The aim of the present study was to determine the expression and diagnostic value of exosomal miR-130a-3p in the serum of patients with differentiated thyroid cancer (DTC). Exosomes were isolated from the serum of patients with DTC and were identified using transmission electron microscopy. A novel exosomal miRNA, miR-130a-3p, was found to be significantly decreased in the serum of patients with DTC compared with those with benign thyroid tumors and healthy controls. Further study revealed that exosomal miR-130a-3p was correlated with the malignant characteristics of DTC, including tumor diameter, lymph node metastasis (LNM) and higher TNM stage. Receiver operating characteristic curve analysis demonstrated that the area under the curve of exosomal miR-130a-3p was better compared with that of TgAb and Tg in patients with DTC. More importantly, the combined use of exosomal miR-130a-3p, TgAb and Tg significantly enhanced the sensitivity and specificity, indicating that exosomal miR-130a-3p is a sensitive biomarker for DTC. A dual luciferase reporter assay indicated that insulin-like growth factor (IGF)-1 was a target gene of miR-130a-3p. Pearson's correlation analysis revealed a negative correlation between serum IGF-1 and serum exosomal miR-130a-3p levels. More importantly, exosomes from patients with DTC increased the expression of IGF-1 and p-PI3K/p-AKT, but these effects were abolished by siRNA targeting IGF-1 in TPC-1 cells. Taken together, the findings of the present study indicated that reduced exosomal miR-130a-3p levels were associated with the risk of DTC and may be used as a biomarker for the diagnosis of DTC.
Differentiated thyroid cancer (DTC), which accounts for 90% of all TCs, is characterized by an innocuous clinical course (
Exosomes are small membrane vesicles measuring 50–100 nm in diameter that are secreted from cells and are key regulators of intercellular communication (
The main focus of the present study was miR-130a-3p, which has been found to be differentially expressed in different tumors, including breast cancer and non-small cell lung cancer (
A total of 80 patients with thyroid diseases admitted to the Affiliated Hangzhou First People's Hospital between March 2018 and August 2019 were selected as the study subjects. The inclusion criteria were as follows: i) Age ≥18 years and ii) DTC diagnosis confirmed by clinical examination, thyroid function tests, imaging examinations, needle aspiration biopsy and surgical results. The exclusion criteria were as follows: i) History of thyroid surgery and ii) hyper- or hypothyroidism and chronic lymphocytic thyroiditis. According to the needle aspiration and surgical biopsy results, the 80 patients with thyroid diseases were divided into those with DTC and those with benign thyroid nodules. Of the patients with DTC, 29 were male and 11 were female, with a mean age of 64.87±4.13 years (range, 45–72 years). Of the patients with benign thyroid nodules, 28 were male and 12 were female, and the mean age was 65.02±5.01 years (age range, 43–74 years). In addition, 50 healthy controls were recruited at the physical center of the Affiliated Hangzhou First People's Hospital over the same time period. Blood samples (5 ml) were collected from the elbow vein and centrifuged at 1,500 × g for 15 min. Serum was separated and stored in a refrigerator at −70°C for future use. The present study was approved by the Ethics Committee of the hospital, and all patients signed an informed consent form.
The Invitrogen™ Exosome Isolation Kit (4484450; Thermo Fisher Scientific, Inc.) was used to isolate exosomes according to the manufacturer's instructions. In brief, after a 10-min treatment with Proteinase K, the exosome isolation reagent was added to the plasma and the solution was incubated for 30 min at 4°C. The precipitated exosomes were recovered by standard centrifugation at 10,000 × g for 5 min at room temperature. The pellet was then resuspended in PBS, and the exosomes were prepared for downstream analysis. The presence of isolated extracellular vesicles was validated using an HT-7700 transmission electron microscope (Hitachi High-Technologies Corporation) (scale bar, 50 nm; magnification, ×40).
To extract RNA from the exosomes, Total Exosome RNA & Protein Isolation Kit (4484450; Thermo Fisher Scientific, Inc.) was used in strict accordance with the manufacturer's instructions. In brief, the exosome pellet was resuspended in ice cold Exosome Resuspension Buffer and the sample was incubated for 5–10 min at room temperature to allow the pellet to dissolve. The sample was gently pipetted up and down. Then, 1X PBS was added to the exosome sample in an RNase-free tube and the RNA was isolated and purified. The concentration and purity of RNA samples were determined by measuring the optical density (OD)260/OD280 ratio.
