Abstract
BackgroundMicro RNAs (miRs) have emerged as key regulators during oncogenesis. They have been found to regulate cell proliferation, differentiation, and apoptosis. Mir-125b has been identified as an oncomir in various forms of tumours, but we have previously proposed that miR-125b is a suppressor of lymph node metastasis in cutaneous malignant melanoma. Our goal was therefore to further examine this theory.MethodsWe used in-situ-hybridization to visualise miR-125b expression in primary tumours and in lymph node metastasis. Then using a miRVector plasmid containing a miR-125b-1 insert we transfected melanoma cell line Mel-Juso and then investigated the effect of the presence of a stable overexpression of miR-125b on growth by western blotting, flow cytometry and β-galactosidase staining. The tumourogenicity of the transfected cells was tested using a murine model and the tumours were further examined with in-situ-hybridization.ResultsIn primary human tumours and in lymph node metastases increased expression of miR-125b was found in single, large tumour cells with abundant cytoplasm. A stable overexpression of miR-125b in human melanoma cell line Mel-Juso resulted in a G0/G1 cell cycle block and emergence of large cells expressing senescence markers: senescence-associated beta-galactosidase, p21, p27 and p53. Mel-Juso cells overexpressing miR-125b were tumourigenic in mice, but the tumours exhibited higher level of cell senescence and decreased expression of proliferation markers, cyclin D1 and Ki67 than the control tumours.ConclusionsOur results confirm the theory that miR-125b functions as a tumour supressor in cutaneous malignant melanoma by regulating cellular senescence, which is one of the central mechanisms protecting against the development and progression of malignant melanoma.
Highlights
Micro RNAs have emerged as key regulators during oncogenesis
In this paper we show that upregulation of Micro RNAs (miRs)-125b induces senescence and might constitute one of the possible mechanisms of the suppressive effect of miR-125b in MM
Plasmid transfection Mel-Juso cells were transfected with a miRVector plasmid containing a miR-125b-1 [UCSC Genome Bioinformatics: uc010rzr.1] [37] insert and a blasticidin resistance gene [38] or the control miRNA Vector plasmid with a blasticidin resistance gene but without any insert (Source BioScience, LifeSciences, Nottingham, UK). 300.000 cells were seeded out 24 h before transfection to reach a confluency of 70– 90%. 1 μg of the plasmid was mixed with 400 μl OPTIMEM (Invitrogen) and 5 μl Lipofectamine RNAiMax (Invitrogen)
Summary
Micro RNAs (miRs) have emerged as key regulators during oncogenesis. They have been found to regulate cell proliferation, differentiation, and apoptosis. Replicative cellular senescence is the principal mechanism limiting proliferation of normal human cells [1,2]. Activation of oncogenes provides a potent senescence signal (oncogene-induced-senescence, OIS) which is considered to be an early protective mechanism against development of cancer [3,5,6,7]. Cellular senescence may be a mechanism inhibiting cancer progression, since senescence-like state can be induced in established tumours and cancer cell lines by a variety of mechanisms including chemotherapeutic agents or radiation (therapy-induced senescence, TIS) [11,12]
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