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Effect of culture conditions on microRNA expression in primary adult control and COPD lung fibroblasts in vitro

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Abstract

In vitro cell cultures, including lung fibroblasts, have been used to identify microRNAs (miRNAs) associated with chronic obstructive pulmonary disease (COPD) pathogenesis. However, culture conditions may affect miRNA expression. We examined whether miRNA expression in primary adult lung fibroblasts varies with cell density or passage in vitro and whether culture conditions confound the identification of altered miRNA expression in COPD lung fibroblasts. Primary adult control and COPD lung fibroblasts were cultured until passage 3 or 8, after which cells were further cultured for 3 or 7 d (low vs. high density). Then, cells at low density were cultured with serum-free media, and those at high density were cultured with serum-free media in the absence or presence of interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) for 24 h. RNA was extracted to perform miRNA microarray from which 1.25-fold differential expression and 10% false discovery rate were applied to identify “invariant” and “variant” miRNA for the various culture conditions. Of the 2226 miRNAs evaluated, 39.0% for cell density, 40.7% for cell passage, and 29.4% for both conditions were identified as “invariant” miRNAs. Furthermore, 38.1% of the evaluated miRNAs were “invariant” for cell passage with IL-1β and TNF-α. Differentially expressed miRNAs between control and COPD lung fibroblasts were identified with and without IL-1β and TNF-α, and of these, 32 out of the 34 top-ranked miRNAs exceeded the differences due to culture conditions. Thus, culture conditions may affect miRNA expression of adult human lung fibroblasts. Nevertheless, in vitro cultures can be used to assess differential miRNA expression in COPD lung fibroblasts.

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Acknowledgments

The authors thank Lillian Richards for excellent secretarial support. This study was supported in part by the Larson Endowment, University of Nebraska Medical Center.

Author contribution

JI designed and conducted experimental work, lead data interpretation, and manuscript preparation; AN assisted with experimental design and work; SI, YG, MF, XW, BH, XL, DD, and SIR assisted with experimental design, data interpretation, and manuscript preparation; LS and BH led the statistical analyses; and CF-B provided cell cultures from non-COPD and COPD patients.

Grants

This study is supported by a grant from the Dean, College of Medicine, University of Nebraska Medical Center.

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Authors and Affiliations

  1. Pulmonary, Critical Care, Sleep and Allergy Medicine, University of Nebraska Medical Center, Omaha, NE, USA

    Jun Ikari, Amy J. Nelson, Shunichiro Iwasawa, Yoko Gunji, Maha Farid, Xingqi Wang, Hesham Basma, Xiangde Liu & Stephen I. Rennard

  2. College of Public Health, Department of Biostatistics, University of Nebraska Medical Center, Omaha, NE, USA

    Lynette M. Smith

  3. Channing Division of Network Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Dawn L. DeMeo

  4. University of Pittsburgh, Pittsburgh, PA, USA

    Carol Feghali-Bostwick

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  1. Jun Ikari
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  2. Lynette M. Smith
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Correspondence to Stephen I. Rennard.

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Editor: T. Okamoto

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Supplemental Fig. 1

Cell numbers of culture conditions. After low (passage 3 (LP)) and high (passage 8 (HP)) passage, control (n = 5, open bar) and COPD (n = 5, closed bar) lung fibroblasts were cultured for 3 d (low density (LD)) or 7 d (high density (HD)) with 10% FCS-containing DMEM after which cells were cultured with serum-free DMEM for 24 h. Cells were harvested, and total cell number was counted. *p < 0.05 compared with the values of LD in the passage. # p < 0.05; ## p < 0.01 compared with control and COPD lung fibroblasts in the same culture condition. (PDF 32 kb)

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Ikari, J., Smith, L.M., Nelson, A.J. et al. Effect of culture conditions on microRNA expression in primary adult control and COPD lung fibroblasts in vitro. In Vitro Cell.Dev.Biol.-Animal 51, 390–399 (2015). https://doi.org/10.1007/s11626-014-9820-8

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