Identification of serum exosomal lncRNA MIAT as a novel diagnostic and prognostic biomarker for gastric cancer

Abstract Background Accumulating evidence has demonstrated that long non‐coding RNAs (lncRNAs) MIAT is significantly upregulated in many cancer types including gastric cancer (GC). However, the potential clinical significance of serum exosomal MIAT in GC is unknown. Methods In this study, a total of 109 GC patients, 48 gastric adenoma patients, and 50 healthy individuals were recruited. Serum exosomal MIAT levels were detected in all participants using quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR). Results The exosomes we extracted from the serum samples were positive for TSG101, CD63, and Flotillin‐1, which were known exosome markers. Serum exosomal MIAT levels were significantly higher in GC patients than in gastric adenoma patients and healthy controls. Interestingly, gastric adenoma patients with higher serum exosomal MIAT expression were more prone to develop GC. In addition, serum exosomal MIAT levels were significantly decreased in post‐treatment blood samples compared to pre‐treatment samples, while markedly increased in the cases suffering recurrence. Moreover, serum exosomal MIAT upregulation was significantly associated with worse clinical variables and shorter survival. Furthermore, serum exosomal MIAT was identified as an independent prognostic factor for GC. Conclusions Collectively, serum exosomal lncRNA MIAT might serve as a promising novel biomarker for monitoring the progression of GC.

biomarkers for early diagnosis and prognosis prediction of GC is urgently required.
Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs longer than 200 nucleotides and lack protein-coding capacity. 4 LncRNAs have been found to play important roles in a variety of biological processes such as chromatin remodeling, cell differentiation, cell growth, and proliferation. 5,6 Emerging evidence has demonstrated that lncRNAs are closely involved in GC tumorigenesis and progression. For instance, Yan et al revealed that lncRNA SNHG6 was upregulated both in GC tissues and cell lines. Downregulation of SNHG6 significantly suppressed the malignant behaviors of GC cells, indicating SNHG6 played an oncogenic role in GC. 7 The expression level of lncRNA OLC8 was markedly upregulated in GC specimens and cell lines. In addition, suppression of lncRNA OLC8 inhibited the oncogenic activities of GC cells both in vitro and in vivo. 8 The expression level of RP11-555H23.1 was significantly decreased in GC tissues, and its reduction was closely correlated with tumor-node-metastasis (TNM) stage. 9 Gao et al reported that ln-cRNA NBAT-1 was significantly downregulated in GC tissues, and NBAT-1 overexpression markedly decreased the proliferative capacity of cancer cells, suggesting that NBAT-1 might act as a tumor suppressor in GC. 10 Exosomes are membrane vesicles and can be found in serum, plasma, urine, and other body fluids. 11 LncRNAs are found in circulating exosomes and might be used as candidate biomarkers for the detection and prognosis prediction of cancers. 12 For instance, the expression of serum exosomal lncRNA PCSK2-2:1 was significantly reduced in patients with GC, and downregulation of serum exosomal lncRNA PCSK2-2:1 was associated with adverse clinical parameters of GC. 13 Similarly, serum exosomal lnc-GNAQ-6:1 level was lower in GC patients and exhibited good performance for discriminating GC patients from healthy volunteers. 14 Myocardial infarction associated transcript (MIAT) was first identified as a lncRNA in 2006 and mapped to human chromosome 12q12.1. 15 MIAT was upregulated both in GC tissues and cell lines.
In vitro and in vivo analyses revealed that MIAT downregulation significantly suppressed the oncogenic activities of GC cells. 16,17 However, the potential clinical value of circulating exosomal MIAT in GC remained unclear. In this study, we first detected serum exosomal MIAT levels in GC patients, and then, the correlations between serum exosomal MIAT expression and the prognosis of GC were further evaluated.

