutant miR-219-5p binding site, 25 ng of the phRL-SV40 control vector (Promega, Madison, WI, U.S.A.) in 24-well plates. Twenty-four hours post-transfection, firefly luciferase activity was measured using a Dual Luciferase Assay Kit (Promega, Madison, WI, U.S.A.). The normalization to Renilla luciferase reference plasmid was performed to evaluate the relative reporter gene activity.

At the end of each experiment, the culture medium was replaced with fresh serum-free RPMI 1640 medium. Ten microliters of CCK8 solution was added into a well of 96-well plates. The plates were placed in the incubator for 1–2 h. Finally, the absorbance at 460 nm was measured by the use of an ELISA reader. The results were shown as fold-change of control.

Apoptosis was measured using a TUNEL staining assay kit (Roche, Basel, Switzerland) according to the manufacturer’s protocols. The percentage of apoptotic cells were analyzed by flow cytometry (BD, C6, U.S.A.). Relative apoptosis was expressed as percentage of control. In transplanted tumor tissues, mRNA and protein levels of Bax and Bcl-2 were determined to evaluate apoptosis.

Isolation of total RNA from cells and tissues was performed using TRIzol reagent (Invitrogen, U.S.A.) according to the instructions. cDNA was synthesized using a Revert-Aid First Strand cDNA Synthesis Kit (Thermo Scientific, U.S.A.) according to the manufacturer’s protocols. Real-time qPCR was conducted on a Bio-Rad CFX96 Detection System (Bio-Rad, U.S.A.) using the SYBR Green Master kit (Takara, China). For the measurement of miR-219a-5p level, total RNA was polyadenylated by poly (A) polymerase (Ambion, Austin, TX, U.S.A.) according to the manufacturer’s instructions. Reverse transcription was performed using an ImPro-II Reverse Transcriptase (Promega, Madison, WI, U.S.A.), according to the manufacturer’s instructions. β-actin and U6 were used as internal controls. 2^−ΔΔC_t method was used to evaluate the relative level of mRNA.

Total protein was extracted from cell lines and tumor tissues using RIPA lysis buffer supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 mM Na₃VO₄). After the quantification of protein by BCA method, the samples were separated by SDS/PAGE and transferred on to a PVDF membrane. The membranes were blocked with 5% BSA for 1 h at room temperature. Then the membranes were incubated with specific primary antibodies (γH2A histone family member X (γH2AX); Santa Cruz Biotechnology, 1:500), CD164 (Thermo Fisher Scientific, 1:1000), β-actin (Sigma–Aldrich, 1:1000)) overnight at 4°C. After washing with TBST, the membranes were incubated with HRP–conjugated secondary antibodies at 37°C for 1 h. Protein bands were visualized using enhanced chemiluminescence reagent (Thermo Fisher Scientific, U.S.A.).

All animal experiments were approved by Institutional Animal Care and Use Committee of Anyang Tumor Hospital. Twenty-four male BALB/c nude mice (4–6 weeks) were obtained from Charles River Laboratories (Beijing, China) and housed in a certified, specific pathogen-free level animal facility. The animal experiment was performed in Anyang Tumor Hospital. A total of 4 × 10⁶ A549 radioresistant cells transfected with miR-219a-5p and pcDNA-CD164 were injected into the mouse mammary fat pads to develop xenograft tumors. The irradiation treatment was performed as previously reported [15]. After the experiment, animals were anesthetized using 2% isoflurane and killed by cervical dislocation. Tissues were then collected for further determination.

Data were expressed as mean ± S.D. Each in vitro experiment was repeated at least three times. Statistical analysis was performed using GraphPad Prism 6.0 software. Differences were analyzed using a one-way analysis-of-variance (ANOVA) test followed by Tukey’s post hoc test. P<0.05 was considered to be statistically significant.

