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, 16 (3), 471-482
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Exploration of Serum Exosomal LncRNA TBILA and AGAP2-AS1 as Promising Biomarkers for Diagnosis of Non-Small Cell Lung Cancer

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Exploration of Serum Exosomal LncRNA TBILA and AGAP2-AS1 as Promising Biomarkers for Diagnosis of Non-Small Cell Lung Cancer

Yao Tao et al. Int J Biol Sci.

Abstract

Non-small cell lung cancer is the most common type of cancer with a poor prognosis, and development of an effective diagnostic method is urgently needed. Exosomal lncRNAs, a class of transcripts longer than 200 nucleotides packaged into exosomes, have been defined as an ideal diagnostic biomarker for cancer. However, little is known about the clinical utility of exosomal lncRNAs in NSCLC. Here, we aimed to identify exosomal lncRNAs as promising biomarkers for NSCLC diagnosis. First, serum exosomes from NSCLC patients were successfully isolated by a polymer precipitation kit and then identified by TEM, NTA and western blot analysis. A total of nine candidate lncRNAs were detected by qRT-PCR in a training set. The two exosomal lncRNA TBILA and AGAP2-AS1 were screened out for the higher levels in NSCLC patients than that of healthy controls in a validation set. And there was a significant positive correlation between these exosomal lncRNAs levels and tumor size, lymph node metastasis and TNM stage. Additionally, we validated that these exosomal lncRNAs were stable in serum. Next, we evaluated the diagnostic efficiency of exosomal lncRNAs in NSCLC patients by ROC curve analysis. The data showed that individual TBILA or AGAP2-AS1 exhibited better diagnostic efficiency in NSCLC patients with different tumor pathologic subtypes and early stage, whereas the combination of lncRNAs did not provide better results than individual lncRNAs. Notably, the combination of two exosomal lncRNAs and the serum tumor biomarker Cyfra21-1 widely used in clinical practices further improved the diagnostic accuracy for NSCLC patients. This study suggests that exosomal lncRNA TBILA and AGAP2-AS1 may be promising biomarkers for diagnosis of NSCLC.

Keywords: AGAP2-AS1.; Biomarker; Exosomes; Long non-coding RNAs; Non-small cell lung cancer; TBILA.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Identification of exosomes in serum. (A) The morphology of serum exosomes was visualized by transmission electron microscopy (TEM). (B) The size distribution and concentration of serum exosomes were assessed by nanoparticle tracking analysis (NTA). (C) The specific exosome marker protein (CD9 and CD63) and non-exosomal protein (Tubulin) in serum exosomes and exosomes-depleted serum (EDS) were detected by western blot analysis.
Figure 2
Figure 2
The levels of three exosomal lncRNAs in NSCLC patients in the validation set. (A) qRT-PCR analysis of three exosomal lncRNAs in NSCLC patients, stage I NSCLC patients and healthy controls. (B) qRT-PCR analysis of three exosomal lncRNAs in lung ADC patients, SCC patients and healthy controls (n=100). * p < 0.05, ** p < 0.01, *** p < 0.001. n.s, no signification, p > 0.05.
Figure 3
Figure 3
Comparison of three exosomal lncRNAs expression in preoperative and postoperative serum samples of NSCLC patients (n=10). (A) TBILA. (B) AGAP2-AS1. (C) SOX2OT. * p < 0.05; n.s, no signification.
Figure 4
Figure 4
Evaluation of the stability of exosomal lncRNA TBILA and AGAP2-AS1 in serum samples. (A) qRT-PCR analysis of lncRNAs levels in serum exosomes and exosomes-depleted serum (EDS) from NSCLC patients (B) The exosomes suspensions were treated with or without RNase A (2 μg/mL) for 20 min at 37°C, following by qRT-PCR analysis of lncRNAs. (C) The exosomes suspensions were placed at room temperature for different times, following by qRT-PCR analysis of lncRNAs. ** p < 0.01; *** p < 0.001; n.s, no signification.
Figure 5
Figure 5
Diagnostic efficiency of individual or combined exosomal lncRNAs for NSCLC. (A-B) ROC curve analysis for exosomal lncRNAs in distinguishing NSCLCs overall or stage I NSCLC patients versus healthy controls. (C-D) ROC curve analysis for exosomal lncRNAs for ADC patients or SCC patients versus healthy controls.
Figure 6
Figure 6
Diagnostic efficiency of the combination of exosomal lncRNAs and serum Cyfra21-1 for NSCLC. (A-B) ROC curve analysis for the combination of exosomal lncRNAs and Cyfra21-1 in distinguishing NSCLCs overall or stage I NSCLC patients versus healthy controls. (C-D) ROC curve analysis for the combination of exosomal lncRNAs and Cyfra21-1 for ADC patients or SCC patients versus healthy controls.
Figure 7
Figure 7
Schematic diagram illustrates the multi-step process of identifying potential exosomal lncRNAs in serum for diagnosis of NSCLC.

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