Project description:The present study focused on establishing the role of microRNA-139-5p (miRNA-139-5p) in the phenotypic expression of basal tone in rat internal anal sphincter (IAS) vs. lack of tone in truly phasic smooth muscle of anococcygeus (ASM), via RhoA-associated kinase (RhoA/ROCK2).
Project description:A comprehensive –omic, computational, and physiological approach was employed to examine the (previously unexplored) role of microRNAs (miRNAs) as regulators of IAS smooth muscle contractile phenotype and basal tone. MicroRNA profiling, genome wide expression, validation and network analyses were employed to assess changes in mRNA and miRNA expression in IAS smooth muscles from young vs. aging rats. Multiple miRNAs, including rno-miR-1, rno-miR-340-5p, rno-miR-185, rno-miR-199a-3p, rno-miR-200c, rno-miR-200b, rno-miR-31, rno-miR-133a and rno-miR-206 were found to be up-regulated in aging IAS. qRT-PCR confirmed the up-regulated expression of these miRNAs and down regulation of multiple, predicted targets (Eln, Col3a1, Col1a1, Zeb2, Myocd, SRF, Smad1, Smad2, RhoA/ROCK2, Fn1, Sm22-v2, Klf4, and Acta2) involved in regulation of SM contractility. Subsequent studies demonstrated an aging-associated increase in the expression of miR-133a, corresponding decreases in RhoA, ROCK2, MYOCD, SRF and SM22α protein expression, RhoA-signaling, and a decrease in basal and agonist (U-46619 (thromboxane A2 analog))-induced increase in the IAS tone. Moreover, in vitro transfection of miR-133a caused a dose-dependent increase of IAS tone in strips, which was reversed by anti-miR-133a. Lastly, in vivo perianal injection of anti-miR-133a reversed the loss of IAS tone associated with age. This work establishes the important regulatory effect of miRNA-133a on basal and agonist-stimulated IAS tone. Moreover, reversal of age-associated loss of tone via anti-miR delivery strongly implicates miR dysregulation as a causal factor in the aging-associated decrease in IAS tone, and suggests miR-133a is feasible therapeutic target in aging-associated rectoanal incontinence.
Project description:Aging causes a functional decline in tissues throughout the body that may be delayed by caloric restriction (CR). However, the cellular profiles and signatures of aging, as well as those ameliorated by CR, remain unclear. Here, we built comprehensive single-cell and single-nucleus transcriptomic atlases across various rat tissues undergoing aging and CR. CR attenuated aging-related changes in cell type composition, gene expression, and core transcriptional regulatory networks. Immune cells were increased during aging, and CR favorably reversed the aging-disturbed immune ecosystem. Computational prediction revealed that the abnormal cell-cell communication patterns observed during aging, including the excessive proinflammatory ligand-receptor interplay, were reversed by CR. Our work provides multi-tissue single-cell transcriptional landscapes associated with aging and CR in a mammal, enhances our understanding of the robustness of CR as a geroprotective intervention, and uncovers how metabolic intervention can act upon the immune system to modify the process of aging.
