Identification of microRNAs Involved in mTOR Nutrient Signaling during Adipocyte Differentiation

Abstract

Aims: In this study, we specifically aimed at identifying distinct miRNAs that are governing nutrient-driven adipogenesis via the mTOR pathway. Methodology: The murine pre-adipocyte cell line 3T3-L1 was differentiated in the presence and absence of mTOR inhibitor rapamycin. MiRNA profiles were generated using two different array platforms. Differential regulated miRNAs were subjected to in silico target prediction. Candidate miRNA-target pairs were studied via luciferase assays, transfection assays and functional assays. Results: This study confirmed 31 previously known and revealed 7 additional miRNAs, namely miR-34a, -96, -106b, -148b, -214*, ‑678 and -690 as being involved in adipocyte differentiation. Furthermore, we identified a subset of 16 miRNAs showing both responsiveness to rapamycin and being predicted to target genes within the mTOR pathway. Within mTOR signaling, the novel miRNA-target pairs miR183-Tsc1 (tuberous sclerosis 1), miR378-Tsc1 and miR103-Pik3r1 (phosphatidylinositol 3-kinase regulatory subunit alpha), were confirmed via luciferase-based binding assays. Accordingly we observed down-regulation of Tsc1 on transcript level in cells transfected with miR-183 or -378 (18.3 % P = .004 and 40.4 % P < .0001). However, on protein level we could detect Tsc1 up-regulation (35.7 % and 39.8 %), which is consistent with an inhibitory influence on mTOR signaling (33.6 % and 32.9 %) and on adipocyte differentiation of these miRNAs. Conclusion: Our findings reveal a novel role for a set of miRNAs during adipocyte differentiation and mTOR signaling. Especially the attenuating effect of miR-183 and -378 on adipocyte differentiation and mTOR signaling suggests their potential as biomarkers for nutrient-driven dysregulation of adipogenesis.