Decreased serum exosomal miR‐29a expression and its clinical significance in papillary thyroid carcinoma

Abstract Background Aberrant levels of circulating microRNAs (miRNAs) are potential biomarkers in papillary thyroid carcinoma (PTC) diagnosis and therapy. The aim of this study was to evaluate serum exosomal miR‐29a expression as a non‐invasive biomarker for PTC diagnosis and prognosis. Methods Quantitative reverse transcription polymerase chain reaction was applied to measure serum exosomal miR‐29a expression levels in blood samples of 119 patients with PTC and 100 control subjects. Results Serum exosomal miR‐29a expression levels were significantly decreased in PTC cases. In addition, receiver operating characteristic (ROC) analysis revealed serum exosomal miR‐29a could well differentiate PTC from normal controls. Moreover, serum exosomal miR‐29a levels increased progressively and significantly 30 days and 90 days after surgery. Furthermore, PTC patients with lower serum exosomal miR‐29a expression had higher risk of recurrence. Decreased serum exosomal miR‐29a expression was significantly associated with worse clinical variables including tumor size, extrathyroidal extension, and TNM stage, as well as shorter survival. Finally, both univariate and multivariate identified serum exosomal miR‐29a as an independent prognostic indicator for overall survival. Conclusion These results demonstrated that serum exosomal miR‐29a might serve as a potential biomarker for PTC diagnosis and prognosis.

including cell proliferation, metabolism, differentiation, and apoptosis. 7 miRNAs can function as either oncogenes or tumor suppressor genes, depending on their specific gene targets. 8 For instance, the expression of miR-98-5p was reduced in PTC. Knockdown of miR-98-5p promoted the malignant activities of PTC cells, and vice versa.
These findings indicate that miR-98-5p plays a tumor suppressive role in PTC. 9 Exosomes are small membrane-enclosed extracellular vesicles of approximately 30-100 nm and widely distributed in the blood, urine, and other bodily fluids. 10,11 Increasing evidence has revealed that most of serum-circulating miRNAs are enriched in exosomes and can be stably detected. 12 Therefore, serum exosomal miRNAs may be useful biomarkers for cancer diagnosis and prognosis prediction.
In terms of PTC, miR-29a has been reported to play a role in the invasion and metastasis of this malignancy. Wang and colleagues showed that miR-29a was dramatically reduced in PTC tissues and cells. Restoration of miR-29a significantly attenuated cancer cell proliferation, invasion, migration in vitro, and tumor growth in vivo by regulating DPP4. 13 Likewise, a reduction in miR-29a expression was observed in PTC tissues and its downregulation was strongly associated with worse clinical features. miR-29a upregulation or AKT3 inhibition greatly restrained carcinogenesis and metastasis of PTC in vitro and in vivo. 14 However, the clinical significance of serum exosomal miR-29a as a potential biomarker for PTC has not yet explored. In this study, we investigated whether serum exosomal miR-29a expression was associated with the clinical outcome of patients with PTC.

| Patients and serum
The study was approved by the Ethics Committee of Qiqihar Medical University. All examinations were carried out after obtaining written informed consent from patients and healthy volunteers. In this study, a total of 119 patients with PTC, comprising 57 men and 62 women, who underwent surgical resection in our hospital were enrolled.

| Exosomal RNA isolation and quantitative reverse transcription PCR
For exosomal RNA extraction, total RNA was extracted from serum samples and culture media using a Qiagen miRNeasy Mini kit (Qiagen).
The quality of total RNAs was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies). The reverse transcription reaction was performed using the TaqMan MicroRNA Reverse Transcription Kit were performed in triplicate. The relative serum exosomal miR-29a expression levels were calculated using the 2 −ΔΔCt method, and spiked-in Cel-miR-39 was used as a normalizer for serum samples.

| Western Blot
The proteins were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Santa Cruz Biotechnology). After blocking with 5% skimmed milk for 1 hour at room temperature, membranes were incubated with anti-TSG101 (Santa Cruz Biotechnology) and anti-CD63 (Abcam) overnight at 4°C. Then, the membranes were incubated with secondary antibodies for 1 hour at room temperature.
The signal was visualized using a Western Blotting Luminol Reagent (Santa Cruz Biotechnology).

| Serum exosomal miR-29a expression in PTC patients and its diagnostic value
The Western blot results demonstrated that the exosomes we isolated from the serum samples were positive for the exosomal biomarkers TSG101 and CD63 ( Figure 1A). Then, the expression levels of serum exosomal miR-29a were detected in 119 patients with PTC and 100 healthy controls using qRT-PCR. As shown in Figure 1B, serum exosomal miR-29a levels in PTC subjects were markedly downregulated compared with those in healthy controls (P < .001).

