Extracellular disposal of tumor-suppressor miRs-145 and -34a via microvesicles and 5-FU resistance of human colon cancer cells

Abstract

The dysregulation of microRNA (miRNA) expression causes various kinds of diseases. Especially, alterations in miRNA expression levels are frequently observed in human tumor cells and are associated with cancer pathogenesis. Earlier we established Fluorouracil (5-FU)-resistant human colon cancer DLD-1 cells (DLD-1/5FU) from parental 5-FU- sensitive DLD-1 cells. In the present study, we examined the expression of miRNA in each cell line and in its extracellular microvesicles (MVs) before and after treatment with 5-FU. The nascent RNAs of anti-oncogenic miR-34a and -145 labeled with EU in both cells were proved to be transferred into MVs in both cell lines. The levels of miR-34a and -145 in the cells and in their MVs were not largely different in the two cell lines, and a substantial amount of both miRNAs was secreted by both cell lines even in the steady-state condition. The exposure of both cell lines to 5-FU significantly increased the intracellular levels of miR-145 and miR-34a in the 5-FU-sensitive DLD-1 cells, whereas the level of neither miR was elevated in the DLD-1/5FU cells. Interestingly, the amount of miR-145 detected in the small MVs shed into the medium of the parental cells was reduced after the treatment with 5-FU. On the other hand, the intracellular expression of miR-34a in the DLD-1/5FU cells was down-regulated compared with that in the parental DLD-1 cells even in the steady-state condition. As to the miR-34a secreted into MVs, the increase in the level in DLD-1/5FU cells was greater than that in the parental DLD-1 cells after the treatment with 5-FU. Thus, the intra- and extracellular miR-145 and -34a were closely associated with 5-FU resistance, and the resistance was in part due to the enhanced secretion of miR-145 and -34a via MVs, resulting in low intracellular levels of both miRNAs.

Figure 1.

Characteristics of microvesicles from human colon cancer DLD-1 cells as determined by Nanoparticle Tracking Analysis (NTA). MVs were isolated by centrifugation and filtered through the 0.22-μm filter of the ExoMir kit. The Nanosight LM10 nanoparticle characterization system (NanoSight, NanoSight Ltd., Amesbury, UK) equipped with blue-laser (638 nm) illumination was used for real-time characterization of the vesicles. The results are presented at the average value of two independent experiments in human colon cancer DLD-1 (A) and 5-FU resistant DLD-1/5FU (B) cells. The number of MVs (E6 particles/mL) and the size distribution (particle diameter, nm) are shown on the y axis and x axis, respectively.

Figure 2.

Tracing EU-labeled miR-145 and -34a from donor cells into MVs. Amounts of EU-labeled nascent miR-145 and -34a in DLD-1 and DLD-1/5FU cells and in their secreted MVs are shown. The relative quantities of EU-labeled miRNA are given, with the amount of the expression in DLD-1 cells indicated as “1”. The results are presented as the average value of three independent experiments. MV(L) and MV(S) are MVs filtered through the 1st filter and 2nd filter, respectively, of the ExoMir kit.

Figure 3.

Detection of miR-145 levels in DLD-1 and DLD-1/5FU cells and in their MVs. Levels of miR-145 in the cells (the upper panel) and in their MVs (the lower panel) trapped by the 1st (L) and 2nd (S) filters of the ExoMir kit. The upper panel shows the relative expression of miRNA in the cells before and after the treatment with 5-FU (10 μM) for 48 h; and the relative quantities are given relative to the amount in DLD-1 cells indicated as “1”, as in the case of MVs. The results are presented as the average value of three independent experiments. MV(L) and MV(S) were MVs filtered through 1st filter and 2nd filter, respectively, of the ExoMir kit. “+”: treatment with 5-FU; “−”: treatment with DMSO alone. * p < 0.05 is considered to be significant.

Figure 4.

Detection of miR-34a levels in DLD-1 and DLD-1/5FU cells and in their MVs. Levels of miR-34a in the cells and in the MVs trapped by the two filters of the ExoMir kit. The upper panel shows the relative expression of miRNAs before and after the treatment with 5-FU (10 μM) for 48 h, with the quantities given being relative to the amount in DLD-1 cells indicated as “1”. Lower panel shows the relative expression of the miRNAs in MVs from DLD-1 and DLD-1/5FU cells before and after the treatment with 5-FU (10 μM) for 48 h. The results are presented at the average value of three independent experiments. “+”: treatment with 5-FU; “−”: treatment with DMSO alone.* p < 0.05 is considered to be significant.

Figure 5.

Schematic diagram of machinery for drug resistance in DLD-1/5FU cells.