Circulating Serum Exosomal Long Non-Coding RNAs FOXD2-AS1, NRIR, and XLOC_009459 as Diagnostic Biomarkers for Colorectal Cancer

Abstract

Background: Exosomes derived from cancer cells encapsulate various kinds of tumor-specific molecules and thus can interact with adjacent or distant cells to mediate information exchange. Long non-coding RNAs (lncRNAs) in exosomes have the potential as diagnostic and prognostic biomarkers in different types of cancers. The current study was aimed to identify circulating exosomal lncRNAs for the diagnosis of colorectal cancer (CRC).

Methods: Exosomes were isolated from the serum by ultracentrifugation and verified by transmission electron microscope (TEM), qNano, and immunoblotting. Exosomal lncRNAs FOXD2-AS1, NRIR, and XLOC_009459 were selected by lncRNA microarray and validated by qPCR in 203 CRC patients and 201 healthy donors. The receiver operating characteristic curve (ROC) was used to assess the diagnostic efficiency of serum exosomal lncRNAs.

Results: Exosomal FOXD2-AS1, NRIR, and XLOC_009459 (TCONS_00020073) levels were significantly upregulated in 203 CRC patients and 80 early-stage CRC patients compared to 201 healthy donors, possessing the area under the curve (AUC) of 0.728, 0.660, and 0.682 for CRC, as well as 0.743, 0.660, and 0.689 for early-stage CRC, respectively. Notably, their combination demonstrated the markedly elevated AUC of 0.736 for CRC and 0.758 for early-stage CRC, indicating their potential as diagnostic biomarkers for CRC.

Conclusions: Our data suggested that exosomal lncRNAs FOXD2-AS1, NRIR, and XLOC_009459 act as the promising biomarkers for the diagnostics of CRC and early-stage CRC.

Keywords: CRC; biomarker; diagnosis; exosomes; lncRNA.

Figure 1

Identification of the differential exosomal lncRNAs (A) TEM images showed that exosomes were oval or bowl-shaped capsules without the nucleus. (B) QNano results suggested that the isolated exosome enriched from serum was about 50–150 nm in diameter. (C) Exosome markers TSG101 and CD9 were detected in the exosomes isolated from the serum; GM130, a negative marker of the exosome, was absent in our isolated exosome. (D) Hierarchical clustering analysis of differentially expressed lncRNAs among CRC patients and healthy donors using lncRNAs microarray. (E) Differential expression analysis between CRC patients and healthy donors were expressed with hybridization signals. The red dot represents upregulated lncRNAs expression in colorectal cancer, and the green dot represents downregulated lncRNAs expression.

Figure 2

Stability of Exosomal lncRNAs FOXD2-AS1 and NRIR and XLOC_009459. (A) qRT-PCR analysis of the three lncRNAs in the exosomes or exosomes deleted with Rnase A (B–D) The expressions of the three serum exosomal lncRNAs when incubated at room temperature (ns, not significant).

Figure 3

Exosomal lncRNAs FOXD2-AS1, NRIR, and XLOC_009459 were upregulated in CRC patients (A–F) Mann-Whitney U test indicated that the expression levels of serum exosomal FOXD2-AS1 and NRIR and XLOC_009459 were significantly upregulated in CRC patients (n=203) and early CRC patients (n=80) as compared to healthy donors (n=201) and benign intestinal diseases (BIDs) (n=20). The expression levels of FOXD2-AS1, NRIR, and XLOC_009459 showed no difference between benign intestinal diseases (BIDs) (n=20) and healthy donors (n=201). (G–I) The expression levels of exosomal FOXD2-AS1 and NRIR and XLOC_009459 were significantly different in 21 preoperative (Pre-OP) patients and paired postoperative (Post-OP) patients. ns, no significance.

Figure 4

Exosomal lncRNAs FOXD2-AS1 and NRIR and XLOC_009459 as diagnostics biomarker for CRC (A–D) Individual and combined diagnosis value of serum exosomal FOXD2-AS1 and NRIR and XLOC_009459 in 203 CRC patients and 201 healthy donors. (E–H) An individual and combined diagnosis value of exosomal FOXD2-AS1 and NRIR and XLOC_009459 in 80 early-stage CRC patients and 201 healthy donors.