| Increased serum exosomal miR-29a levels in PTC patients after surgery
Next, we detected serum exosomal miR-29a levels in patients with PTC at 30 days, 90 days following surgery. Serum exosomal miR-29a levels in blood samples collected 30 days after surgery were significantly higher than those in blood samples collected before surgery (P < .001, Figure 3A), and serum exosomal miR-29a levels in blood samples collected 90 days after surgery were significantly elevated compared with those collected 30 days after surgery (P < .001 Figure 3A). Thus, serum exosomal miR-29a expression levels were gradually and significantly increased at 30-and 90-days following surgery and could be useful for monitoring treatment response and postoperative progression.
It is important that 5 of 54 (9.2%) PTC subjects in high serum exosomal miR-29a expression group were diagnosed with recurrence after surgery, and 25 of 65 (38.5%) PTC cases in low serum exosomal miR-29a group were confirmed to have recurrence after surgery, suggesting that PTC patients with lower serum exosomal miR-29a levels had a higher risk of recurrence ( Figure 3B). The AUC value of serum exosomal miR-29a for discriminating recurrence cases from non-recurrence cases was 0.753. The sensitivity and specificity were 77.3% and 66.2%, respectively ( Figure 3C).

| Serum exosomal miR-29a expression and clinicopathological features
The association between serum exosomal miR-29a expression and clinicopathological parameters was then assessed. Patients with F I G U R E 2 A, receiver operating characteristic (ROC) curve to assess the diagnostic accuracy of serum exosomal miR-29a level in all papillary thyroid carcinoma (PTC) patients compared with controls. B, ROC curve to assess the diagnostic accuracy of serum exosomal miR-29a level in I/II stage PTC patients compared with III/IV stage PTC patients PTC expressing serum exosomal miR-29a at levels above the median level were assigned to the high expression group (n = 54), and those cases with expression less than the median value were assigned to the low expression group (n = 65). As shown in Table 1, serum exosomal miR-29a downregulation was strongly correlated with tumor size (P = .0299), extrathyroidal extension (P = .0041), and TNM stage (P = .0009). Whereas, no significant association was found between serum exosomal miR-29a expression and age (P = .7693), gender (P = .7497), tumor location (P = .1036), and lymph node metastasis (P = .0624).

| Decreased serum exosomal miR-29a expression predicted poor prognosis of PTC patients
To analyze the correlation of serum exosomal miR-29a with prognosis of patients with PTC, the Kaplan-Meier method was used to plot OS and RFS curves. As shown in Figure 4A, the OS rate was sig-  Table 2).

| DISCUSS ION
Screening for the early detection of PTC was crucial to improve patient survival and facilitate cancer prevention. In this study, serum exosomal miR-29a levels in PTC cases were significantly decreased compared with those in controls. In addition, serum exosomal miR-29a was a promising biomarker for screening patients PTC from normal controls and early-stage PTC from advanced-stage PTC.
Moreover, serum exosomal miR-29a levels in PTC cases at 90 days F I G U R E 3 A, Serum exosomal miR-29a expression levels were progressively and significantly increased at 30-and 90-d following surgery. B, papillary thyroid carcinoma (PTC) patients with lower serum exosomal miR-29a levels were more prone to develop a recurrence. C, The AUC value of serum exosomal miR-29a for discriminating recurrence cases from non-recurrence cases was 0.753. The sensitivity and specificity were 77.3% and 66.2%, respectively following surgery were significantly higher than those in PTC cases at 30 days following surgery, or in patients with PTC before surgery.
Furthermore, low serum exosomal miR-29a expression was closely associated with aggressive clinical parameters, shorter OS/RFS and identified as an independent prognostic biomarker in patients with PTC. The data suggested that serum exosomal miR-29a might function as a tumor suppressor in PTC.
miR-29a had also been reported as a tumor suppressor by regulating the expression of cancer-related target genes in some malignancies. In gastric cancer (GC), miR-29a expression was markedly downregulated in GC tissues and enforced miR-29a expression significantly suppressed cell proliferation and induced cell cycle arrest through silencing p42.3 or VEGF-A expression. 15 29 which was contradictory to the findings in the lung cancer tissues. 19 One possible explanation for the different expression of miR-29a in different types of cancers or even in the same cancer type is that the concrete role of miR-29a in tumorigenesis might depend on the specific tumor microenvironment and the downstream targets in regulates.
We have revealed that serum exosomal miR-29a levels were significantly lower in patients with PTC than in healthy controls. Low serum exosomal miR-29a expression was associated with worse prognosis. Taken together, serum exosomal miR-29a was a reliable novel biomarker of PTC diagnosis and prognosis.