Use of agomir and antagomir technologies to alter satellite cell proliferation in vitro, mirna expression and muscle fiber hypertrophy in intrauterine growth restricted lambs

Authors: GREENE, M. - Clemson University; WORLEY, G. - Clemson University; UDOKA, A.N. S. - Clemson University; POWELL, R. - Clemson University; BRUCE, T. - Clemson University; Klotz, James; BRIDGES, JR., W. - Clemson University; DUCKETT, S. - Clemson University

Submitted to: Frontiers in Molecular Biosciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/18/2023
Publication Date: 11/3/2023
Citation: Greene, M.A., Worley, G.A., Udoka, A., Powell, R.R., Bruce, T., Klotz, J.L., Bridges, Jr., W.C., Duckett, S.K. 2023. Use of agomir and antagomir technologies to alter satellite cell proliferation in vitro, mirna expression and muscle fiber hypertrophy in intrauterine growth restricted lambs. Frontiers in Molecular Biosciences. 10. Article 1286890. https://doi.org/10.3389/fmolb.2023.1286890.
DOI: https://doi.org/10.3389/fmolb.2023.1286890

Interpretive Summary: MicroRNA (miRNA) are a class of small non-coding RNA that regulate protein expression by binding with messenger RNA (mRNA) and either inhibit that mRNA from being translated into a protein or label it for degradation. Several miRNA have been reported to be muscle specific. Use of mimics/agomirs or inhibitors/antagomirs to overexpress or knock down expression of miRNA can alter growth and development of muscle cells in vitro. miRNA treatments have been successfully used to regulate mRNA expression in cancer treatments and has the potential to alter muscle growth, but has not been tested in livestock species. The objectives of this study were to examine the use of mimic/agomir and antagomir technologies to alter miRNA expression on muscle cell proliferation in vitro and muscle hypertrophy in vivo during early growth in lambs subjected to intra-uterine growth restriction. This work demonstrated that the use of mimic/agomir and antagomir technologies was successful in altering endogenous miRNA expression in both the in vitro and in vivo experiments; however, additional research is needed to further refine dose levels, timing of injections, and alternative methods of delivering miRNA treatments to increase muscle hypertrophy. This research will benefit other researchers interested in manipulating the mechanisms associated muscle growth and development and researchers interested in remedies to treat growth restriction at the muscle level.

Technical Abstract: Two experiments were conducted using mimic/agomiR (AGO) or antagomir (ANT) technologies to alter miRNA expression and examine change in myoblast proliferation in vitro and muscle hypertrophy in vivo. In experiment 1, several mimics (miR-127 or -299a) or anatgomiRs (miR-22-3p, -29a, -133, or -27) were transfected to alter miRNA expression and measure myoblast proliferation. AntagomiR-22-3p increased (P<0.05) myoblast proliferation compared to other antagomir treatments or controls. miR-127 mimic (100nM) mimic treatments increased (P < 0.05) myoblast proliferation compared to others or controls. For experiment 2, pregnant ewes (n = 18) were nutrient restricted during late gestation to produce intrauterine growth restricted (IUGR) lambs. On d 2 of age, female lambs were randomly assigned to one of two miRNA treatments: 1) agomiR-127 (AGO127, n =8) or 2) antagomiR-22-3p (ANT22, n = 8). miRNA treatments (AGO127 or ANT22) were injected intramuscularly on the left side of longissimus muscle (LM) every 3-d starting at the 10th rib and moving posterior by 1.27 cm at each injection for a total of 7 injections. A sham control (PBS only) was administered similarly on the right LM to serve as the in-animal control. Lambs were harvested 4 d after the last injection and samples were collected from the injection region. Data were analyzed using paired t-tests to compare each miRNA treatment to its SHAM within animal. The use of antagomiR or mimic/agomiR treatments down-regulated or up-regulated, respectively, miRNA expression for that miRNA of interest. Lamb body weights and average daily gains did not differ between AGO/SHAM127 and ANT/SHAM22. Expression of predicted target KIF3B mRNA for miR-127 was up-regulated and ACVR2a mRNA was up-regulated for miR-22-3p. ANT22 injection also up-regulated the major regulator of protein synthesis (mTOR). Proteomic analyses identified 11 proteins for AGO127 and 9 proteins for ANT22 that were that were differentially expressed. Muscle fiber type and cross-sectional area were altered for ANT22 treatments to transition fibers to a more oxidative state. The use of agomiR and antagomir technologies allow us to alter miRNA expression in vitro and in vivo to enhance myoblast proliferation and alter muscle fiber hypertrophy in IUGR lambs during early postnatal growth.