| Patients and serum collection
A total of 109 patients with GC, 48 patients with gastric adenoma, and 50 healthy volunteers were enrolled in this study. Of all 109 GC cases, 81 were males and 28 were females. None of these cases have received prior treatment before first-time blood sampling. The pathological staging of GC was assessed according to the seventh edition of the Union for International Cancer Control (UICC) tumornode-metastasis (TNM) staging system. The exclusion criteria for GC were as follows: (a) with a secondary malignancy, (b) presence of severe heart disease such as acute myocardial infarction, arrhythmia, and heart failure, and (c) presence of systemic diseases such as liver failure, multiple organ dysfunction syndrome, and chronic kidney disease. The clinical characteristics of GC patients were summarized in Table 1

| Isolation of exosomes
Exosomes were isolated from the serum samples using the ExoQuick Exosome Precipitation Solution (System Biosciences) according to the manufacturer's instructions. Briefly, followed by thawing on ice, the serum was centrifuged at 3000 g for 10 minutes and filtrated with a 0.22 μm syringe filter (Millipore) to eliminate possible cell debris. Then, the supernatant was mixed with 125 μL ExoQuick reagent, and the mixture was incubated at 4°C for 30 minutes. The exosomes were pelleted by centrifuging the mixture at 1500 g for 30 minutes. The pellets containing exosomes were resuspended in phosphate-buffered saline (PBS) and stored at −80°C for further experiments.

| Western blot
Protein samples (20 µg) were separated on 8%-12% sodium dodecyl sulfate polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes. Followed by blocking with 5% non-fat milk in TBST for 1 hour at room temperature, the membranes were probed with primary antibodies of CD63 (Abcam), TSG101 (Abcam), and Flotillin-1(Abcam) in the cold room overnight. Followed by washing three times in TBST, the membranes were incubated with appropriated secondary antibodies for 1h at room temperature. The blots were visualized by SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Inc).

| Total RNA extraction and quantitative realtime reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was isolated from serum samples using mirVana™ miRNA Isolation Kit (Ambion). Reverse transcription was performed with  factors for OS P value <.05 was considered statistically significant.

| Serum exosomal MIAT was upregulated in GC
Our Western blot results demonstrated that the exosomes we extracted from the serum samples were positive for exosome markers including TSG101, CD63, and Flotillin-1. However, no or weak signal was detected in the supernatant samples ( Figure 1A). As shown in Figure 1B, the expression level of serum exosomal MIAT was markedly higher in GC patients than in gastric adenoma patients (P < .001) and healthy controls (P < .001). In addition, serum exosomal MIAT level was higher in patients with gastric adenoma than in healthy volunteers (P = .011). Moreover, ROC curve analysis revealed that serum exosomal MIAT well-discriminated GC patients from healthy  Figure 1C).
All gastric adenoma patients were divided into two subgroups using the median value of serum exosomal MIAT levels. As presented in Figure 1D, gastric adenoma patients in the high serum exosomal MIAT subgroup suffered a significantly higher risk to progress into GC (10/24) than those in the low serum exosomal MIAT subgroup (3/24).

| High serum exosomal MIAT was correlated with aggressive clinical variables
Then, the associations between serum exosomal MIAT levels and  Table 1). In addition, GC patients with poorly differentiated tumor grade (P = .001), or with lymph node metastasis (P < .001), or at the advanced stages (P < .001) had significantly higher serum exosomal MIAT levels than those with well/ moderate differentiated tumor grade, or without lymph node metastasis, or at the early stages (Figure 2A-2C).

| Changes in serum exosomal MIAT level before and after treatment
The serum exosomal MIAT levels in paired blood samples from all 109 GC patients were compared before and 1 month after treatment. As showed in Figure 3A, serum exosomal MIAT levels were significantly decreased in the post-treatment samples (P < .001). A total of 57 cases relapsed during the follow-up. Interestingly, serum exosomal MIAT levels were also markedly downregulated in these relapsed cases one month after treatment. However, the levels of serum exosomal MIAT were significantly increased at the timepoint of relapse. (P < .001, Figure 3B). As presented in Figure 3C, GC patients in the high serum exosomal MIAT subgroup had a higher recurrence rate (36/54) than those in the low serum exosomal MIAT subgroup (21/55).

| GC patients with higher serum exosomal MIAT suffered shorter OS and RFS
The survival analysis showed that the GC patients in high serum exosomal MIAT expression group had shorter OS than those in low-high serum exosomal MIAT expression group (P = .022, Figure 4A). Similarly, GC patients with higher serum exosomal MIAT levels suffered worse RFS compared to those with lower serum exosomal MIAT levels (P = .002, Figure 4B).

| Serum exosomal MIAT was an independent prognostic factor for GC
The multivariate analysis showed that serum exosomal MIAT ex-  (Table 2).  clinical parameters and worse prognosis. Therefore, serum exosomal lncRNA MIAT might serve as a promising novel biomarker for monitoring the progression of GC.

ACK N OWLED G M ENTS
This study was funded in part or by Sichuan Provincial Department of