To investigate the potential role of miR-219a-5p in the regulation of radiosensitivity in NSCLC, we compared the expression of miR-219a-5p between NSCLC and non-tumor specimens and compared the expression of miR-219a-5p between radiosensitive and radioresistant tissues. The results showed that miR-219a-5p expression in serum and lung tissue of tumor patients was significantly lower than that in matched control samples (Figure 1A,C). In addition, miR-219a-5p expression in serum and lung tissue of radioresistant patients was significantly lower than that in radiosensitive samples (Figure 1B,D). We also determined the cell viabilities and expression levels of miR-219a-5p in a series of NSCLC cell lines after 5 Gy of γ-ray irradiation. We revealed that the expression level of miR-219a-5p was negatively correlated with the viability of NSCLC cells treated by irradiation (Figure 1E,F). The results indicated that dysregulation of miR-219a-5p expression may be associated with radioresistance in NSCLC.

Radioresistant NSCLC cell lines were generated using gradually increasing doses of irradiation in A549 and H358 cells. The radioresistant cell lines were defined as A549-RR and H358-RR and the parental radiosensitive cell lines were defined as A549-RS and H358-RS. The radiosensitive and radioresistant A549 and H358 cells were exposed to 0, 2, 4, 6, 8 and 10 Gy, at a dose rate of 2 Gy/min. The results confirmed the establishment of radioresistant A549 and H358 cells (Figure 2A,B). In Figure 2C, we showed that miR-219a-5p expression was decreased in radioresistant A549-RR and H358-RR cells, compared with the parental radiosensitive cells. To evaluate the role of miR-219a-5p in the development of radioresistance in A549 and H358 cells, the radioresistant in A549-RR and H358-RR cells were transfected with miR-219a-5p mimics (Supplementary Figure S1). As shown in Figure 2D,E, transfection of miR-219a-5p significantly decreased cell viability under basal condition and reduced cell viability in response to different doses of irradiation. Up-regulation of miR-219a-5p increased irradiation-induced cytotoxicity by three- to five-folds (Figure 2F,G). In addition, radiosensitive A549-RS and H358-RS cells were transfected with anti-miR-219a-5p (Supplementary Figure S2). The results showed that inhibition of miR-219a-5p in A549-RS and H358-RS cells significantly increased basal cell viability and increased cell viability after different doses of irradiation treatment. The results indicated that miR-219a-5p could regulate radiosensitivity in NSCLC cells.

To investigate the mechanism of miR-219a-5p-induced regulation of radiosensitivity of NSCLC cells, we performed informatics analysis. We found that there was a putative binding site of miR-219a-5p in the 3′UTR of CD164 (Figure 3A). In addition, CD164 expression in lung tissue of tumor patients was significantly higher than that in matched control samples (Figure 3B). CD164 expression in lung tissues of radioresistant patients was significantly lower than that in radiosensitive samples (Figure 3C). Transfection of miR-219a-5p in radioresistant A549-RR and H358-RR cells induced a significant decrease in CD164 mRNA (Figure 3D) and protein (Figure 3E) expression. Luciferase reporter assay showed that in radioresistant A549-RR and H358-RR cells, miR-219a-5p could decrease CD164-WT luciferase activity but not CD164-MUT luciferase activity (Figure 3F,G). The results indicated that miR-219a-5p directly regulated CD164 mRNA level. To investigate the possible role of CD164 in miR-219a-5p-induced regulation of radiosensitivity in A549 and H358 cells, A549-RR and H358-RR cells were co-transfected with miR-219a-5p and pcDNA-CD164 (Supplementary Figure S3). We showed that miR-219a-5p-induced decrease in cell viability in irradiation-treated cells was inhibited by pcDNA-CD164 (Figure 3H,I). In addition, overexpression of CD164 inhibited irradiation-induced decrease in cell viability in A549-RR and H358-RR cells (Figure 3H,I). These results suggested that miR-219a-5p enhanced radiosensitivity through inhibition of CD164 via directly targeting its 3′UTR.

