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Abstract
DUSP9 belongs to the family of protein phosphatases that negatively regulate MAP kinases. A significant decrease in the level of DUSP9 mRNA in clinical kidney carcinoma (KC) samples compared to normal tissue was first reported by Cheburkin and co-authors in 2002 and than confermed by many other research groups. We showed that suppression of mRNA expression of DUSP9 occurs already in the early stages of KC and that inactivation of DUSP9 in KC can be regulated at the epigenetic level and, moreover, differentially, depending on the gender of the patient.
The aim of this work was to study the effect of exogenous expression of DUSP9 on proliferation and migration of human KC cells.
Materials and methods. DUSP9-negative human KC cell line ACHN was used in the work. The transfection of cells was carried out by an expression plasmid constructed by us, which contained cDNA of DUSP9 and EGFP genes. Transfection efficiency was evaluated by immunofluorescence and Western-blot. The number of viable cells was assessed by MTT-test and the rate of cell migration by Scratch-wound assay. Extracellular vesicles from cell culture conditioned medium were isolated using SubXTM technology and characterized by nanoparticle tracking analysis and Western-blot.
Results. Transfection efficiency varied from 70% to 80%. The metabolic activity of the cells expressing DUSP9 and the cells transfected by the vector without insertion differed slightly 48 hours after transfection. The expression of DUSP9 significantly decreased the rate of cell migration compared to the control. DUSP9 protein was detected in the exosomal fraction of serum-free conditioned medium from the cells transfected by the DUSP9-expressing vector.
Conclusions. Using the ACHN cell line as a model of kidney carcinoma, we have shown that exogenous expression of DUSP9 slows down cell migration. The result indicates that one of the possible roles of DUSP9 in renal carcinogenesis may be the regulation of the metastasis process.
References
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