Serum exosomal miR‐378 upregulation is associated with poor prognosis in non–small‐cell lung cancer patients

Abstract Background Deregulated circulating microRNAs (miRNAs) are potential biomarkers for the early detection and prognosis prediction of non–small‐cell lung cancer (NSCLC). The aim of the present study was to investigate the expression pattern of serum exosomal miR‐378 in NSCLC and its correlation with clinical variables. Methods Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) was performed to detect serum exosomal miR‐378 levels in 103 patients with NSCLC and 60 control subjects. Results Our results showed that serum exosomal miR‐378 was significantly overexpressed in NSCLC patients, and serum exosomal miR‐378 upregulation was clearly associated with positive lymph node metastasis and advanced TNM stage. In addition, receiver operating characteristic (ROC) analysis demonstrated that combination of serum exosomal miR‐378 expression and carcinoembryonic antigen (CEA) had a high discriminating power to differentiate NSCLC subjects from controls. Moreover, serum exosomal miR‐378 levels in 73 NSCLC cases were significantly decreased after radiotherapy and could be used as an indicator of radiotherapeutic response in NSCLC. Furthermore, survival analyses revealed that patients with higher serum exosomal miR‐378 expression had poor overall survival. Multivariate analysis showed that serum exosomal miR‐378 expression was independently associated with overall survival. Conclusions Collectively, serum exosomal miR‐378 has strong potential as a promising non‐invasive biomarker for screening and monitoring NSCLC.

Exosomes are small, cell-secreted vesicles with a diameter about 30-100 nm which carry proteins, RNAs, miRNAs, or lipids. Exosomes are widespread in the blood, serum, urine, and other bodily fluids. 5,6 MicroRNAs (miRNAs) are mall non-coding RNAs which post-transcriptionally regulate protein expression by binding to the 3′-UTR region of mRNAs. MiRNAs can function as tumor suppressors or oncogenes by regulating the expression of cancer-related gene and are associated with the tumorigenesis and development of many cancers. 7,8 Previous studies have showed that most of serum miRNAs are enriched in exosomes and stably detected. 9 Thus, serum exosomal miRNAs can be used as potential biomarkers for the diagnosis and prognosis of many cancer types, including NSCLC.
MiR-378 is located on chromosome 5q32, and its role in progression of NSCLC has been explored by some previous studies. Ji and colleagues showed that miR-378 expression was markedly higher in lung cancer tissues than in non-cancerous tissues, and miR-378 underexpression inhibited cancer cell proliferation and induced cell apoptosis by targeting FOXG1, RBX1, and CRKL. 10,11 Similarly, Skrzypek et al reported enforced miR-378 expression or HMOX1 downregulation significantly promoted cancer cell proliferation and migration in vitro and in vivo, and decreased miR-378 expression exerted opposite effects. 12 However, the clinical significance of serum exosomal miR-378 as a biomarker in patients with NSCLC remains poorly known. In this study, we detected the serum exosomal miR-378 expression levels in 103 NSCLC patients and assessed its potential value for NSCLC diagnosis and prognosis.

| Patients and serum sample preparation
A total of 103 patients diagnosed with NSCLC, 32 patients with nonmalignant respiratory diseases (NMRD), and 60 healthy volunteers were recruited in our study. The NSCLC patients receiving any radiotherapy or chemotherapy prior to blood sample collection were excluded. The group comprised 62 men and 41 women, with a me- Up to 5 mL blood samples were withdrawn from all the participants and stored in tubes containing ethylenediaminetetraacetic acid (EDTA).
Moreover, paired serum samples from 73 NSCLC patients treated with radiotherapy were collected six weeks later. The samples were separated by centrifugation at 1600 g for 10 minutes. Subsequently, the supernatant sera were divided into small aliquots and stored at −80°C.

| Exosome isolation
The exosomes were isolated from the serum samples using ExoQuick™ Kit (System Biosciences, Palo Alto) according to the manufacturer's instructions. Briefly, the serum samples were centrifuged at 12 000 g for 5 minutes. Then, the supernatant was mixed with ExoQuick™ solution.
Follow by incubation at 4°C for 30 minutes, the mixture was centrifuged at 2000 g for 30 minutes, and the exosome was resuspended in nuclease-free water. The exosomes were stored at −80°C until further use.