Transfection of anti-miR-219a-5p in radiosensitive A549-RS and H358-RS cells induced a significant increase in CD164 mRNA (Figure 4A) and protein (Figure 4B) levels. Luciferase reporter assay showed that in radiosensitive A549-RS and H358-RS cells, anti-miR-219a-5p could increase CD164-WT luciferase activity but not CD164-MUT luciferase activity (Figure 4C,D). Radiosensitive A549-RS and H358-RS cells were also co-transfected with anti-miR-219a-5p and shCD164 (Supplementary Figure S4). We showed that anti-miR-219a-5p-induced increase in cell viability in irradiation-treated cells was inhibited by shCD164 (Figure 4E,F). In addition, down-regulation of CD164 promoted irradiation-induced decrease in cell viability (Figure 4E,F). These results suggested that inhibition of miR-219a-5p promoted radioresistance in NSCLC cells through regulation of CD164.

To evaluate the role of miR-219a-5p in the regulation of radiosensitivity of NSCLC in vivo, we established xenograft tumor mice using transfected radioresistant A549-RR cells. As shown in Figure 5A,B, irradiation decreased the tumor size and weight in a degree. In the miR-219a-5p transfection group, the tumor size and weight significantly decreased after irradiation, compared with mice treated by irradiation alone. In addition, transfection of pcDNA-CD164 significantly inhibited the effect of miR-219a-5p on tumor growth. In Figure 5C,D, we showed that irradiation resulted in a decrease in miR-219a-5p expression and increase in CD164 mRNA and protein levels. miR-219a-5p significantly increased the mRNA and protein levels of CD164 in tumors. These results indicated that miR-219a-5p increased the radiosensitivity of NSCLC cells through targeting CD164 in vivo.

To explore the mechanism of miR-219a-5p/CD164-induced regulation of radiosensitivity in NSCLC cells, we determined the role of miR-219a-5p and CD164 in the regulation of apoptosis in radioresistant A549-RR and H358-RR cells. As shown in Figure 6A,B, miR-219a-5p transfection significantly increased irradiation-induced apoptosis in radioresistant A549-RR and H358-RR cells. Up-regulation of CD164 repressed miR-219a-5p-induced effect on apoptosis (Figure 6A,B). Overexpression of CD164 could also decrease apoptosis induced by irradiation in radioresistant A549-RR and H358-RR cells (Figure 6A,B). In xenograft tumor tissues, irradiation-induced increase in Bax expression and decrease in Bcl-2 expression were enhanced by miR-219a-5p (Figure 6C–E). This effect of miR-219a-5p was inhibited by CD164 (Figure 6C–E). Moreover, we also determined the effect of up-regulation of miR-219a-5p and CD164 on the expression of γ-H2A histone family member X (γ-H2AX). We found that irradiation had little effect on γ-H2AX expression in both cells and tumor tissues (Figure 6F–I). In response to irradiation, γ-H2AX expression could be significantly increased by either up-regulation of miR-219a-5p or down-regulation of CD164 (Figure 6F–I). Furthermore, miR-219a-5p-induced increase in γ-H2AX expression was significantly inhibited by up-regulation of CD164 (Figure 6F–I). These results demonstrated that apoptosis and DNA damage were involved in miR-219a-5p/CD164-induced regulation of radiosensitivity in NSCLC cells.

Radioresistance is a major obstacle in the treatment of NSCLC [16]. Emerging evidence supports the notion that dysregulation of miRNAs plays an important role in the occurrence of radioresistance in NSCLC [17]. However, the mechanism of miRNAs dysregulation-induced radioresistance in NSCLC is far from being completely understood. In recent years, abnormal expression of miR-219 is associated with the development of various types of tumors. It has been shown that miR-219 plays a tumor suppressive role in glioma [18], malignant melanoma [19], esophageal squamous cell carcinoma [20], colorectal cancer [14] and epithelial ovarian cancer [21]. In particular, miR-219 level is down-regulated in NSCLC and inhibits cell growth and metastasis [22]. However, there is no literature reporting the possible role of miR-219 in radioresistance of NSCLC.

