Identification of miRNAs Present in Cell- and Plasma-Derived Extracellular Vesicles-Possible Biomarkers of Colorectal Cancer

Abstract

Globally, an increasing prevalence of colorectal cancer (CRC) prompts a need for the development of new methods for early tumor detection. MicroRNAs (also referred to as miRNAs) are short non-coding RNA molecules that play a pivotal role in the regulation of gene expression. MiRNAs are effectively transferred to extracellular vesicle (EVs) membrane sacs commonly released by cells. Our study aimed to examine the expression of miRNAs in four CRC cell lines and EVs derived from them (tumor EVs) in comparison to the normal colon epithelium cell line and its EVs. EVs were isolated by ultracentrifugation from the culture supernatant of SW480, SW620, SW1116, HCT116 and normal CCD841CoN cell lines and characterized according to the MISEV2023 guidelines. MiRNAs were analyzed by small RNA sequencing and validated by quantitative PCR. The performed analysis revealed 22 common miRNAs highly expressed in CRC cell lines and effectively transferred to tumor EVs, including miR-9-5p, miR-182-5p, miR-196b-5p, miR-200b-5p, miR-200c-3p, miR-425-5p and miR-429, which are associated with development, proliferation, invasion and migration of colorectal cancer cells, as well as in vesicle maturation and transport-associated pathways. In parallel, normal cells expressed miRNAs, such as miR-369 and miR-143, which play a role in proinflammatory response and tumor suppression. The analysis of selected miRNAs in plasma-derived EVs and tumor samples from CRC patients showed the similarity of miRNA expression profile between the patients' samples and CRC cell lines. Moreover, miR-182-5p, miR-196-5p, miR-425-5p and miR-429 were detected in several EV samples isolated from patients' plasma. Our results suggest that miR-182-5p, miR-196b-5p and miR-429 are differentially expressed between EVs from CRC patients and healthy donors, which might have clinical implications.

Keywords: colorectal cancer; colorectal cancer cell lines; miRNA expression profile; miRNAs; tumor-derived extracellular vesicles.

Figure 1

EV characterization. WB images of CD9 (A) and CD63 (B) protein expression in EV lysates obtained from healthy donors (HD), colorectal cancer (CRC) patients and cell lines; lanes from left to right: 1-2-HD, 3-4-CRC patients, 5-protein ladder, 6-CCD841CoN, 7-SW480, 8-SW620, 9-SW1116 and 10-HCT116. Molecular weights for CD9 are 22, 24 and 35 kDa, and for CD63-28–35 kDa. WB image of GM130 (C) protein expression in cell line lysates and their EVs, HD and CRC peripheral blood mononuclear cells (PBMC) and EVs lysates; lanes from left to right: 1-CCD841CoN, 2-SW480, 3-SW620, 4-SW1116, 5-HCT116, 6-CCD841CoN EVs, 7-SW480 EVs, 8-SW620 EVs, 9-SW1116 EVs, 10-HCT116 EVs, 11-protein ladder, 12-HD PBMC, 13-CRC PBMC, 14-HD EVs and 15-CRC EVs. The molecular weight for GM130 is 140 kDa. The uncropped blots and molecular weight markers are shown in Supplementary Figure S1. The size profile of EVs (results from one representative measurement for each case are shown) measured using NTA (D); MEMGlow staining of EVs plasma membrane (results from one representative measurement for each case are shown) (E). For (D,E), graphs from left to right: cell lines: CCD841CoN, SW480, SW620, SW1116 and HCT116; HD, CRC patients. Color legend: green—stained EVs, red—unstained EVs, gray—PBS with and without MEMGlow.

Figure 2

Validation of NGS results (left panel) of selected miRNA expression using qPCR (right panel) in the cell lines (A) or EVs (B). MiRNA expression of miR-19a-3p, miR-143-3p and miR-369-3p in cell lines (A) or the expression of miR-143-3p, miR-146a-5p and miR-181a-5p in EVs (B) was analyzed using RT-qPCR and normalized to U6 expression using the 2^−ΔΔCT method. Data were analyzed using the Kruskal–Wallis test with Dunn’s post hoc test, and medians ± IQR are shown. * p < 0.05, ** p < 0.01.

Figure 3

The differential expression of miRNAs using Venn diagrams. Comparison of miRNAs that were at least two times more expressed in tumor cell lines, compared to the normal CCD841CoN cell line (A) and in tumor EVs, compared to EVs isolated from control CCD841CoN cell line (B). Common miRNAs, showing 2FC difference for both tumor cell lines and tumor EVs, compared to the normal cell line or its EVs, respectively (C).

Figure 4

KEGG and GO analysis of selected top 22 miRNAs and visualization of targeted genes. KEGG pathway analysis results of the top 12 pathways associated with at least 10 analyzed miRNAs (A). GO analysis of cellular components showing 22 analyzed miRNAs significantly associated with EVs development and processing (B). Visualization of genes targeted by selected seven miRNAs: miR-9-5p, miR-196b-5p, miR-182-5p, miR-200b-5p, miR-200c-3p, miR-429 and miR-425-5p (C).

Figure 5

qPCR analysis of selected miRNAs: miR-9-5p (A), miR-182-5p (B), miR-196b-5p (C), miR-200b-5p (D), miR-200c-3p (E), miR-425-5p (F) and miR-429 (G), and in all analyzed cell lines and their EVs. miRNA expression was analyzed using RT-qPCR and normalized to U6 expression using the 2^−ΔΔCT method. Data were analyzed using the Kruskal–Wallis test with Dunn’s post hoc test, and medians ± IQR are shown. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

qPCR analysis of selected miRNA expression in CRC patients‘ samples, in comparison to healthy donors. The expression of seven selected miRNAs in tumor samples obtained from CRC patients (A). miR-182-5p, miR-196-5p, miR-425-5p and miR-429 expression in EVs isolated from patients’ plasma (B). The analysis was performed in CRC patients (n = 9) and healthy donors (n = 9). miRNA expression was analyzed using RT-qPCR and normalized to U6 expression using the 2^−ΔΔCT method. Data were analyzed using the Kruskal–Wallis test with Dunn’s post-hoc test (A) or Mann–Whitney test (B), and medians ± IQR are shown. Asterisks mark significant differences: * p < 0.05, ** p < 0.01.