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. 2022 Apr 4;12:852952. doi: 10.3389/fonc.2022.852952

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Copyright © 2022 Moubarak, Koetz-Ploch, Mullokandov, Gaziel, de Pablos-Aragoneses, Argibay, Kleffman, Sokolova, Berwick, Thomas, Osman, Brown and Hernando

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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miR-124a overexpression inhibits established metastases. (A) The 113/6-4L melanoma cells were transduced with GFP-Luciferase followed by TRIPZ-Scr or TRIPZ-miR-124a lentiviral infections. Efficient miRNA-124 overexpression after doxycycline treatment in vitro was assessed by RT-qPCR (unpaired t test; *****p = 0.000005). (B) Schematic representation of in vivo metastasis assay with inducible overexpression of miR-124a: The 113/6-4L-GFP-Luciferase cells transduced with doxycycline-inducible control (TRIPZ-miR-Scr) or (TRIPZ-miR-124a) tagged with a tRFP reporter were selected with puromycin, and ultrasound-guided intracardiac injection was performed in NSG mice (n = 11 per group). Mice were initiated on doxycycline (200 mg/kg) chow 8 days post-injection. (C) RFP and GFP fluorescence images of brain, lung, liver, and kidneys in all mice are shown, and the areas of metastatic lesions were plotted using ImageJ (red dots: RFP+ metastases; green dots: GFP+ metastases). Unpaired t test was used for statistical significance of differences in brain (***p = 0.0006 for RFP+ metastases in miR124a vs. miR-Scr group; ns, p = 0.274 for GFP+ in miR124a vs. miR-Scr group), lung (**p = 0.0041 for RFP+ metastases in miR124a vs. miR-Scr group; ***p = 0.00056 for GFP+ in miR124a vs. miR-Scr group), liver (*****p < 0.000001 for RFP+ metastases in miR124a vs. miR-Scr group; ns, p = 0.056 for GFP+ in miR124a vs. miR-Scr group), and kidney (***p = 0.00033 for RFP+ metastases in miR124a vs. miR-Scr group; ns, p = 0.058 for GFP+ in miR124a vs. miR-Scr group) metastases. (D) Expression of miR-124a in RFP-/GFP+ and RFP+/GFP+ liver metastatic foci dissected from two representative mice (#16 and #18) from the miR124a group is measured by quantitative RT-qPCR (unpaired t test; *p = 0.018 for mouse 16 and ***p = 0.0011 and **p = 0.005 for mouse 18). (E) Representative images of the liver and kidney sizes of two mice from both groups are shown. ns, non significant.