In the present study, we verified the expression pattern of miR-219a-5p in patients and identified that miR-219a-5p levels in serum and lung tissues in tumor patients were increased, compared with matched control samples. Moreover, miR-219a-5p levels in serum and lung tissues in radioresistant patients were higher than that in radiosensitive patients. We also used cells and xenograft tumor mice to verify the role of miR-219a-5p. We revealed that up-regulation of miR-219a-5p significantly increased the radiosensitivity in radioresistant NSCLC cells in vitro and in vivo. Down-regulation of miR-219a-5p notably decreased the radiosensitivity in radiosensitive NSCLC cells. The results suggest that, in addition to previously reported tumor-suppressive role, miR-219a-5p also plays a role in promoting radiosensitivity in NSCLC.

A previous study has shown that miR-219 inhibits the proliferation, migration and invasion of medulloblastoma cells by targeting CD164 [23]. It is also shown that inhibition of CD164 expression in colon cancer cell line HCT116 reduces cancer cell proliferation, mobility, and metastasis in vitro and in vivo [24]. CD164 could promote tumor progression and predict the poor prognosis of bladder cancer [25]. CD164 has also been found to promote lung tumor-initiating cells with stem cell activity and determine tumor growth and drug resistance via Akt/mTOR signaling [26]. These results indicate that CD164 plays a tumor-promoting role. In the present study, we found that CD164 expression was higher in radioresistant patients, compared with that in radiosensitive patients. Overexpression of CD164 significantly inhibited miR-219a-5p-induced increase in radiosensitivity in NSCLC cells in vitro and in vivo. miR-219a-5p could also directly regulate the mRNA 3′UTR of CD164. The results suggested that miR-219a-5p could regulate radiosensitivity in NSCLC via directly regulating CD164 expression.

Apoptosis is an important mechanism of irradiation-induced cytotoxicity [27]. In the current study, we also examined the role of miR-219a-5p and CD164 in the regulation of apoptosis in the context of irradiation. Our results showed that miR-219a-5p significantly enhanced irradiation-induced apoptosis in vitro and in vivo. CD164 played an inhibitory effect on apoptosis in the context of irradiation. Overexpression of CD164 could inhibit miR-219a-5p-induced enhancement of apoptosis in response to irradiation. Moreover, we evaluated the role of miR-219a-5p and CD164 in the regulation of DNA damage in irradiation-treated NSCLC cells. Our results showed that the DNA damage marker γ-H2AX was increased by up-regulation of miR-219a-5p and down-regulation of CD164. Up-regulation of CD164 could inhibit miR-219a-5p-induced up-regulation of γ-H2AX in irradiation-treated cells. The results demonstrated that apoptosis and DNA damage may be important mechanisms underlying miR-219a-5p/CD164-induced regulation of radiosensitivity in NSCLC cells.

In summary, our results revealed that miR-219a-5p enhanced radiosensitivity in NSCLC cells in vitro and in vivo. miR-219a-5p could inhibit CD164, promote DNA damage and apoptosis and enhance irradiation-induced cytotoxicity (Figure 7). Our data highlight the importance of miR-219a-5p/CD164 pathway in the regulation of radiosensitivity in NSCLC and provide novel targets for potential intervention.

The data will be available on reasonable request.

The authors declare that there are no competing interests associated with the manuscript.

This work was supported by the Anyang Tumor Hospital, The Fourth Affiliated Hospital of Henan University of Science and Technology.

Conceived and designed the study: T.W. and L.J.F. Performed the study: T.W., S.C., X.N.F. and L.J.F. Analyzed the results: T.W., S.C., X.N.F. and L.J.F. Contributed reagents/materials/analysis tools: T.W. and L.J.F. Wrote the manuscript: T.W., S.C., X.N.F. and L.J.F. All authors reviewed and agreed to the publication of the manuscript.

Bax

BCL2-associated X

Bcl-2

B-cell lymphoma-2

CCK-8

cell counting kit-8

HRP

horseradish peroxidase

miRNA

microRNA

NC

negative control

NSCLC

non-small cell lung carcinoma

qPCR

quantitative ploymerase chain reaction

RIPA

radio immunoprecipitation assay

TUNEL

terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling

γ-H2AX

γ-H2A histone family member X