Circulating exosome-derived miR-191-5p is a novel therapeutic biomarker for radiotherapy in esophageal squamous cell carcinoma patients

Background: Exosomes are nano-sized extracellular vesicles and are detectable in most body fluids. Circulating exosomal microRNAs are an easily obtained, and they could be minimally invasive biomarker for cancer treatment. Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive carcinomas. Radiotherapy is one of the most important treatment option for ESCC, and it would thus be extremely crucial to predict therapeutic sensitivity and the patient prognosis in advance. Methods: A search for miRNAs with a therapeutic biomarker in ESCC was performed using the miRNA expression signatures obtained from ESCC plasma before radiotherapy. miR-191-5p was selected because it was found to be associated with the prognosis in ESCC based on the findings of previous reports. As a result, we decided to perform more studies to elucidate the significance of miR-191-5p. Gain-of-function analyses were performed to evaluate the functional significance of miR-191-5p in ESCC progression. The effects of miR-191-5p on ESCC radiosensitivity were determined by cell proliferation, a clonogenic survival assay and an apoptosis assay. A gene set enrichment analysis was used to investigate the downstream signaling pathway related to the miR-191-5p functions. The 5-year progression-free survival (PFS) rate was used to directly compare the usefulness of these biomarkers for determining the patient prognosis between the miR-191-5p high expression patients and low expression patients. Results: A subset of seven microRNAs (miR-628, miR-363, miR-191-5p, miR-185, miR-148a, miR-320d, miR-30e) was identified to be candidates of therapeutic biomarker for ESCC patients underwent radiotherapy in a global microRNA expression analysis. A high miR-191-5p expression promoted ESCC cell proliferation, invasion and migration and induced G0/G1 to S/G2M transition. miRNA-191-5p overexpression promoted cell survival and reduced cell apoptosis after irradiation. Mechanistically, miR-191-5p may directly target death-associated protein kinase 1 (DAPK1) radiosensitivity in ESCC plasma exosomes. The usefulness of circulating exosomal miR-191-5p as a biomarker for ESCC treatment was examined. but was markedly higher in exosomes than in normal esophageal keratinocytes. These results suggest that miR-191-5p may be secreted into the circulation in greater amounts than is expressed in tumor cells, via exosomes. Furthermore, the plasma exosomes miR-191-5p expression may be a better reflection of a cancer-bearing patient’s status than the miR-191-5p expression in tumor tissue assessed by a biopsy. However, further investigations on these points are still needed.

Conclusion: Tumor-derived exosomal miR-191-5p is a potential non-invasive biomarker for predicting the prognosis in esophageal cancer patients after radiotherapy.

Background
Esophageal cancer is the seventh-most common cancer and the sixth leading cause of mortality worldwide [1]. Esophageal squamous cell carcinoma (ESCC) comprises over 90% of all esophageal malignancies in Asia. Although the treatment of esophageal cancer has progressed in recent years, its prognosis remains poor, with a 5-year survival rate of < 20% [2].
Exosomes are nano-sized extracellular vesicles (50-100 nm in diameter) and detectable in most body fluids, such as plasma, urine, saliva, and ascites [3]. The role of exosomes in cancer progression is gradually being recognized by the study of their various molecular constituents, including proteins and nucleic acids. Tumor-released exosomes have been widely reported to regulate tumor progression, invasion, metastasis, and resistance to chemotherapy [4].
MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression through posttranscriptional regulation [5]. MicroRNAs exert various function by targeting different genes. They act as oncogenes or tumor suppressor genes and also play an important role in predicting the therapeutic response. Several studies have suggested that circulating exosomal microRNAs, such as miRNA-21 [6] and miRNA-1246[7], may be useful as clinical diagnostic or prognostic biomarkers for esophageal cancer. However, the exosomal microRNAs from blood samples associated with therapeutic efficacy have been poorly investigated in ESCC.
Radiotherapy plays a critical role in both neoadjuvant therapy and as radical treatment itself for resectable and unresectable locally advanced esophageal cancer [2]. The identification of molecules involved in ESCC radiosensitivity would be of great significance for the treatment of ESCC.
Furthermore, plasma exosome tests that can be performed through blood tests would be minimally invasive by avoiding the need for a biopsy and be extremely versatile.
In the present study, we identified miRNAs related to radiosensitivity in ESCC plasma exosomes. The usefulness of circulating exosomal miR-191-5p as a biomarker for ESCC treatment was examined. Extraction of exosomes from the plasma.