| Extraction of RNA and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
The total RNA was isolated from serum exosomes using the Qiagen miRNeasy Kit (Qiagen). The quantity and purity of the isolated RNA were evaluated using a NanoDrop ND-1000 Spectrophotometer (NanoDrop). The cDNA was reverse-transcribed using the TaqMan™ MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Inc). qRT-PCR was carried out using

| Statistical analysis
All statistical analyses were performed by GraphPad Prism

| Upregulation of serum exosomal miR-378 in NSCLC samples
Real-time PCR was performed to measure the expression levels of serum exosomal miR-378 in different groups. As shown in Figure 1, serum exosomal miR-378 levels were significantly higher in NSCLC subjects compared to the patients with NMRD (P < .0001) and the healthy controls (P < .0001). No significant difference was found for the serum exosomal miR-378 level between healthy individuals and patients with NMRD.

| Relationship between serum exosomal miR-378 level and clinical features of NSCLC
Then, the association between serum exosomal miR-378 expression level and the clinical features of NSCLC was explored. All patients were divided into high and low expression groups based on the median expression levels of serum exosomal miR-378. According to the criterion, 56 cases and 47 cases were classified as high and low expression groups, respectively. As illustrated in Table 1

| Diagnostic implication of serum exosomal miR-378 and CEA
Receiver operating characteristic (ROC) curves were constructed to assess the diagnostic accuracy of serum exosomal miR-378 and carcinoembryonic antigen (CEA), which was widely used as a serum The data showed that combination of these two markers was more F I G U R E 1 Serum exosomal miR-378 levels were higher in NSCLC patients than in NMRD patients and healthy controls specific and sensitive in classifying NSCLC from healthy control samples than single marker alone.

| Dynamics of serum exosomal miR-378 level as a predictor of radiotherapeutic response in NSCLC
To assess whether radiotherapy affected serum exosomal miR-378 expression, the pre-and post-radiotherapy serum exosomal miR-378 levels were compared in 73 patients who received radiotherapy. We found that the expression levels of serum exosomal miR-378 were greatly reduced in post-radiotherapy samples compared to those in matched pre-radiotherapy samples (P = .0011; Figure 3A). According to the radiotherapeutic response, 55 patients were classified into responding group (complete remission or partial response), and 18 patients were classified into non-responding group (stable disease or progressive response). A comparison of serum exosomal miR-378 levels before and after radiotherapy showed that serum exosomal miR-378 levels were remarkably decreased in responding group (P = .0008; Figure 3A). However, no significant difference was found in non-responding group (P = .4126; Figure 3B).

| Relationship between serum exosomal miR-378 level and NSCLC prognosis
Follow-up data were available for all NSCLC subjects, and Kaplan-Meier survival analysis with log-rank test was used to explore the associations between serum exosomal miR-378 expression levels and OS. Patients in the high serum exosomal miR-378 expression group had significantly worse outcomes compared with patients in the low serum exosomal miR-378 expression group (P < .001; Figure 4A).
For TNM stage I/II or III/IV patients, patients in high serum exosomal miR-378 expression group had shorter OS than those in low serum exosomal miR-378 expression group (P = .042; Figure 4B and P = .022; Figure 4C).

| D ISCUSS I ON
Non-small-cell lung cancer is still one of the leading causes for cancer-related death in China. In this study, we found serum exosomal miR-378 levels were significantly higher in NSCLC cases than in the patients with NMRD as well as in healthy controls. In   27 These data suggested that miR-378 might act as either an oncomiR or tumor suppressor gene in cancer. The biological functions of miRNAs might largely depend on its downstream targets. Therefore, it is reasonable to observe the phenomenon that miR-378 plays a contradictory role in different types of cancers.
In summary, we have revealed an increase in serum exosomal miR-378 levels in NSCLC patients. The combination of serum exosomal miR-378 and CEA represented a very promising screening test for NSCLC diagnosis, and higher serum exosomal miR-378 levels were associated with unfavorable prognosis of NSCLC. Collectively, serum exosomal miR-378 might be a reliable biomarker for the diagnosis and prognosis of NSCLC.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.