TY - JOUR
T1 - Knockdown of Drosha in human alveolar type II cells alters expression of SP-A in culture
T2 - A pilot study
AU - Silveyra, Patricia
AU - Chroneos, Zissis C.
AU - Diangelo, Susan L.
AU - Thomas, Neal J.
AU - Noutsios, Georgios T.
AU - Tsotakos, Nikolaos
AU - Howrylak, Judie A.
AU - Umstead, Todd M.
AU - Floros, Joanna
N1 - Funding Information:
This work was supported by grants from NIH (HL-34788, JF), Children’s Miracle Network (PS), and Sigma Delta Epsilon-Graduate Women in Science (Adele Lewis Grant Fellowship, PS).
Funding Information:
Dr. Silveyra’s research is supported by the Eu-nice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under BIRCWH award number K12HD055882, “Career Development Program in Women’s Health Research at Penn State.” The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
PY - 2014/9
Y1 - 2014/9
N2 - 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.
AB - 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.
KW - Alveolar type II cells
KW - Lung epithelium
KW - MiRNAs
KW - Surfactant protein A
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UR - http://www.scopus.com/inward/citedby.url?scp=84906046244&partnerID=8YFLogxK
U2 - 10.3109/01902148.2014.929757
DO - 10.3109/01902148.2014.929757
M3 - Article
C2 - 25058539
AN - SCOPUS:84906046244
SN - 0190-2148
VL - 40
SP - 354
EP - 366
JO - Experimental Lung Research
JF - Experimental Lung Research
IS - 7
ER -