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. 2023 Jul 14;24(14):11464.
doi: 10.3390/ijms241411464.

miR-124 as a Liquid Biopsy Prognostic Biomarker in Small Extracellular Vesicles from NSCLC Patients

Darío Sanchez-Cabrero  1   2 , Álvaro Garcia-Guede  1   3 , Miranda Burdiel  1   3 , Olga Pernía  1   3 , Julián Colmenarejo-Fernandez  1   3 , Laura Gutierrez  1   2 , Oliver Higuera  2 , Isabel Esteban Rodriguez  1   4 , Rocío Rosas-Alonso  1   3 , Carlos Rodriguez-Antolín  1   3 , Itsaso Losantos-García  5 , Olga Vera  1   3 , Javier De Castro-Carpeño  1   2 , Inmaculada Ibanez de Caceres  1   3
Affiliations

miR-124 as a Liquid Biopsy Prognostic Biomarker in Small Extracellular Vesicles from NSCLC Patients

Darío Sanchez-Cabrero et al. Int J Mol Sci. .

Abstract

Despite advances in non-small cell lung cancer (NSCLC) research, this is still the most common cancer type that has been diagnosed up to date. microRNAs have emerged as useful clinical biomarkers in both tissue and liquid biopsy. However, there are no reliable predictive biomarkers for clinical use. We evaluated the preclinical use of seven candidate miRNAs previously identified by our group. We collected a total of 120 prospective samples from 88 NSCLC patients. miRNA levels were analyzed via qRT-PCR from tissue and blood samples. miR-124 gene target prediction was performed using RNA sequencing data from our group and interrogating data from 2952 NSCLC patients from two public databases. We found higher levels of all seven miRNAs in tissue compared to plasma samples, except for miR-124. Our findings indicate that levels of miR-124, both free-circulating and within exosomes, are increased throughout the progression of the disease, suggesting its potential as a marker of disease progression in both advanced and early stages. Our bioinformatics approach identified KPNA4 and SPOCK1 as potential miR-124 targets in NSCLC. miR-124 levels can be used to identify early-stage NSCLC patients at higher risk of relapse.

Keywords: NSCLC; liquid biopsy; miR-124; sEVs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Cumulative survival in terms of relapse and exitus in NSCLC patients with a follow-up of 60 months in early stages and 34 months in advanced stages: (A) Early-stage patients. N = 16. (B) Advanced-stage patients. N = 51.
Figure 2
Figure 2
Candidate miRNA levels in tumor tissue (T), adjacent non-tumor tissue (NT), and serum (Cir) samples. The relative levels of miR-7, miR-148, miR-10a, miR-335, miR-124, miR-9, and miR-132 measured via qRT-PCR and calculated using 2−ΔCt are represented. Box-plot graph shows the median with quartile Q1 and Q3. Error bars represent the minimum and maximum values. Mean comparison using Wilcoxon test. *: p < 0.05; **: p < 0.005; NS: not significant.
Figure 3
Figure 3
Association between the expression levels of the miRNAs and relapse or exitus in 16 early-stage NSCLC patients. (A) Comparison of miRNA levels in tissue and miRNAs with relapse event. (B) Comparison of miRNA levels in tissue with exitus event. (C) Comparison of miRNA levels in circulation with relapse event. (D) Comparison of miRNA levels in circulation with exitus event. Bar-plot graphs show the confidence levels (1-p-value) of the mean comparison Mann–Whitney U test between the no-event group vs. the event group: NR: no relapse, R: relapse, NE: no exitus, E: exitus. The p-value of significant analyses and the association of miRNA expression with risk are indicated. The dotted line indicates 95% confidence, i.e., p-value = 0.05. Dot-plot graphs show the relative expression (mean and SD) measured via qRT-PCR and calculated using 2−ΔCt of significant analyses.
Figure 4
Figure 4
Implications of miR-124- and miR-132-like liquid biopsy markers in advanced-stage NSCLC disease. (A) Comparison of relative expression detection levels of miR-124 and miR-132 in free-circulating serum samples from early stages (Es); EV plasma samples of another early-stage cohort and of advanced stages (As). These data were measured via qRT-PCR and calculated using 2-ΔCt. Mean comparison via Student’s t or Mann–Whitney U test. Kaplan–Meier survival analysis in 51 advanced-stage NSCLC samples using the mean of the expression levels of miR-124 (B) and miR-132 (C) as the cut-off point for the subgroups. Terms of relapse (progression-free survival, PFS) and exitus (overall survival, OS) are represented, and a p-value < 0.05 was considered to be significant. *: p < 0.05; **: p < 0.005; ***: p < 0.001. HR: hazard ratio.
Figure 5
Figure 5
In silico identification of miR-124 target genes with potential implications in NSCLC. (A) Bioinformatic analysis workflow to discover KPNA4 and SPOCK1 as potential target genes of miR-124. (B,C) In silico survival analysis of KPNA4 (B) and SPOCK1 (C) expression in two cohorts, Kaplan–Meier plotter (left, n = 1925), and TCGA (right, n = 1027) of NSCLC patients in terms of progression-free survival (PFS), disease-free survival (DFS), and overall survival (OS).
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