Effect of immunosuppression in miRNAs from extracellular vesicles of colorectal cancer and their influence on the pre-metastatic niche

Abstract

Colorectal cancer (CRC) occurs with more aggressiveness in kidney transplant recipients compared to the general population. Immunosuppressive therapy plays a crucial role in the development of post-transplant malignancy. Concretely, cyclosporine A (CsA) has intrinsic pro-oncologic properties, while several studies report a regression of cancer after the introduction of rapamycin (RAPA). However, their effect on the extracellular vesicle (EV) content from CRC cell lines and their relevance in the pre-metastatic niche have not yet been studied. Here, we investigated the effect of RAPA and CsA in EV-miRNAs from metastatic and non-metastatic CRC cell lines and the role of relevant miRNAs transferred into a pre-metastatic niche model. EV-miRNA profiles showed a significant upregulation of miR-6127, miR-6746-5p, and miR-6787-5p under RAPA treatment compared to CsA and untreated conditions in metastatic cell lines that were not observed in non-metastatic cells. From gene expression analysis of transfected lung fibroblasts, we identified 22 shared downregulated genes mostly represented by the histone family involved in chromatin organization, DNA packaging, and cell cycle. These results suggest that EV-miR-6127, miR-6746-5p and miR-6787-5p could be a potential epigenetic mechanism induced by RAPA therapy in the regulation of the pre-metastatic niche of post-transplant colorectal cancer.

Figure 1

Characterization of extracellular vesicles from HCT116 and SW480 cell lines under rapamycin and cyclosporine A. Characterization of EVs released by HCT116 and SW480 CRC cell lines. (a,c) NTA measurement shows the concentration and size distribution of (a) HCT116EVs and (c) SW480EVs. (b,d) Images from cryo-electron microscopy of purified (b) HCT116EVs and (d) SW480EVs (scale bars 0.2 and 0.1 μm. (e) Bead-based flow cytometry analysis of HCT116EVs and SW480EVs stained with EV markers: CD9, CD81, and CD63. (f,g) EV production in Untr, RAPA, and CsA treatment was calculated as EVs/ml per total cells in HCT116 and SW480. Data are expressed as the mean ± SD. (n = 4). *P < 0.05 versus Untr (untreated) by Student’s t-test.

Figure 2

Differential expression of EV-miRNAs in HCT116 compared to SW480 under rapamycin and cyclosporine A. (a) Heat map of miRNA analysis. miRNA array analysis revealed six miRNAs significantly upregulated and downregulated under RAPA and CsA treatment compared to untreated cells in HCT116, respectively. No significant expression was observed in SW480 under the same conditions. Colour intensity levels indicate FC (from −4.2 to 6.9). (b) Heat map of Gene Ontology (GO) biological process enrichment of miR-6127, miR-6746-5p and miR-6787-5p. Gene Ontology displayed only results with false discovery rate < 0.05. Colour intensity levels indicate fold enrichment (from 1.7 to 1.2). FC = fold change.

Figure 3

Validation of differentially expressed miRNAs in HCT116 and SW480 cell lines and their EVs with qRT-PCR. (a) miR-6127, miR-6746-5p, and miR-6787 relative expression in HCT116EVs and SW480EVs was normalized to EV production in Untr, RAPA, and CsA treatment. Data are expressed as the mean ± SD. (n = 3). *P < 0.05 versus Untr (untreated) and ≠P < 0.05 versus CsA treatment. (b) miR-6127, miR-6746-5p, and miR-6787 relative expression in HCT116 and SW480 in Untr, RAPA, and CsA treatment. Data are expressed as the mean ± SD. (n = 6). *P < 0.05 versus Untr (untreated) and ≠ P < 0.05 versus CsA by Student’s t-test.

Figure 4

Epigenetic genes are transcriptionally downregulated in lung fibroblasts (IRM90) by miR-6127, miR-6746-5p, miR-6787-5p, and miR-mix. IRM90 was transfected with miR-6127, miR-6746-5p miR-6787-5p and miR-mix for 24 h (n = 3). (a) The GO-Enrichment analysis from four lists (miR-6127, miR-6746-5p and miR-6787 and miR-mix) of downregulated genes of transfected IMR90 versus untreated. (b) Venn diagram of significantly downregulated transcript lists of miRNA transfections revealed 22 genes in common. (c) GO terms of the common genes show biological processes mainly related to epigenetic genes. GO = Gene Ontology.

Figure 5

No significant changes in the cell cycle of lung fibroblasts were induced by miR-6127, miR-6746-5p, miR-6787-5p, and miR-mix. Cell cycle profiles of IRM90 untransfected and transfected with miR-6127, miR-6746-5p, miR-6787-5p, and miR-mix for 48 h (n = 3). The x-axis represents the DNA content of the nuclear population, whereas the y-axis identifies the events. The percentage of cells present in G0/G1, S, and G2/M phases are shown.