Changes in microRNA and mRNA expression with differentiation of human bronchial epithelial cells - PubMed (original) (raw)

doi: 10.1165/rcmb.2012-0368OC.

Milena Sokolowska, Steven Kern, A Sally Davis, Sara Alsaaty, Jeffery K Taubenberger, Junfeng Sun, Rongman Cai, Robert L Danner, Michael Eberlein, Carolea Logun, James H Shelhamer

Affiliations

Changes in microRNA and mRNA expression with differentiation of human bronchial epithelial cells

Asuncion Martinez-Anton et al. Am J Respir Cell Mol Biol. 2013 Sep.

Abstract

We studied the changes in expression of microRNAs (miRNAs or miRs) and mRNA in normal human bronchial epithelial cells as they differentiate from an undifferentiated monolayer to a differentiated pseudostratified epithelium after 28 days of air-liquid interface (ALI) culture. After 28 days in ALI, the epithelial cells differentially expressed basal, ciliated, and goblet cell markers. Using Affymetrix microarrays, 20 human miRNAs were found to be up-regulated, whereas 35 miRNAs were found to be down-regulated in differentiated cells compared with undifferentiated cells. An analysis of changes in global mRNA expression revealed that 1,201 probe sets demonstrated an 8-fold change (FC) or greater at Day 28 of ALI culture. Of these, 816 were up-regulated and 385 were down-regulated. With differentiation, miR-449a increased (FC, 38.15), and was related to changes in mRNA for cell division cycle 25 homolog A (FC, 0.11). MiR-455 decreased (FC, 0.12) and was related to changes in mRNA for the epithelial cell marker, mucin 1 (FC, 136). Transfection with anti-miR-449 or miR-455-3p resulted in changes in target protein expression (cell division cycle 25 homolog A and mucin 1, respectively), whereas transfection with reporter genes with 3'-untranslated regions of these targets confirmed control of expression through that structure. Therefore, changes in specific miRNAs during human airway epithelial cell differentiation control gene and protein expression important for differentiation.

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Figures

<i>Figure 1.</i>

Figure 1.

Confocal images of confluent and fully differentiated normal bronchial epithelial cells. Top row shows the confluent monolayer on its membrane labeled as follows: (A) ciliated cell (CC) marker, anti–β-tubulin (red); (B) basal cell (BC) marker, anti–cytokeratin 5 (green); (C) goblet cell (GC) marker, lectin jacalin (cyan); (D) fluorescence merge with above three markers along with DRAQ5 nuclear marker (blue). The central row (E_–_H) shows Day 28 air–liquid interface (ALI) fully differentiated, pseudostratified epithelium on its membrane labeled as above for the confluent monolayer. The bottom row includes (I_–_K) hematoxylin and eosin–stained sections of the differentiated pseudostratified epithelium on its membrane for each of the three donors studied (original magnification ×1,000) and (L) a max projection of a differential interference contrast stack composed of a slice where the cilia are in focus and a slice with intracellular goblet cell mucins in focus imaged at the same physical slide location as in (E_–_H). All confocal images were taken at 630× magnification at a zoom of 2.3× and are maximum projections of z stacks unless otherwise noted. (scale bars, 10 μm).

<i>Figure 2.</i>

Figure 2.

Principal components analysis (PCA) of microRNA (miRNA or miR) (A) and mRNA (B) normalized expression data from differentiating normal human bronchial epithelial cells. The PCA of the data revealed strong separation between all the groups and good homogeneity within each group, for both miRNA and gene arrays. Expression data from three independent cultures from a single donor indicate that the predominant difference among the culture conditions is the difference between differentiated cells at Day 28 (star) and subconfluent (triangle) and confluent (circle) cells. (C and D) Summary of the number of probe sets that displayed a 2-fold change (miRs) and 8-fold change (mRNA) (increase or decrease) between the different groups: subconfluent; confluent; Day-28 normal human bronchial epithelial (NHBE) cells grown in an ALI culture system for 28 days.

<i>Figure 3.</i>

Figure 3.

Heat map showing significant changes of miRNA expression between undifferentiated confluent cells and Day-28 ALI differentiated NHBE cells. Red represents increased expression, whereas green represents decreased expression. The column on the left shows the list of miRNAs presenting fold changes ≥2.0. Labels on the bottom of the heat map (1–3) refer to three independent cultures from the same donor. hsa, homo sapiens.

<i>Figure 4.</i>

Figure 4.

Cell division cycle (CDC) homolog 25A and mucin (MUC) 1 gene (A and B) and protein (C and D) expression in undifferentiated (confluent) and differentiated (Day 28) NHBE cells. Bar graphs indicate fold changes in expression of CDC25A (A) and MUC1 (B) mRNA relative to confluent cells. Results are expressed as mean ± SEM (Student’s t test: *P < 0.05) from three different donors. Western blot analysis of CDC25A (C) and MUC1 (D) protein levels in undifferentiated (confluent) and differentiated (Day 28) NHBE cells. The Western blot is representative of data from two separate experiments. β-actin expression is shown as a loading control.

<i>Figure 5.</i>

Figure 5.

Effect of functional inhibition of miR-449a on CDC25A expression. NHBE cells were transduced when subconfluent with lentiviral particles expressing an anti–miR control or anti–miR-449a. Cells were harvested after 14 days of ALI culture. Bar graphs indicate fold changes in expression of miR-449a (A) and CDC25A (B) after anti–miR-449a transduction relative to negative control cells. Results from three independent cultures from a single donor are expressed as means ± SEM (Student’s t test: *P < 0.05). (C) Western blot analysis of CDC25A protein levels after anti–miR-449a transduction compared with nontransduced (negative control) and anti–miR control–transduced cells. The Western blot is representative of data from two independent experiments. β-actin expression is shown as a loading control. (D) A549 cells were transfected with the CDC25A 3′-untranslated region (UTR)–luciferase construct in the presence or absence of 20 nM of miR control or miR-449a precursor. Luciferase activity was measured at 48 hours post-transfection. Results are expressed as means ± SEM of three independent experiments, each assayed in triplicate (Student’s t test: *P < 0.05, compared with negative control cells).

<i>Figure 6.</i>

Figure 6.

Effect of functional overexpression of miR-455-3p on MUC1 expression. NHBE cells were transduced when subconfluent with lentiviral particles expressing miR control or miR-455-3p, and harvested after 14 days of ALI culture. (A) Bar graphs indicate fold changes in expression of miR-455-3p (A) and MUC1 (B) after miR-455-3p transduction relative to negative control cells. Results from three independent cultures from a single donor are expressed as means ± SEM (Student’s t test: *P < 0.05 compared with negative control [Neg CTL]; &P < 0.05 compared with miR-CTL). (C) Western blot analysis of MUC1 protein levels after miR-455-3p transduction compared with nontransduced (negative control) and miR control–transduced cells. The Western blot is representative of data from two independent experiments. β-actin expression is shown as a loading control. (D) A549 cells were transfected with the MUC1 3′-UTR–luciferase construct in the presence or absence of 20 nM of miR control or miR-455-3p precursor. Luciferase activity was measured at 48 hours post-transfection and normalized to the internal Renilla luciferase control. Results are expressed as means ± SEM of three independent experiments, each assayed in triplicate (Student’s t test: *P < 0.05, compared with negative control cells).

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