shRNA-MEDIATED KNOCKDOWN OF 14-3-3g; REVEALS AN ESSENTIAL ROLE IN REGULATING ADIPOGENIC DIFFERENTIATION OF UCMSCs

SANTONI, D. N.; BUSTOS, D. M.; RIVERA, L.; UHART, M.

Salta

Conferencia; Joint LV AnnualSAIB Meeting and XIV PABMB Congress; 2019

SAIB-PABMB

Human umbilical cord derived-mesenchymal stem cells (UCMSCs) are self-renewing multipotent progenitors that can differentiateinto cells of the mesoderm-lineage, which makes them attractive targets for regenerative medicine. However, the lack ofunderstanding of the molecular mechanisms that regulate cell differentiation is currently an obstacle to such applications. In thissense, 14-3-3 proteins have received tremendous attention since these phospho-serine/threonine binding proteins have a pivotal rolein the regulation of metabolism and signal transduction networks. A recently published work in our lab has shown that both at themRNA and protein level, 14-3-3 β and γ were the isoforms that most changed after adipogenic differentiation of 3T3-L1, a wellestablished mourine pre-adipocyte cell line. Since we have developed a method for UCMSCs isolation and also obtained an efficientadenoviral transduction of these cells, the aim of this study was to analyze the effect of decreased expression of 14-3-3γ by usingshRNA on the adipogenic potential of UCMSCs. The recombinant adenoviruses (Adv) were E1/E3-deleted type 5 Advs expressingshRNA for 14-3-3γ, under the control of the small nuclear RNA (snRNA) U6 promoter. The Pac I-digested vector was used totransfect 293A cells to produce Adv-shRNA 14-3-3γ stock. Then, the Advs were amplified by infecting the 293A cells with the crudeviral lysate. UCMSCs were isolated and expanded from Wharton Jelly of human umbilical cord using a culture explant method. Fortransduction experiments, cells at 80-90% confluence were incubated with Adv-shRNA 14-3-3γ for 2 hs at 37oC and then thetransduction media was replaced with standard growth media (high glucose DMEM; 10% FBS). To investigate the effects of 14-3-3γin adipogenesis, we induced UCMSCs (transduced or not- with Adv-shRNA 14-3-3γ) with adipogenic differentiation medium (ADM;an optimized drug cocktail that includes high glucose DMEM, 10% FBS, Dexamethasone, Insulin, Rosiglitazone and IBMX) for 10days. We also used untreated and Adv-GFP transduced cells as controls. Lipid droplets accumulation was examined using Oil Red Ostaining. UCMSCs transduced with Adv-shRNA 14-3-3γ exhibited an increased lipid droplets accumulation, compared to controlcells. This data suggest an essential role for 14-3-3γ in the process of adipogenesis in UCMSCs. Our finding proposes the existenceof a previously unknown regulatory mechanism of UCMSCs adipogenic differentiation, and that 14-3-3γ could be an interestingcandidate to be evaluated as therapeutic target molecule to treat chronic diseases, such as obesity and type 2 diabetes.