RNA was isolated from exosomes using the Total Exosome RNA & Protein Isolation kit (cat. no. 4484450; Thermo Fisher Scientific, Inc.). RNA reverse transcription was performed according to the instructions of the QuantiTect Reverse Transcription Kit (Thermo Fisher Scientific, Inc.). SYBR Green Super mix (Bio-Rad Laboratories, Inc.) was used for qPCR according to the manufacturer's instructions. The PCR thermocycling conditions were as follows: 95°C for 30 sec, followed by 45 cycles of 5 sec at 95°C and 30 sec at 60°C. Relative miRNA expression was normalized to U6 expression using the 2−∆∆Cq method (
293T cells and TPC-1 cells were purchased from American Type Culture Collection and cultured in RPMI-1640 (Cytiva) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.), streptomycin (100 mg/ml) and penicillin (100 U/ml) at 37°C in a humidified atmosphere containing 5% CO2.
Total protein was isolated from TPC-1 cells using a total protein extraction kit (Beijing Solarbio Science & Technology Co., Ltd.). Protein concentration was determined using a BCA protein assay kit (cat. no. 23225, Pierce; Thermo Fisher Scientific, Inc.) was used. A total of 20 µg protein was separated using 12% SDS-PAGE (10%) and the proteins were subsequently transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% fat-free milk at room temperature for 2 h and subsequently incubated with primary antibodies against IGF-1 (1:1,000; cat. no. ab133542; Abcam), p-PI3K (1:1,000; cat. no. 17366; Cell Signaling Technology, Inc.), p-Akt (1:1,000; cat. no. 4060; Cell Signaling Technology, Inc.) and GAPDH (1:5,000; cat. no. 5174, Cell Signaling Technology, Inc.) overnight at 4°C. Following the primary incubation, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Beijing Zhongshan Golden Bridge Biotechnology Co.) for 2 h at room temperature, followed by three washes with TBST (0.1% of Tween-20). Enhanced chemiluminescence (EMD Millipore) was used to determine the protein concentrations, according to the manufacturer's protocol. Signals were detected using the SuperLumia ECL Plus HRP Substrate kit (cat. no. AMJ-KT0002, AmyJet Scientific,
The potential target gene of miR-130a-3p was predicted using the TargetScan database (
A dual-luciferase reporter assay was performed using a Dual Luciferase Reporter Assay System (Promega Corporation), according to the manufacturer's instructions. Following transfection, the medium was discarded and the cells were washed with 100 µl 1×PBS. Subsequently, 5×PLB was diluted with ddH2O to 1×PLB and left to stand at room temperature prior to use. A total of 50 µl 1×PLB was added to each well and shaken for 20 min. Subsequently, 10 µl supernatant was added to a 96 well microplate and 100 µl of premixed luciferase assay regent II was added to each well. After 2 sec, the reaction intensity of luciferase was detected. At the end of the assay, 100 µl of pre mixed stop & glo regent was added to each well, and the data were measured to detect the luciferase reaction intensity (RLU1: Firefly luciferase activity; RLU2:
Data are expressed as the mean ± standard deviation. Each experiment was carried out with three replicates. Multiple comparisons were performed using one-way analysis of variance followed by Tukey's multiple comparison test. Receiver operating characteristic (ROC) curve analysis was carried out to explore the diagnostic value of exosomal miR-130a-3p in the serum of patients with DTC. P<0.05 was considered to indicate a statistically significant difference. The data were analyzed using SPSS software, version 20.0 (IBM Corp.).
As shown in
After isolation of exosomes from the serum of patients with DTC, the level of exosomal miR-130a-3p was quantified. As shown in
The level of exosomal miR-130a-3p was then analyzed according to the clinical characteristics of patients with DTC. As shown in
Previous studies have indicated that TgAb and Tg are important biomarkers for the diagnosis of DTC (
The aforementioned findings prompted us to further explore the possible target gene of miR-130a-3p. Based on TargetScan, a conserved binding site was identified in the 3′UTR of IGF-1, a well-known oncogenic gene in TC (
It was next investigated whether exosomal miR-130a-3p targets IGF-1. As shown in
DTC is characterized by high incidence rate and complex etiology, which may be associated with a number of factors, such as diet, environment and heredity (
Exosomes are small vesicles that are released by cancer cells and transfer mRNAs, miRs and proteins from donor to recipient cells (
TgAb is an important thyroid tissue antibody and an indicator for the diagnosis of thyroid diseases (
Furthermore, the possible target gene of miR-130a-3p in the progression of DTC was investigated. To the best of our knowledge, the present study was the first to identify IGF-1 as a target gene of miR-130a-3p. The oncogenic role of IGF-1 has been widely reported in various cancers (
An increasing number of exosomal miRs have been suggested to be implicated in TC (
However, there were certain limitations to the present study. First, the sample size was relatively small, and further studies with a large sample size are required to validate the findings. Second, the diagnostic value of exosomal miR-130a-3p was not compared with that of other known exosomal miRs. In a future study, it would be interesting to compare their diagnostic value, which may help improve sensitivity and specificity. Third, the clinical and surgical implications of the present study require further consideration. Abnormal expression of miR-130a-3p has also been identified in other tumors, including breast cancer and non-small cell lung cancer (
In summary, reduced levels of exosomal miR-130a-3p were found to be associated with the risk of DTC and may be used as a biomarker for the diagnosis of DTC.
