Exosomal long noncoding RNA CRNDE-h as a novel serum-based biomarker for diagnosis and prognosis of colorectal cancer

Abstract

Cancer-secreted long non-coding RNAs (lncRNAs) are emerging mediators of cancer-host cross talk. The aim of our study was to illustrate the clinical significance of the lncRNA CRNDE-h in exosomes purified from the serum of patients with colorectal cancer (CRC). The study was divided into four parts: (1) The exosome isolated methods and lncRNA detected methods which accurately and reproducibly measure CRC-related exosomal CRNDE-h in serum were optimized in preliminary pilot stage; (2) The stability of exosomal CRNDE-h was evaluated systematically; (3) The origin of exosomal CRNDE-h was explorated in vitro and in vivo; (4) The diagnostic and prognostic value of exosomal CRNDE-h for CRC were validated in 468 patients. In pilot study, our results indicated that exosomal CRNDE-h was detectable and stable in serum of CRC patients, and derived from tumor cells. Then, the increased expression of exosomal CRNDE-h was successfully validated in 148 CRC patients when compared with colorectal benign disease patients and healthy donors. Exosomal CRNDE-h level significantly correlated with CRC regional lymph node metastasis (P = 0.019) and distant metastasis (P = 0.003). Moreover, at the cut-off value of 0.020 exosomal CRNDE-h level of serum, the area under ROC curve distinguishing CRC from colorectal benign disease patients and healthy donors was 0.892, with 70.3% sensitivity and 94.4% specificity, which was superior to carcinoembryogenic antigen. In addition, high exosomal CRNDE-h level has a lower overall survival rates than that for low groups (34.6% vs. 68.2%, P < 0.001). In conclusion, detection of lncRNA CRNDE-h in exosome shed a light on utilizing exosomal CRNDE-h as a noninvasive serum-based tumor marker for diagnosis and prognosis of CRC.

Keywords: CRNDE-h; biomarker; colorectal cancer; exosome; long noncoding RNA.

Figure 1. Characterization of serum exosome. A.

Serum exosomes were analyzed under electron microscopy which displayed the same morphology. Exosomes were highlighted using red arrows. Scale bar = 100nm. B. Exosome-enriched protein CD63 and Hsp70 were analyzed by western blotting in exosomal (E) and exosome-depleted supernatant (EDS) samples in serum.

Figure 2. Standard curves and reference gene expression in exosome. A.

Standard curves for GAPDH, UBC, and CRNDE-h. B. Comparison of GAPDH and UBC reference genes expression among NC (n=10), IBD (n=10), HP (n=10), adenoma (Ad; n=10), and CRC (n=10) detected by RT-qPCR. E represents the reaction efficiency.

Figure 3. General characterization of the exosomal CRNDE-h. A.

Exosomal CRNDE-h levels amplified from exosome-depleted supernatant (EDS), serum exosome (E) and whole serum (S). Comparison of the CRNDE-h expression level between exosome group and isolated nucleic acid (Exo.RNA) group when they were subjected to 3 h in RNase A B. low (pH = 1), high (pH = 13) pH solutions C. multiple freeze-thaws D. room temperature incubation E. and -80°C F. *P<0.01.

Figure 4. Exosomal CRNDE-h can enter serum at measurable levels

Levels of exosomal CRNDE-h expressed in six colorectal cells A. and culture medium B. C. Levels of exosomal CRNDE-h in xenograft model and controls. D. Spearman's correlation analysis between exosomal CRNDE-h levels in tumor tissues and matched serum samples of CRC. E. Levels of exosomal CRNDE-h in CRC patients’ serum varied in before surgery (Pre-operation) and 14 days after tumor resection (Post-operation). *P<0.01.

Figure 5. Quantitative analyses of exosomal CRNDE-h in validation phase

Relative expression of exosomal CRNDE-h in NC (n=80), HP (n=80), IBD (n=80), adenoma (Ad; n=80), and CRC (n=148). Yellow line represents the optimal cut-off value as 0.020 for discriminating CRC from colorectal benign disease groups and normal colonoscopy. Red line represents the median value and the gray line means the 25% and 75% interquartile range. **P<0.001.

Figure 6. Diagnostic performance of serum exosomal CRNDE-h in CRC

ROC curves for detection of CRC using exosomal CRNDE-h A., CEA B., and exosomal CRNDE-h combined with CEA C., as assessed by AUC. D. ROC curves for detection of adenoma patients using exosomal CRNDE-h.

Figure 7. Kaplan–Meier curves for overall survival according to serum exosomal CRNDE-h A.

and combined exosomal CRNDE-h and CEA B. The optimal cut-off value (0.020) of exosomal CRNDE-h was used to categorize the CRC patients into low or high, and low or high CEA based on the cut-off value (5ng/ml).