Exosomal miR-665 as a novel minimally invasive biomarker for hepatocellular carcinoma diagnosis and prognosis

Abstract

Recent studies have shown that circulating microRNAs are potential biomarkers for various types of malignancies. The aim of this study was to investigate the feasibility of using serum exosomal microRNAs (miRNAs) as novel serological biomarkers for hepatocellular carcinoma (HCC) diagnosis and prognosis. Exosomes are small membranous vesicles (30-100 nm). Exosomal miR-665 levels in HCC patients were significantly higher than those in healthy subjects (P < 0.05), and exosomal miR-665 levels were significantly upregulated in tumours larger in size (> 5 cm), in tumours with local invasion and in those at an advanced clinical stage (stage III/IV) of HCC (P = 0.0042, 0.0197, and 0.0276, respectively). The survival time of the exosomal miR-665 high-expression group (n = 17) was significantly shorter than that of the low-expression group (n = 13) (P = 0.036). In addition, we found that HCC cell-derived exosomes promoted hepatoma cell proliferation and upregulated the expression level of proteins in the MAPK/ERK pathway in vitro and in vivo. This study suggests that serum exosomal miR-665 may be a novel minimally invasive biomarker for HCC diagnosis and prognosis.

Keywords: ERK; HCC; biomarker; exosomal miRNA-665; tumor growth.

Figure 1. Characterization of isolated exosomes

Exosomes were purified from the serum of HCC patients (A), LO2 (B), MHCC-97L (C) and MHCC-97H (D) cell culture supernatant and examined by TEM. (E) Size distribution analysis of purified exosomes using a Nanosight instrument. (F) Exosome markers (CD9, CD63) were analysed using Western blot. The data demonstrated that extracts were enriched with exosomal marker proteins CD9 and CD63.

Figure 2. Exosomal miR-665 expression is significantly higher in HCC

(A) A heatmap representing the differential miRNA expression in exosomes derived from three cell lines. The colour “red” indicates high relative expression, and “green” indicates low relative expression. (B) RT-PCR validated the reliability of the miRNA microarray, the average fold increase of exosomal miR-665 in MHCC-97H and MHCC-97L cells vs. the normal liver cell line (LO2) were 10.1 and 5.8, respectively. *P < 0.05; **P < 0.01. (C) The levels of exosomal miR-665 in serum samples from 30 patients with HCC and 10 healthy controls were determined by RT-PCR. (D) miR-665 expression in HCC, adjacent and normal liver tissue. The absolute 2^−ΔΔCT values are presented. (E) miR-665 expression was detected by RT-PCR in the exosomes derived from multiple hepatoma cell lines (HepG2, PLC and Hep3B).

Figure 3. Overall survival curves for 30 HCC patients who underwent hepatectomy

The patients with high exosomal miR-665 expression exhibited significantly poorer long-term prognosis after hepatectomy.

Figure 4. Exosomal miR-665 promotes cell proliferation in HCC cells

Micrographs of MHCC-97L (A) and MHCC-97H cells (B) infected with lentivirus-green fluorescent protein, captured after 72 h under a fluorescence microscope. Scale bar represents 500 μm. (C) miR-665 expression in the lentivirus group and control group. *P < 0.05. (D) Cell viability was assessed using an MTT assay. HCC exosomes promote cell proliferation in HCC cells; after the application of anti-miR-665, the cell proliferation-promoting effects of exosomes were downregulated. Equal volume of PBS was used as a control. *P < 0.05.

Figure 5. Exosomal miR-665 upregulated the expression of HCC cell proliferation-related proteins

(A) The expression levels of p-ERK, CyclinD1 and p-STAT3 were detected by Western blot; an equal volume of PBS was used as a control. Exosomal miR-665 could promote HCC cell proliferation through activating the MAPK/ERK pathway; the effects of exosomes decreased with the application of anti-miR-665 in HCC cells. (B) The expression levels of VEGFR and EGFR were detected by Western blot. HCC-derived exosomes contained many different molecules (VEGFR and EGFR). *P < 0.05.

Figure 6. Exosomal miR-665 promoted xenograft tumour growth in vivo

The size of tumours at the end of the experiment from mice treated with PBS (Control, A), MHCC97H-exosome (B) and MHCC97H-exosome anti-miR-665 (C, D, E) Representative images and tumour volume changes in the mice bearing MHCC97H-exosome or MHCC97H- exosome anti-miR-665. n = 5, significant difference between groups are shown as *P < 0.05.

Figure 7. MHCC97H-exosome anti miR-665 downregulated the expression of P-ERK in vivo

Tumours from mice treated with PBS (Control, A), MHCC97H-exosome (B) and MHCC97H-exosome anti-miR-665 (C) were paraffin-embedded and sectioned, followed by staining by using immunohistochemistry method. (D) The number of P-ERK-positive cells notably decreased in the MHCC97H-exosome anti-miR-665 group compared with the number in the MHCC97H-exosome group. *P < 0.05.