title: Knockdown of Drosha in human alveolar type II cells alters expression of SP-A in culture: a pilot study.
abstract: Human surfactant protein A (SP-A) plays an important role in surfactant metabolism and lung innate immunity. SP-A is synthesized and secreted by alveolar type II (ATII) cells, one of the two cell types of the distal lung epithelium (ATII and ATI). We have shown that miRNA interactions with sequence polymorphisms on the SP-A mRNA 3'UTRs mediate differential expression of SP-A1 and SP-A2 gene variants in vitro. In the present study, we describe a physiologically relevant model to study miRNA regulation of SP-A in human ATII. For these studies, we purified and cultured human ATII on an air-liquid interface matrix (A/L) or plastic wells without matrix (P). Gene expression analyses confirmed that cells cultured in A/L maintained the ATII phenotype for over 5 days, whereas P-cultured cells differentiated to ATI. When we transfected ATII with siRNAs to inhibit the expression of Drosha, a critical effector of miRNA maturation, the levels of SP-A mRNA and protein increased in a time dependent manner. We next characterized cultured ATII and ATI by studying expression of 1,066 human miRNAs using miRNA PCR arrays. We detected expression of >300 miRNAs with 24 miRNAs differentially expressed in ATII versus ATI, 12 of which predicted to bind SP-A 3'UTRs, indicating that these may be implicated in SP-A downregulation in ATI. Thus, miRNAs not only affect SP-A expression, but also may contribute to the maintenance of the ATII cell phenotype and/or the trans-differentiation of ATII to ATI cells, and may represent new molecular markers that distinguish ATII and ATI.
authors: 1Center for Host Defense, Inflammation, and Lung Disease (CHILD) Research, Department of Pediatrics, The Pennsylvania State University College of Medicine , Hershey, Pennsylvania , USA.
authors: Patricia
authors: P
authors: Silveyra
authors: Zissis C
authors: ZC
authors: Chroneos
authors: Susan L
authors: SL
authors: DiAngelo
authors: Neal J
authors: NJ
authors: Thomas
authors: Georgios T
authors: GT
authors: Noutsios
authors: Nikolaos
authors: N
authors: Tsotakos
authors: Judie A
authors: JA
authors: Howrylak
authors: Todd M
authors: TM
authors: Umstead
authors: Joanna
authors: J
authors: Floros
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D035683:MicroRNAs / Q000235:genetics; D010641:Phenotype; D010865:Pilot Projects; D011650:Pulmonary Alveoli / Q000378:metabolism*; D037662:Pulmonary Surfactant-Associated Protein A / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D043244:Ribonuclease III / Q000235:genetics* / Q000378:metabolism*
chemical_list: D020413:3' Untranslated Regions; D035683:MicroRNAs; D037662:Pulmonary Surfactant-Associated Protein A; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C517918:DROSHA protein, human; D043244:Ribonuclease III
keywords: alveolar type II cells; lung epithelium; miRNAs; surfactant protein A