Not applicable.
The present study was supported by the Zhejiang Provincial Natural Science Foundation of China (grant no. Q17H030001) and the Zhejiang Medical and Health Research Project (grant no. 2020KY700).
The datasets used and/or analyzed in the present study are available from the corresponding author upon reasonable request.
GY performed the experiments and analyzed the data. WK, SZ, YS, JZ and RY collected the patient samples and performed RT-qPCR experiments. HW designed all the experiments, analyzed the data and gave final approval of the version of the manuscript to be published. GY and HW confirmed the authenticity of all the raw data. All the authors have read and approved the final version of the manuscript.
The present study was approved by the Affiliated Hangzhou First People's Hospital (Hangzhou, China; approval no. HZP-20170862) and all patients provided written informed consent prior to the study start.
Not applicable.
The authors declare that they have no competing interests.
Exosomes were isolated from the serum of patients DTC. (A) Transmission electron microscopy revealed that the exosomes isolated from the peripheral serum samples of patients with DTC had a diameter of ~100 nm. (B) Western blot assay identified the protein markers Alix, TSG101 and CD63 in the serum samples of patients with DTC. DTC, differentiated thyroid cancer.
Reverse transcription-quantitative PCR analysis revealed that exosomal miR-130a-3p was significantly decreased in the serum of patients with DTC compared with patients with benign thyroid tumors and healthy controls. ***P<0.001. DTC, differentiated thyroid cancer.
Receiver operating characteristics curve analysis was undertaken to explore the diagnostic efficiency of exosomal miR-130a-3p in patients with DTC. DTC, differentiated thyroid cancer.
IGF-1 is a target gene of miR-130a-3p. (A) Based on TargetScan, a conserved binding site was identified in the 3′UTR of IGF-1, a well-known oncogenic gene in thyroid cancer. (B) Dual luciferase reporter assay demonstrated that miR-130a-3p significantly suppressed the relative luciferase activity of pmirGLO-IGF-1-3′UTR. (C) The serum IGF-1 level was significantly increased in patients with DTC compared with patients wit benign thyroid tumors and healthy controls. (D) Pearson's correlation analysis indicated that serum miR-130a-3p was negatively correlated with exosomal miR-130a-3p. ***P<0.001. DTC, differentiated thyroid cancer; IGF-1, insulin-like growth factor; UTR, untranslated region.
Exosomal miR-130a-3p regulates the malignancy of DTC by targeting IGF-1. (A) Reverse transcription-quantitative PCR analysis demonstrated that an siRNA targeting IGF-1 significantly decreased the mRNA level of IGF-1. (B) Knockdown of IGF-1 decreased the phosphorylation level of AKT, while transfection with miR-130a-3p inhibitor enhanced the phosphorylation level of AKT. (C) Exosomes from patients with DTC increased the expression of IGF-1 and p-PI3K/p-AKT, but these effects were abolished by siRNA targeting IGF in TPC-1 cells. (D) Exosomes from DTC patients strongly enhanced TPC-1 cell migration, but TPC-1 cells treated with shRNA targeting IGF-1 decreased these effects. *P<0.05, **P<0.01 and ***P<0.001. DTC, differentiated thyroid cancer; IGF-1, insulin-like growth factor.
Association between exosomal miR-130a-3p expression and clinicopathological characteristics of patients with differentiated thyroid cancer.
Characteristics | Number of patients, n | Exosomal miR-130a-3p | P-value |
---|---|---|---|
Sex | 0.458 | ||
Male | 29 | 1.40±0.70 | |
Female | 11 | 1.10±0.84 | |
Age, years | 0.888 | ||
≤42 | 23 | 1.25±0.36 | |
>42 | 17 | 1.19±0.94 | |
Tumor diameter, cm | <0.001 | ||
≤2 | 18 | 1.76±0.61 | |
>2 | 22 | 0.86±0.65 | |
Capsular infiltration | 0.876 | ||
Yes | 21 | 1.24±0.42 | |
No | 19 | 1.18±0.87 | |
Lymph node metastasis | <0.001 | ||
Yes | 18 | 1.71±0.66 | |
No | 22 | 0.87±0.57 | |
TNM stage | <0.001 | ||
I/II | 23 | 1.94±0.55 | |
III/IV | 17 | 0.89±0.58 |
TNM, tumor-node-metastasis.