Plasma Exosomal miRNA Expression Profile as Oxaliplatin-Based Chemoresistant Biomarkers in Colorectal Adenocarcinoma

Abstract

Background: Chemotherapy is one of the most common therapies used in the treatment of colorectal cancer (CRC), but chemoresistance inevitably occurs. It is challenging to obtain an immediate and accurate diagnosis of chemoresistance. The potential of circulating exosomal miRNAs as oxaliplatin-based chemoresistant biomarkers in CRC patients was investigated in this study. Methods: Plasma exosomal miRNAs in sensitive and resistant patients were analyzed by miRNA microarray analysis, followed by verification with a quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assay in two independent cohorts. The diagnostic accuracy was determined by ROC curve analysis. Logistic regression analysis and Spearman's rank correlation test were also performed. Finally, bioinformatics was used to preliminarily explore the potential molecular mechanism of the selected miRNAs in chemoresistance. Results: miRNA microarray analysis identified four upregulated miRNAs and 20 downregulated miRNAs in chemoresistant patients compared to chemosensitive patients. Twelve markedly dysregulated miRNAs were selected for further investigation, of which six (miR-100, miR-92a, miR-16, miR-30e, miR-144-5p, and let-7i) were verified to be significantly and consistently dysregulated (>1.5-fold, P < 0.05). The combination of the six miRNAs had the highest AUC (0.825, 95% CI, 0.753-0.897). The expression level of these 6 miRNAs was not correlated with tumor location, stage, or chemotherapy program. Only miR-100 was significantly upregulated in low histological grade. GO analysis and KEGG pathway analysis showed that miRNAs were related to RNA polymerase II transcription and enriched in the PI3K-AKT signaling pathway, AMPK signaling pathway, and FoxO signaling pathway. Conclusions: We identified a panel of plasma exosomal miRNAs, containing miR-100, miR-92a, miR-16, miR-30e, miR-144-5p, and let-7i, that could significantly distinguish chemoresistant patients from chemosensitive patients. The detection of circulating exosomal miRNAs may serve as an effective way to monitor CRC patient responses to chemotherapy. Targeting these miRNAs may also be a promising strategy for CRC treatment.

Keywords: chemoresistance; circulating biomarkers; colorectal cancer; exosome; miRNA profile.

Figure 1

Characterization of exosomes derived from the plasma of chemoresistant and chemosensitive CRC patients. (A) The morphology and size of exosomes by TEM. (B) The size distribution of exosomes, analyzed by NTA. (C) Western blots of the exosomal markers: CD9, Alix, and TSG101. Rs, Resistance; Rp, Response.

Figure 2

An overview of the experimental design.

Figure 3

Heat map showing the significantly upregulated and downregulated miRNAs in the resistance group compared to the response group.

Figure 4

The relative expression of plasma exosomal miRNAs in the control CRC group (n = 47), response group (n = 72), and resistance group (n = 67). (A) Relative expression of plasma exosomal miR-184. (B) Relative expression of plasma exosomal miR-100. (C) Relative expression of plasma exosomal miR-10a. (D) Relative expression of plasma exosomal miR-92a. (E) Relative expression of plasma exosomal miR-16. (F) Relative expression of plasma exosomal miR-30e. (G) Relative expression of plasma exosomal miR-144-5p. (H) Relative expression of plasma exosomal let-7i. Statistics were analyzed by one-way ANOVA, data was shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

Receiver operating curves (ROC) of selected miRNAs for differentiation between responsive patients and resistant patients based on the plasma exosomal miRNA profile.

Figure 6

GO and KEGG pathway analyses of downstream target genes of two upregulated miRNAs. (A-C): GO analysis. (A) Biological processes. (B) Cellular component. (C) Molecular function. (D) The top ten KEGG pathways enriched in the target genes. (E) The analysis of Focal adhesion, FoxO signaling pathway, and PI3K-Akt signaling pathway. (F) The identified target gene PTEN was measured by Western blot in CRC cell lines and tissues. P_S: chemosensitive CRC patients. P_R: chemoresistant CRC patients.

Figure 7

GO and KEGG pathway analyses of downstream target genes of four downregulated miRNAs. (A-C): GO analysis. (A) Biological processes. (B) Cellular component. (C) Molecular function. (D) The top ten KEGG pathways enriched in the target genes. (E) The analysis of PI3K-Akt signaling pathway, MAPK signaling pathway, and pathways in cancer. (F) The identified target gene KRAS was measured by Western blot in CRC cell lines and tissues. P_S: chemosensitive CRC patients. P_R: chemoresistant CRC patients.