Extracellular Vesicle-Associated mir-21 and mir-144 Are Markedly Elevated in Serum of Patients With Hepatocellular Carcinoma

Abstract

Aim: The aim of this study was to observe the possible change of microRNAs (miRNAs) in serum extracellular vesicles (EVs) from hepatocellular carcinoma (HCC) patients. Methods: The serum EVs were purified from 17 healthy donors, 16 chronic hepatitis B (CHB) patients and 24 HCC patients. The sequenced microRNAs in the purified EVs were analyzed to obtain highly differentially expressed genes (DEGs). Finally, the expression pattern of DEGs was validated using qRT-PCR. Results: We found that the expression of hsa-miR-21-5p and hsa-miR-144-3p were significantly higher in EVs and liver cancer tissues compared with serum and the distal liver tissues in HCC patients. The ratio of hsa-miR-144-3p/hsa-miR-21-5p was significantly decreased in the patients with CHB but significantly increased in patients with HCC developed from CHB (P < 0.05). Hsa-144-3p/hsa-miR-21-5p exhibited greater performance than alpha-fetoprotein (AUC 0.780, 95% CI 0.601-0.960, versus AUC 0.626, 95% CI 0.410-0.843) in ROC curve analysis. Conclusion: Extracellular vesicle-associated hsa-miR-21-5p and hsa-miR-144-3p are markedly elevated in serum of patients with HCC. The potential role of these microRNAs in the pathogenesis of HCC is worth of further study.

Keywords: extracellular vesicle; hepatocellular carcinoma; microRNA; qRT-PCR; sequencing analysis.

FIGURE 1

Identification and characterization of extracellular vesicles (EVs) of healthy donors and patients with CHB and HCC. (A) Transmission electron micrograph of purified EVs shows small vesicles with sizes ranging from 30 to 110 nm in diameter (bars 100 nm). Arrow represented EVs. (B) Western blotting analysis of EV-associated proteins CD9 and CD63 in purified EVs from normal individuals, CHB and HCC patients. (C) Morphology of EVs by ZetaView. (a) Extraction by ultracentrifugation. (b) Extraction by Exo-spin EV Purification Kit. Analysis parameters: Max Area: 1000, Min Area: 5, Min Brightness: 20. (D) Particle size and concentration distribution of EVs by ZetaView.

FIGURE 2

Relative expression of miR-21-5p and miR-144-3p of EVs were higher in patients with HCC. (A) The relative expression of miR-21-5p of EVs and EV-free serum in healthy donors and patients with CHB and HCC by qRT-PCR (Normalized by U6). (B) The relative expression of miR-144-3p of EVs and EV-free serum in healthy donors and patients with CHB and HCC by qRT-PCR (Normalized by U6). (C) The ratio of miR-144-3p/miR-21-5p in EVs of healthy donors and patients with CHB and HCC. (D) The ratio of miR-144-3p/miR-21-5p in EV-free serum of healthy donors and patients with CHB and HCC. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

FIGURE 3

Relative expression of miR-21-5p and miR-144-3p of liver cancer tissues were higher in patients with HCC. (A) The relative expression of miR-21-5p in distal tissues of hepatic carcinoma (BON) and hepatocellular carcinoma (BOT) (Normalized by U6). (B) The relative expression of miR-144-3p in distal tissues of hepatic carcinoma (BON) and hepatocellular carcinoma (BOT) (Normalized by U6). ^∗P < 0.05.

FIGURE 4

The general diagnostic efficiency of miR-144-3p/miR-21-5p and AFP for HCC. (A) ROC curves of miR-21-5p, miR-144-3p, and miR-144-3p/miR-21-5p for patients with HCC versus AFP. (B) The area under the ROC curve.