Material And Methods
Each plasma sample was centrifuged at 2,000 g for 20 min at room temperature to remove cells and debris. The supernatant containing the partially clarified plasma was transferred to a new tube and then centrifuged at 10,000 g for 20 min at room temperature. The supernatant was then transferred to a new tube, and proteinase K (Proteinase K For Total Exosome Isolation from plasma; Invitrogen, Carlsbad, Calif., United States) was added to remove the endogenous nucleases. Exosomes were isolated using an exosome isolation reagent (Total Exosome precipitation reagent from plasma; Invitrogen) according to the manufacturer's protocol.
Transmission electron microscope (TEM) observation TEM observation was performed using a carbon-coated copper grid (Excel support film, 200 mesh, RL26A; NISSHIN EM Co., Ltd., Tokyo, Japan) with the negative stain method. Negative staining used a 2% phosphotungstic acid solution (pH 7-7.4). All sample grids were subjected to hydrophilic treatment with glow-discharged treatment before absorption using plasma etching device (SEDE-GE; Meiwafosis Co., Ltd., Tokyo, Japan). One dispersed droplet of the sample and two droplets of phosphotungstic acid solution were prepared on a parafilm consisting of 20 µl each, respectively. The stain protocols were the same as the sample absorption protocols, with staining performed twice. After negative staining, the grids were left to dry in a dry box overnight. The samples were then subjected to TEM observation (H-7650; Hitachi High-Technologies Corporation, Tokyo, Japan) at an acceleration voltage of 80.0 kV.
MiRNA and mRNA isolation and detection by quantitative real-time polymerase chain reaction (qRT-

PCR)
Total RNA was extracted from exosomes using the Total Exosome RNA and Protein Isolation Kit (Invitrogen) according to the manufacturer's protocol. Total cellular RNA was extracted using the miRNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol.
To measure the expression of death-associated protein kinase 1 (DAPK1), cDNA was generated using a High-Capacity RNA-to-DNA™ Kit (Thermo Fisher Scientific) according to the manufacturer's protocol.

Cell cultures and miRNA transfection
The human ESCC cell lines TE11, and KYSE-960 were used in the present study. Immortalized esophageal keratinocyte cells (R2C3) that had been established in our institute were used as a control line [12]. KYSE-960 were obtained from JCRB Cell Bank. TE11 was obtained from Cell Resource Center for Biomedical Research Institute of Development, Aging and Center, Tohoku University, Japan.
The miR-191-5p mimic and negative control mimic were purchased from Thermo Fisher Scientific. The cell lines TE11 and KYSE-960 were seeded into 6-well plates (2.5 × 10 5 cells per well), and after 24 h, cells were transfected with miR-191-5p mimic or negative control mimic using Lipofectamine™ RNAi-investigation.
Cell proliferation and colony formation assays Cells were seeded onto a 96-well plate at a density of 5000 cells per well after transfection with miR-191-5p mimic or negative control mimic in the cell proliferation assay. The cell proliferation was then measured using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) every 24 h.
For irradiation, cells were irradiated every 24 h with 0 or 5 Gy X-ray irradiation after cell adherence.
Cells were seeded onto a 6-well plate at various cell densities (4 × 10 2 -8 × 10 3 ) per well after transfection in the colony formation assay. The plates were then irradiated with a single dose of 0, 2, 4, 6 or 8 Gy X-ray irradiation (MBR-1520R-3; Hitachi) at 24 h after seeding. Following incubation for an additional 10-14 days, the cells were fixed and stained using Diff-Quick Staining (Sysmex Corp., Extraction of exosomes from the cell culture medium A total of 5 × 10 6 cells were incubated with 10% exosome-free FBS medium for 48 h. A total of 30 ml medium was harvested and centrifuged at 2000 g for 30 min to remove cells and debris. Total Exosome Isolation (from cell culture media) reagent (15 ml; Invitrogen) was then added to the samples, which were incubated at 4 ℃ overnight. After incubation, the samples were centrifuged at 10,000 g for 1 h at 4 ℃. Exosomes were then resuspended in phosphate-buffered saline (PBS) for downstream analyses.

Gene set enrichment analyses
The enrichment analysis of The Cancer Genome Atlas (TCGA) database (https://www.cbioportal.org/datasets) was performed with GSEA v4.0.1. A total of 196 RNAseq data and miRNAseq data points from esophageal cancer patients were used to evaluate the miRNA activities in cancer transcriptomes. Data were divided into miR-191-5p high-expression and lowexpression groups based on the median miR-191-5p expression. A STRING analysis (https://stringdb.org/) was used to predict the functional protein association networks of DAPK1.

