Serum extracellular vesicles 3'tRF-ThrCGTand 3'tRF-mtlleGAT combined with tumor markers can serve as minimally invasive diagnostic predictors for colorectal cancer

Abstract

Background: Colorectal cancer (CRC) is a leading cause of morbidity and mortality, and timely diagnosis and intervention are crucial for cancer patients. Transfer RNA-derived fragments (tRFs) play a noncoding regulatory role in organisms. Serum EV(extracellular vesicles), as an integral mediator of intercellular transmission of genetic information vesicles in Transfer RNA-derived fragment (tRF RNA), are expected to be minimally invasive diagnostic and predictive biologic factors of CRC.

Methods: Collect serum samples from 205 CRC patients, and then isolate extracellular vesicles from the serum. Captured the physical morphology of EV through transmission electron microscopy. The particle size was detected by particle size assay, and protein expression on the surface of EV was verified by Western blot. Gene microarrays were screened for differentially expressed tRF-RNA. TRF RNAs were verified by qPCR for differential expression in 205 CRC patients and 201 healthy donors, assessing the CRC diagnostic efficiency by area under the curve (AUC).

Results: Compared with 201 healthy donors, CRC patients experienced significantly down-regulated serum EV 3'tRF-ThrCGT while significantly up-regulated 3'tRF-mtlleGAT. Serum EV 3'tRF-ThrCGT and 3'tRF-mtlleGAT predictive diagnostic efficiency: 0.669 and 0.656, and the combination of CEA and CA724 predictive diagnostic efficiency was 0.938.

Conclusion: The study data showed that 3'tRF-ThrCGT and 3'tRF-mtlelGAT can be minimally invasive diagnostic CRC indicators. The combination of tumor markers CEA and CA724 has important diagnostic significance.

Keywords: 3’tRF-ThrCGT; 3’tRF-mtlleGAT; colorectal cancer; minimally invasive diagnostic predictors; serum EV; tumor markers.

Figure 1

Performance verification of serum exosomes. (A) Identification of the physical morphology of exosomal by transmission electron microscopy (TEM). (B) Measurement of exosomal size distribution using particle size analyzer by qNano. (C) Western blot validated of serum exosomal surface protein markers.

Figure 2

Exosomal miRNAs profile of the CRC patients. Cluster analysis of differentially expressed serum exosomal tRFRNAs between 6 CRC patients and 3 healthy donor.

Figure 3

Serum exosomal 3’tRF-ThrCGT and 3’tRF-mtlleGAT are potential biomarkers of colorectal cancer. The expression levels of serum exosomes 3’RF-ThrCGT (A) and 3’tRF-mtlleGAT (B) in CRC and healthy donors.

Figure 4

Association between the serum exosomal tRFRNA and tumor stage. Expression levels of 3’tRF-ThrCGT (A) and (B) in T and N stages patients. (C) Serum exosomal 3’tRF-ThrCGT expression in early stage (I+II) and advanced stage (III+IV) patients. The expression level is represented using -ΔCT. (D, E) Statistical levels of 3’tRF-mtlleGAT in T and N stages patients (F) Serum exosomal 3’tRF-mtlleGAT expression in stage (**P < 0.01, *P < 0.05, ns, not significant).

Figure 5

Diagnostic role of serum exosomal 3’tRF-ThrCGT and 3’tRF-mtlleGAT expression levels in CRC patients patients. The AUCs of 3’tRF-ThrCGT (A), 3’tRF-mtlleGAT (B), and both (C) in CRC patients relative to healthy donors.

Figure 6

Improved diagnostic capacity of serum exosomes 3’tRF-ThrCGT and 3’tRF-mtlleGAT combined with established tumor markers in CRC patients. The AUCs of 3’tRF-ThrCGT combined with CEA (A), CA724 (B), and both (C). The AUCs of 3’tRF-mtlleGAT combined with CEA (D), CA724 (E), and both (F). The AUCs of 3’tRF-ThrCGT and 3’tRF-mtlleGAT combined with CEA (G), CA724 (H), and both (I).