GPC1 exosome and its regulatory miRNAs are specific markers for the detection and target therapy of colorectal cancer

Abstract

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. However, a biomarker for a sensitive and simple diagnostic test and highly effective target therapy of CRC is still clinically unavailable. This study is to investigate the evidence and significance of plasma GPC1 positive exosomes as a biomarker of CRC. Results showed that GPC1⁺ exosomes were successfully isolated from tissues and plasma. The percentage of GPC1⁺ exosomes and the GPC1 protein expression in exosomes from tumour tissues and plasma of CRC patients before surgical treatment was significantly elevated compared to that in the peritumoural tissues and the plasma of healthy controls. miR-96-5p and miR-149 expression in tumour tissues and plasma of CRC patients as well as in the GPC1⁺ exosomes from CRC patients were significantly decreased compared to that in the peritumoural tissues and the plasma of healthy controls. Two months after surgical treatment, levels of all tested markers significantly normalized. Overexpression of miR-96-5p and miR-149 significantly decreased GPC1 expression in HT-29 and HCT-116 cells, xenograft tumours, plasma in mice bearing HT-29 and HCT-116 tumours, and the secretion of GPC1⁺ exosomes from the HT-29 and HCT-116 cells and xenograft tumours. Overexpression of miR-96-5p and miR-149 significantly decreased cell viability and increased cell apoptosis in HT-29 and HCT-116 cells, and inhibited the growth of xenograft HT-29 and HCT-116 tumours. In conclusion, the increased plasma GPC1⁺ exosomes and reduced plasma miR-96-5p and miR-149 expression are specific markers for the diagnosis of CRC and targets for the therapy of CRC.

Keywords: GPC1; biomarker; colorectal cancer; exosome; miRNA.

Figure 1

Isolation of GPC1⁺ exosomes. (A) Representative Western blots of GPC1 and β‐actin protein expression in tissues. (B) Semi‐quantitative analysis of GPC1 protein expression in human normal colon tissues (n = 89) and CRC tumour tissues (n = 102). **P < 0.001 versus normal tissue. (C) Representative Western blot of CD63 and β‐actin protein expression in CRC tissue lysates and exosomes. (D) A representative TEM of GPC1⁺ exosomes from plasma. (E) Cytometry assay of percentage of GPC1⁺ exosomes in CRC tumour tissues (n = 102) and normal colon tissues (n = 89). **P < 0.001 versus normal tissue. (F) Cytometry assay of percentage of GPC1⁺ plasma exosomes in CRC patients (n = 102) before surgical treatment (B/T), after surgical treatment (A/T), and healthy subjects (control) (n = 80). **P < 0.001 versus other two groups.

Figure 2

Measure of GPC1 expression in exosomes. (A) Representative Western blots of GPC1 protein expression in tumour exosomes and normal tissue exosomes. (B) Semi‐quantitative analysis of GPC1 expression in tissue exosomes in (A). **P < 0.001 versus normal tissue exosomes. n = 102 for CRC, n = 89 for normal tissue. (C) Representative Western blot of GPC1 expression in the plasma of healthy individuals, CRC patients before and after surgical treatment. (D) Semi‐quantitative analysis of GPC1 expression in plasma exosomes in (C). **P < 0.001 versus other two groups. n = 102 for CRC, n = 80 for healthy control.

Figure 3

miR‐96‐5p, miR‐149, and miR‐182‐5p expression in tissues, plasma, and exosomes. miRNA expression was measured by real‐time PCR. (A) miR‐96‐5p expression in normal colon tissues, CRC tumour tissues, normal tissue exosomes and CRC tumour exosomes. **P < 0.001 versus normal tissue or normal tissue exosomes. (B) miR‐96‐5p expression in the plasma of healthy individuals, CRC patients before (B/T) and after surgical treatment (A/T), as well as their plasma exosomes. *P < 0.001, **P < 0.001 versus other two groups. (C) miR‐149 expression in normal colon tissues, CRC tumour tissues, normal tissue exosomes and CRC tumour exosomes. **P < 0.001 versus normal tissue or normal tissue exosomes. (D) miR‐149 expression in the plasma of healthy individuals, CRC patients before (B/T) and after surgical treatment (A/T), as well as their plasma exosomes. ^# P < 0.05, **P < 0.001 versus other two groups. (E) miR‐182‐5p expression in normal colon tissues, CRC tumour tissues, normal tissue exosomes, and CRC tumour exosomes. ^# P < 0.05, *P < 0.01 versus normal tissue or normal tissue exosomes. (F) miR‐182‐5p expression in the plasma of healthy individuals, CRC patients before (B/T) and after surgical treatment (A/T), as well as their plasma exosomes. n = 102 for CRC, n = 89 for normal tissue, n = 80 for control.