Tumor xenografts
Six-week-old female BALB/c Slc-nu/nu mice had 5 × 10 6 TE11 cells injected into their back. Seven days after the injection, the tumor volume was measured by calipers, with measurements subsequently repeated every three days. The mice were randomly divided into a tumor-bearing group (n = 4) and a tumor-free group (n = 4). When the average tumor volume reached 300 mm 3 , mice were anaesthetized with isoflurane, and blood samples were collected by cardiac puncture. Exosomes were then extracted from the plasma for further research.
The present study was performed according to the guidelines on animal experiments and approved by the animal experiment and welfare committee at Chiba University.

Statistical analyses
The statistical analyses were performed with the SPSS 21 software program (SPSS, Chicago, IL, USA) and the GraphPad Prism 7.04 software program (GraphPad Software, Inc., La Jolla, CA, USA). The difference between two groups was analyzed using Student's t-test. Differences in the expression of miR-191-5p in relation to the clinical characteristics were examined using the chi-square test. The Kaplan-Meier method was used to plot survival curves, and the results were compared using the logrank test. Cox's proportional hazards regression model was used to analyze the univariate and multivariate survival. P < 0.05 was considered significant.

Proliferation And Colony Formation In Vitro
The overexpression of miR-191-5p compared with parent cells was confirmed by qRT-PCR in TE11 and KYSE-960 cell lines after miR-191-5p transfection ( Supplementary Fig. 1). As shown in Fig Fig. 2).
As shown in the Fig. 3E and 3F, the cellular apoptosis rate of miR-191-5p -transfected TE11 and KYSE-  Fig. 4A). qRT-PCR and a Western blot analysis showed that the DAPK1 mRNA and protein expression was significantly reduced after miR-191-5p transfection ( Fig. 4B and 4C).

miR-191-5p Expression Analysis Of Tumor-derive Exosome In Vivo
Exosomes were extracted from plasma of tumor-bearing and tumor-free mice. The expression of exosomal miR-191-5p was significantly higher in the tumor-bearing group than in the tumor-free group (p = 0.0014) (Fig. 5A).

