Exosomal MicroRNA-223, MicroRNA-146, and MicroRNA-21 Profiles and Biochemical Changes in Laryngeal Cancer

Abstract

Laryngeal squamous cell carcinoma (LSCC) is one of the most aggressive cancers, and its early diagnosis is urgent. Exosomes are believed to have diagnostic significance in cancer. However, the role of serum exosomal microRNAs, miR-223, miR-146, and miR-21, and phosphatase and tensin homologue (PTEN) and hemoglobin subunit delta (HBD) mRNAs in LSCC is unclear. Exosomes were isolated from the blood serum of 10 LSCC patients and 10 healthy controls to perform scanning electron microscopy and liquid chromatography quadrupole time-of-flight mass spectrometry analyses to characterize them and to undergo reverse transcription polymerase chain reaction to identify miR-223, miR-146, miR-21, and PTEN and HBD mRNA expression phenotypes. Biochemical parameters, including serum C-reactive protein (CRP) and vitamin B12, were also obtained. Serum exosomes of 10-140 nm were isolated from LSCC and controls. Serum exosomal miR-223, miR-146, and PTEN were found to be significantly decreased (p < 0.05), in contrast to serum exosomal miRNA-21 (p < 0.01), and serum vitamin B12 and CRP (p < 0.05) were found to be significantly increased, in LSCC vs controls. Our novel data show that the combination of reduced serum exosomal miR-223, miR-146, and miR-21 profiles and biochemical alterations in CRP and vitamin B12 levels may be useful indicators of LSCC that could be validated by large studies. Our findings also suggest a possible negative regulatory effect of miR-21 on PTEN in LSCC, encouraging a more extensive investigation of its role.

Figure 1

Representative MRI findings in the right section of the neck. Diagnosis; laryngeal tumor. White arrows show the larynx cancer area, orange arrows show the vertebra, and yellow arrows show the larynx.

Figure 2

Representative pathological image of a patient with LSCC diagnosis showing invasiveness, heterogeneity, and amorphous cell formation (100 μm hematoxylin and eosin staining; by ImageScope). Red arrow: infiltrative cancer cells.

Figure 3

Controls (A) and LSCC (B) patient serum exosomes examined by SEM. The white arrow shows particle size. P: particle, Pa: particle size in nm, Pb: particle angle and, Pa R: particle radius. (by Carl Zeiss Evo 40 SEM; Jena Germany).

Figure 4

Diagram depicts the total results from QTOF analysis (A). Diagrams (B) depict the QTOF of each molecule: (a) nummularine, (b) N-caffeoyltryptophan, (c) 1-monopalmitin, (d) hexadecyl acetyl glycerol, (e) pipericine, and (f) cyclohexanecarbonylpentadecylamine (Agilent 6530 QTOF).

Figure 5

Serum exosomal miRNA-223, miRNA-146, and miRNA-21 levels (A), and PTEN and HBD mRNA levels (B) in serum LSCC and healthy controls (*p < 0.05; **p < 0.001; by t-test; GraphPad Prism 7.0). (miRNA levels were normalized to the RNU6 reference control; mRNA levels were normalized to the β-actin reference gene).