title: Goat activin receptor type IIB knockdown by muscle specific promoter driven artificial microRNAs.
abstract: Activin receptor type IIB (ACVR2B) is a transmembrane receptor which mediates signaling of TGF beta superfamily ligands known to function in regulation of muscle mass, embryonic development and reproduction. ACVR2B antagonism has shown to enhance the muscle growth in several disease and transgenic models. Here, we show ACVR2B knockdown by RNA interference using muscle creatine kinase (MCK) promoter driven artificial microRNAs (amiRNAs). Among the various promoter elements tested, the ∼1.26 kb MCK promoter region showed maximum transcriptional activity in goat myoblasts cells. We observed up to 20% silencing in non-myogenic 293T cells and up to 32% silencing in myogenic goat myoblasts by MCK directed amiRNAs by transient transfection. Goat myoblasts stably integrated with MCK directed amiRNAs showed merely 8% silencing in proliferating myoblasts which was increased to 34% upon induction of differentiation at transcript level whereas up to 57% silencing at protein level. Knockdown of ACVR2B by 5'-UTR derived amiRNAs resulted in decreased SMAD2/3 signaling, increased expression of myogenic regulatory factors (MRFs) and enhanced proliferation and differentiation of myoblasts. Unexpectedly, knockdown of ACVR2B by 3'-UTR derived amiRNAs resulted in increased SMAD2/3 signaling, reduced expression of MRFs and suppression of myogenesis. Our study offers muscle specific knockdown of ACVR2B as a potential strategy to enhance muscle mass in the farm animal species.
authors.affiliation: Department of Animal Biotechnology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand 388001, India.
authors.forename: Amrutlal K
authors.initials: AK
authors.lastname: Patel
authors.affiliation: Department of Animal Biotechnology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand 388001, India.
authors.forename: Ravi K
authors.initials: RK
authors.lastname: Shah
authors.affiliation: Department of Animal Biotechnology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand 388001, India.
authors.forename: Utsav A
authors.initials: UA
authors.lastname: Patel
authors.affiliation: Department of Pathology, Case Western Reserve University, Cleveland, OH, USA.
authors.forename: Ajai K
authors.initials: AK
authors.lastname: Tripathi
authors.affiliation: Department of Animal Biotechnology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand 388001, India. Electronic address: cgjoshi@rediffmail.com.
authors.forename: Chaitanya G
authors.initials: CG
authors.lastname: Joshi
mesh_terms: D030301:Activin Receptors, Type II / Q000235:genetics*; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D002478:Cells, Cultured; D055785:Gene Knockdown Techniques / Q000379:methods*; D006041:Goats / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics; D009132:Muscles / Q000378:metabolism*; D032446:Myoblasts; D011401:Promoter Regions, Genetic / Q000235:genetics*
chemical_list: D035683:MicroRNAs; D030301:Activin Receptors, Type II; C425584:activin receptor type II-B
keywords: ACVR2B; Artificial microRNAs; Goat myoblasts; Muscle specific promoter; RNA interference