Figure 4

Silencing of miR‐96‐5p and miR‐149 expression in HT‐29 cells. (A) Representative Western blot of GPC1 protein expression in control virus (control), AdmiR96, and AdmiR149 infected HT‐29 cells. (B) Semi‐quantitative analysis of GPC1 expression in HT‐29 cells in (A). (C) Cytometry assay of the percentage of GPC1⁺ exosomes in the supernatant of cultured HT‐29 cells infected with viruses. (D) Representative Hoechst33342 staining of HT‐29 cells infected with viruses. (E) Percentage of apoptotic HT‐29 cells infected with virus. (F) MTT assay of cell viability in HT‐29 cells infected with viruses. **P < 0.001 versus control, n = 4.

Figure 5

Silencing of miR‐96‐5p and miR‐149 expression in HCT‐116 cells. (A) Representative Western blot of GPC1 protein expression in control virus (control), AdmiR96, and AdmiR149 infected HCT‐116 cells. (B) Semi‐quantitative analysis of GPC1 expression in HCT‐116 cells in (A). (C) Cytometry assay of percentage of GPC1⁺ exosomes in the supernatant of viruses‐infected HCT‐116 cells. (D) Representative Hoechst33342 staining of HCT‐116 cells infected with viruses. (E) Percentage of apoptotic HCT‐116 cells infected with virus. (F) MTT assay of cell viability in HCT‐116 cells infected with viruses. **P < 0.001 versus control, n = 4.

Figure 6

Inhibition of GPC1 expression and secretion of GPC1⁺ exosomes suppressed xenograft HT‐29 tumour growth. (A) Injection of AdmiR96 and AdmiR149 viruses inhibited xenograft HT‐29 tumour growth. The control mice were injected with control virus. (B) Representative Western blots of GPC1 protein expression in the HT‐29 tumour tissues. (C) Semi‐quantitative analysis of GPC1 expression in HT‐29 tumours measured by Western blot. **P < 0.001 versus control, n = 10. (D) Cytometry assay of the percentage of GPC1⁺ exosomes in the plasma of mice bearing HT‐29 tumours. **P < 0.001 versus control, n = 10. (E) Representative Western blots of plasma GPC1 protein levels in mice bearing HT‐29 xenograft tumours and healthy mice. (F) Semi‐quantitative analysis of plasma GPC1 expression in mice bearing HT‐29 xenograft tumours and healthy mice. **P < 0.001 versus healthy mice, *P < 0.05 versus healthy mice, ^# P < 00.01 versus mice bearing HT‐29 xenograft tumours injected with control virus, n = 10.

Figure 7

Inhibition of GPC1 expression and secretion of GPC1⁺ exosomes suppressed xenograft HCT‐116 tumour growth. (A) Injection of AdmiR96 and AdmiR149 viruses inhibited xenograft HCT‐116 tumour growth. The control mice were injected with control virus. #P < 0.05, *P < 0.01, **P < 0.001 versus AdmiR96 and AdmiR149 group, n = 10. (B) Representative Western blot of GPC1 protein expression in the HCT‐116 tumour tissues. (C) Semi‐quantitative analysis of GPC1 expression in HCT‐116 tumours measured by Western blot. **P < 0.001 versus control, n = 10. (D) Cytometry assay of percentage of GPC1⁺ exosomes in plasma of mice bearing HCT‐116 tumours. **P < 0.001 versus control, n = 10. (E) Representative Western blots of plasma GPC1 protein levels in mice bearing HCT‐116 tumours and healthy mice. (F) Semi‐quantitative analysis of plasma GPC1 expression in mice bearing HCT‐116 xenograft and healthy mice. **P < 0.001 versus all other groups, n = 10.