The oncomir face of microRNA-206: A permanent miR-206 transfection study

Abstract

MiR-206 is a remarkable miRNA because it functions as a suppressor miRNA in rhabdomyosarcoma while at the same time, as previously showed, it can act as an oncomiRNA in SMARCB1 immunonegative soft tissue sarcomas. The aim of this study was to investigate the effect of miR-206 on its several target genes in various human tumorous and normal cell lines. In the current work, we created miR-206-overexpressing cell lines (HT-1080, Caco2, iASC, and SS-iASC) using permanent transfection. mRNA expression of the target genes of miR-206 (SMARCB1, ACTL6A, CCND1, POLA1, NOTCH3, MET, and G6PD) and SMARCB1 protein expression were examined with quantitative real-time polymerase chain reaction, immunoblotting, immunocytochemistry, and flow cytometry. MiRNA inhibition was used to validate our results. We found a diverse silencing effect of miR-206 on its target genes. While an overall tendency of downregulation was noted, expression profiles of individual cell lines showed large variability. Only CCND1 and MET were consistently downregulated. MiR-206 had an antiproliferative effect on a normal human fibroblast cell line. A strong silencing effect of SMARCB1 in miR-206 transfected SS-iASC was most likely caused by the synergic influence of the SS18-SSX1 fusion protein and miR-206. In the same cell line, a moderate decrease of SMARCB1 protein expression could be observed with immunocytochemistry and flow cytometry. In the most comprehensive analysis of miR-206 effects so far, a modest but significant downregulation of miR-206 targets on the mRNA level was confirmed across all cell lines. However, the variability of the effect shows that the action of this miRNA is largely cell context-dependent. Our results also support the conception that the oncomiR effect of miR-206 on SMARCB1 plays an important but not exclusive role in SMARCB1 immunonegative soft tissue sarcomas so it can be considered important in planning the targeted therapy of these tumors in the future. Impact statement Mir-206 is a very unique microRNA because it can act as a suppressor miRNA or as an oncomiRNA depending on the tumor tissue. In SMARCB1 negative soft tissue sarcomas miR-206 is overexpressed, so thus in epithelioid and synovial sarcomas it functions as an oncomiRNA. MiR-206 has diverse silencing effects on its target genes. We found that the action of miR-206 is largely cell context dependent. The oncomiR role of miR-206 is crucial but not exclusive in SMARCB1 negative soft tissue sarcomas and miR-206 has an antiproliferative effect on a normal human fibroblast cell line. Expressions of miR-206 targets observed in tumors can only be reproduced in the corresponding tumorous cell lines. This is the first study which examined the permanent effect of miR-206 on its target genes in normal, tumor, and genetically engineered cell lines.

Keywords: SMARCB1; epigenetic regulation; miR-206; permanent microRNA transfection; relative gene expression; synovial sarcoma.

Figure 1.

Classical hematoxylin and eosin (H&E) staining (a) and SMARCB1 immunhistochemistry (b) of a biphasic synovial sarcoma. Epithelial components show positive reaction with SMARCB1 immunostaining, while the spindle cell components are negatively stained (b). miR-206 is highly overexpressed (37,66-fold) in the spindle cell components compared to the epithelial components, measured with q-RT-PCR. Relative miR-206 level was normalized to endogenous RNU6B. As calibrator, normal liver tissue was used (c). (A color version of this figure is available in the online journal.)

Figure 2.

Images of SS-iASC-206 as observed under inverted phase contrast microscope (a) and fluorescence microscope (b). Efficiency of permanent miR-206 transfection was determined after 24 h. GFP (green fluorescent protein) signals were detectable in 75–80% of the permanently transfected cells. (A color version of this figure is available in the online journal.)

Figure 3.

Expression levels of miR-206 target genes. (a) The left column charts represent the SMARCB1, ACTL6A, CCND1, POLA1, NOTCH3, SNAI1, MET, and G6PD mRNA expression in the miR-206 transfected SS-iASC-206 and iASC-206 cell lines. (b) The right column charts illustrate these gene expressions after inhibiting miR-206. Bars represent means ± SD; *P < 0.05.

Figure 4.

Expression levels of miR-206 target genes. (a) The left column charts represent the SMARCB1, ACTL6A, CCND1, POLA1, NOTCH3, SNAI1, MET, and G6PD mRNA expression in the miR-206-transfected Caco2–206 and HT-1080–206 cell lines. (b) The right column charts illustrate these gene expressions after inhibiting miR-206. Bars represent means ± SD; *P < 0.05.

Figure 5.

SMARCB1 immunocytochemistry and flow cytometry analysis of SS-iASC and SS-iASC-206. (a) Positive SMARCB1 staining of nuclei of not transfected SS-iASC cell line. (b and c) Between the strongly SMARCB1-immunopositive nuclei, decreased SMARCB1 protein expression was observed in individual nuclei of SS-iASC-206; (*) marks the decreased nuclear SMARCB1 expression. (d) Measurement of SMARCB1 expression by flow cytometry: gating strategy on the living cells using forward and side scattered light scattergram. (e) Fluorescence intensity of SMARCB1 in SS-iASC-206 and SS-iASC cell populations labeled with Alexa Flour® 647 conjugated goat anti-mouse IgG. The red histogram demonstrates miR-206 transfected SS-iASC-206, while the blue histogram shows the not transfected SS-iASC population. (A color version of this figure is available in the online journal.)