title: Identification of a microRNA (miR-663a) induced by ER stress and its target gene PLOD3 by a combined microRNome and proteome approach.
abstract: MicroRNAs (miRs) regulate gene expression to support important physiological functions. Significant evidences suggest that miRs play a crucial role in many pathological events and in the cell response to various stresses.
  
  
  
  With the aim to identify new miRs induced by perturbation of intracellular calcium homeostasis, we analysed miR expression profiles of thapsigargin (TG)-treated cells by microarray. In order to identify miR-663a-regulated genes, we evaluated proteomic changes in miR-663a-overexpressing cells by two-dimensional differential in-gel electrophoresis coupled to mass spectrometric identification of the differentially represented proteins. Microarray and proteomic analyses were supported by biochemical validation.
  
  
  
  Results of microarray revealed 24 differentially expressed miRs; among them, miR-663a turned out to be by ER stress and under the control of the PERK pathway of the unfolded protein response. Proteomic analysis revealed that PLOD3, which is the gene encoding for collagen-modifying lysyl hydroxylase 3 (LH3), is regulated by miR-663a. Luciferase reporter assays demonstrated that miR-663a indeed reduces LH3 expression by targeting to 3'-UTR of PLOD3 mRNA. Interestingly, miR-663a inhibition of LH3 expression generates reduced extracellular accumulation of type IV collagen, thus suggesting the involvement of miR-663a in modulating collagen 4 secretion in physiological conditions and in response to ER stress.
  
  
  
  The finding of the ER stress-induced PERK-miR-663a pathway may have important implications in the understanding of the molecular mechanisms underlying the function of this miR in normal and/or pathological conditions.
authors.affiliation: Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, 84034, Fisciano, Salerno, Italy.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Amodio
authors.affiliation: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, 80131, Naples, Italy.|CEINGE Biotecnologie Avanzate, 80145, Naples, Italy.
authors.forename: Emanuele
authors.initials: E
authors.lastname: Sasso
authors.affiliation: Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147, Naples, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: D'Ambrosio
authors.affiliation: Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147, Naples, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Scaloni
authors.affiliation: Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, 84034, Fisciano, Salerno, Italy.
authors.forename: Ornella
authors.initials: O
authors.lastname: Moltedo
authors.affiliation: Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, 84034, Fisciano, Salerno, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Franceschelli
authors.affiliation: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, 80131, Naples, Italy.|CEINGE Biotecnologie Avanzate, 80145, Naples, Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: Zambrano
authors.affiliation: Dipartimento di Medicina, Chirurgia e Odontoiatria "Scuola Medica Salernitana", Università degli Studi di Salerno, 84034, Baronissi, Salerno, Italy. premondelli@unisa.it.|Dipartimento di Medicina e Chirurgia, Università degli Studi di Salerno, Via Salvador Allende, I-84034, Baronissi, Salerno, Italy. premondelli@unisa.it.
authors.forename: Paolo
authors.initials: P
authors.lastname: Remondelli
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000235:genetics; D045744:Cell Line, Tumor; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000235:genetics*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology / Q000235:genetics; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000235:genetics* / Q000378:metabolism; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D013312:Stress, Physiological / Q000235:genetics; D059467:Transcriptome; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism
chemical_list: C552243:MIRN663 microRNA, human; D035683:MicroRNAs; D020543:Proteome; C475767:PLOD3 protein, human; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; C460640:EIF2AK3 protein, human; D019892:eIF-2 Kinase
keywords: ER stress; Lysyl hydroxylase 3 (LH3); MicroRNA; Mir 663a; PLOD3; Proteomics