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MicroRNA-25 Modulates Proliferative Signaling Via the Mammalian Target of Rapamycin Pathway in Asthmatic Airway Smooth Muscle Cells In Vitro
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A5810 - MicroRNA-25 Modulates Proliferative Signaling Via the Mammalian Target of Rapamycin Pathway in Asthmatic Airway Smooth Muscle Cells In Vitro
Author Block: M. A. Hernandez, M. A. Ba, K. Hernandez, E. Hernandez, M. Mendoza, J. Evasovic, C. A. Singer; Pharmacology, University of Nevada, Reno School of Medicine, Reno, NV, United States.
Rationale. Bioinformatic analyses predict that microRNA-25 (miR-25) targets the 3’ untranslated region (UTR) of tuberous sclerosis complex 1 (TSC1), an upstream repressor of mammalian target of rapamycin complex 1 (mTORC1). Extensive studies in cancer cells have shown that rapamycin, an inhibitor of mTORC1, attenuates cell proliferation. Previous data from our lab establishes that miR-25 is down-regulated in airway smooth muscle (ASM) cells in vitro upon receiving a pro-inflammatory stimulus. Inhibition of miR-25 promotes ASM cell proliferation and correlates with enhanced Akt phosphorylation in vitro. Our laboratory has further demonstrated that miR-25 expression was inversely correlated with TSC1 transcript levels in the trachea of a mouse model of smooth muscle-targeted miR-25 expression (TgSM-miR25). Therefore, these experiments test the hypothesis that miR-25 targets TSC1 to attenuate dysregulated ASM cell proliferation via mTOR signaling in asthmatic ASM.
Methods. Asthmatic and non-asthmatic ASM cells were grown in culture. RNA and protein were extracted from proliferating, 2-day and 7-day growth arrested cells. The role of miR-25 expression on TSC1, mTORC1 and S6 Kinase, a downstream effector of the mTOR pathway, was assessed via qPCR and western blot analyses.
Results. Expression of miR-25 was down-regulated by ~70% in proliferating asthmatic ASM cells, and inversely correlated with TSC1 mRNA and protein levels. Phosphorylation of mTOR (p. mTOR) at the serine-2448 (Ser-2448) repressor site was reduced in asthmatic ASM cells, while P70S6K phosphorylation was increased in proliferating and 7-day growth arrested asthmatic ASM cells. There was also reduced Raptor expression in asthmatic ASM cells, indicative of increased mTORC1 activity in asthmatic ASM cells.
Conclusions. Together, these data suggest that mTOR activity is augmented in asthmatic airway ASM cells due to reduced Ser-2448 phosphorylation. The steady P70S6K phosphorylation levels between 7-day growth arrested asthmatic ASM cells and proliferating control supports the idea that not all asthmatic ASM cells reach mature differentiated phenotype, and remain in a proliferative state. Further experiments will test the effect of miR-25 expression on the mTORC1 signaling pathway. These studies will clarify the role of miR-25 on proliferative targets during asthma pathogenesis, advancing the establishment of miR-25 as a novel therapeutic candidate for asthma.
Author Block: M. A. Hernandez, M. A. Ba, K. Hernandez, E. Hernandez, M. Mendoza, J. Evasovic, C. A. Singer; Pharmacology, University of Nevada, Reno School of Medicine, Reno, NV, United States.
Rationale. Bioinformatic analyses predict that microRNA-25 (miR-25) targets the 3’ untranslated region (UTR) of tuberous sclerosis complex 1 (TSC1), an upstream repressor of mammalian target of rapamycin complex 1 (mTORC1). Extensive studies in cancer cells have shown that rapamycin, an inhibitor of mTORC1, attenuates cell proliferation. Previous data from our lab establishes that miR-25 is down-regulated in airway smooth muscle (ASM) cells in vitro upon receiving a pro-inflammatory stimulus. Inhibition of miR-25 promotes ASM cell proliferation and correlates with enhanced Akt phosphorylation in vitro. Our laboratory has further demonstrated that miR-25 expression was inversely correlated with TSC1 transcript levels in the trachea of a mouse model of smooth muscle-targeted miR-25 expression (TgSM-miR25). Therefore, these experiments test the hypothesis that miR-25 targets TSC1 to attenuate dysregulated ASM cell proliferation via mTOR signaling in asthmatic ASM.
Methods. Asthmatic and non-asthmatic ASM cells were grown in culture. RNA and protein were extracted from proliferating, 2-day and 7-day growth arrested cells. The role of miR-25 expression on TSC1, mTORC1 and S6 Kinase, a downstream effector of the mTOR pathway, was assessed via qPCR and western blot analyses.
Results. Expression of miR-25 was down-regulated by ~70% in proliferating asthmatic ASM cells, and inversely correlated with TSC1 mRNA and protein levels. Phosphorylation of mTOR (p. mTOR) at the serine-2448 (Ser-2448) repressor site was reduced in asthmatic ASM cells, while P70S6K phosphorylation was increased in proliferating and 7-day growth arrested asthmatic ASM cells. There was also reduced Raptor expression in asthmatic ASM cells, indicative of increased mTORC1 activity in asthmatic ASM cells.
Conclusions. Together, these data suggest that mTOR activity is augmented in asthmatic airway ASM cells due to reduced Ser-2448 phosphorylation. The steady P70S6K phosphorylation levels between 7-day growth arrested asthmatic ASM cells and proliferating control supports the idea that not all asthmatic ASM cells reach mature differentiated phenotype, and remain in a proliferative state. Further experiments will test the effect of miR-25 expression on the mTORC1 signaling pathway. These studies will clarify the role of miR-25 on proliferative targets during asthma pathogenesis, advancing the establishment of miR-25 as a novel therapeutic candidate for asthma.
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