Overall and progression-free survival (PFS) of the ESCC patients after RT
The clinical characteristics of the patients are shown in Table 2. Patients were divided into a highexpression group (n = 33) and low-expression group (n = 34) according to the expression of miR-191-5p. The clinical characteristics did not marked differ between the two groups. Patients who underwent RT with a low miR-191-5p expression showed a longer PFS than those with a high expression (p = 0.026, log-rank test) (Fig. 6A). The disease-specific survival rate of the low-expression group was higher than in the high-expression group, although not to a significant degree (p = 0.052, log-rank test) (Fig. 6B). The univariate and multivariate analyses of the factors associated with the PFS of RT patients indicated that the exosomal miR-191-5p expression and cStage were independent prognostic factors (Table.3). resulting in the termination of cell division and proliferation, cell necrosis, or apoptosis [12].
Radioresistance can develop through various pathways, including cancer stem cells [13], reactive oxygen species (ROS) [14], and the tumor microenvironment (TME) [15], all of which are related to the response to the irradiation. It remains the main reason for the limited efficacy of radiotherapy in esophageal cancer patients. It would thus be extremely crucial to predict therapeutic sensitivity and the ESCC patient prognosis in advance. The present study therefore explored the molecular factors of the exosome responsible for conferring radioresistance in order to develop new strategies to increase patients' sensitivity to irradiation.
MiRNAs are widely reported to be involved in radioresistance of esophageal cancer. The upregulation of miR-624 [16], miR-205 [17] and miR-21 [18] induces radioresistance in esophageal cancer, while that of miR-338 [19], miR-301 [20] and miR-381 [21] has been reported to increase the radiosensitivity in esophageal cancer. This study is the first report to identify the different expression profiles of circulating exosomal miRNAs in esophageal cancer patients who were sensitive or resistant to CRT and to identify miR-191-5p as significantly overexpressed in radioresistant patients.
Accumulating data in recent years has indicated that miR-191-5p is abnormally expressed in more than 20 different cancers and is a major player in some of these entities [22]. In hepatocellular carcinoma, for example, miR-191-5p was shown to act as a potential therapeutic target because it promoted proliferation and invasion and reduced apoptosis of cholangiocarcinoma cells through the miR-191/TET1/p53 pathway [23]. It was also reported that miR-191-5p was abnormally expressed in osteosarcoma and promoted cell proliferation, migration, and invasion through the EGR1 and PI3K/AKT pathways [24]. Furthermore, a high miR-191-5p expression has been reported to be associated with a bad prognosis in pancreatic cancer [25], AML [26], and breast cancer [27].
In the present study, miR-191-5p was confirmed to promote ESCC cell proliferation, invasion and migration and induce G0/G1 to S/G2M transition in the cell cycle. In addition, the overexpression of miR-191-5p promoted the cell survival and decreased cell apoptosis after irradiation. A clonogenic survival assay indicated that miR-191-5p decreased radiosensitivity 0.78-and 0.65-fold in TE11 and KYSE-960 cells, respectively. Therefore, these results indicate that miR-191-5p synergistically elicits radiation resistance by inhibiting radiation-induced apoptosis.
DAPK1 acts as a positive mediator of apoptosis induced by many apoptotic signals, including DNAdamaging agents, different death stimuli [28]. Previous studies have shown that DAPK1 is a tumorsuppressive gene and is suppressed in a variety cancers [29]. GSEA results showed that the MAPK pathway was upregulated in the miR-191-5p low-expression group and suppressed in the miR-191-5p high-expression group. DAPK1 has also been found to be a key factor regulating the MAPK pathway [28]. In a previous study, miR-191-5p inhibited TNF-α-induced apoptosis of ovarian endometriosis and endometrioid carcinoma by targeting DAPK1 [30]. DAPK1 acts as an upstream activator of JNK, which is the vital downstream signal of the MAPK pathway [31]. The inhibition of JNK activation, not ERK or p38, by miR-191-5p transfection has been confirmed in our study. Taken together, these previous findings provide important insight into the fact that miR-191-5p directly targets DAPK1 to regulate irradiation-induced apoptosis by decreasing the JNK activation. In the present study, we reported for the first time that DAPK1 act as a direct target of miR-191-5p and described its potential signaling pathway in ESCC. However, further studies will be required to obtain more evidence concerning the relationship between DAPK1 and the JNK signaling pathway.
The difference in the expression of miR-191-5p between tumor cells and tumor cell-derived exosomes was investigated in vitro in the present study. Interestingly, the miR-191-5p expression was slightly lower in cancer cells than in normal cells but was markedly higher in exosomes than in normal esophageal keratinocytes. These results suggest that miR-191-5p may be secreted into the circulation in greater amounts than is expressed in tumor cells, via exosomes. Furthermore, the plasma exosomes miR-191-5p expression may be a better reflection of a cancer-bearing patient's status than the miR-191-5p expression in tumor tissue assessed by a biopsy. However, further investigations on these points are still needed.
Patients who underwent RT with a low expression of miR-191-5p in circulating exosomes showed a longer PFS than those with a high expression of miR-191-5p. While there is no notable evidence that miR-191-5p affects the disease-specific survival of esophageal cancer, miR-191-5p was reported to be frequently overexpressed in ESCC tissues and significantly related to an advanced clinical stage, metastasis, and poor survival rate of ESCC [32]. Therefore, circulating exosomal miRNA-191 may act as a potential prognostic biomarker in ESCC patients after RT.

Conclusion
In conclusion, the tumor-derived exosome miR-191-5pmi expression was suggested to be a useful non-invasive biomarker for predicting the prognosis in patients with esophageal cancer after radiation therapy. In addition, the present study provides evidence that miR-191-5p directly targets DAPK1 to regulate irradiation-induced apoptosis by decreasing the JNK activation. All authors read and approved the final manuscript.

Ethics approval and consent to participate
The present study was approved by the Ethics Committee of Graduate School of Medicine, Chiba University. Written informed consent was obtained from all of the patients.   The cells were seeded in a 6-well plate at a density of 800 cells per well after transfection.
After 10 days, the cells were stained using Diff-Quick Staining. The data are presented as the means ± SEM. C: Migration and invasion were detected by transwell assays. A total of 5 ×104 transfected cells were seeded into the upper chamber with FBS-free medium, while medium with 10% FBS was used in the lower chamber. After incubation for 24 (migration assay) or 36 h (invasion assay), the cells on the lower surface were fixed and stained. The data are presented as the means ± SEM. *P < 0.05 and **P < 0.01 compared with the negative control.    Kaplan-Meier analysis of the progression-free survival after radiotherapy(A) and the diseasespecific survival(B) was performed in 67 esophageal cancer patients according to the miR-191-5p expression. Patients who underwent RT with a low miR-191-5p expression showed a longer PFS than those with a high expression (p=0.026, log-rank test).

Supplementary Files
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