Changes in microRNA and mRNA Expression with Differentiation of Human Bronchial Epithelial Cells

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.

Validation of Normal Human Bronchial Epithelial Cells as a Model for Influenza A Infections in Human Distal Trachea

“…Previously, it has been used to identify goblet cells in both NHBE cells and tracheal tissue, where we and others have shown it to label equivalently to Muc5Ac in these cells (Fig. S1) (Chandrasekaran et al 2008;Martinez-Anton et al 2013;Vermeer et al 2003). In our hands, CK5 consistently labeled all cells at the basal aspect of the NHBE epithelium that have a round, non-apical cell morphology and are also CK8 negative, consistent with a basal cell (data not shown).…”

“…Because we had previously observed an up-regulation in genes that localize to club (Clara) cells-specifically, multiple secretoglobins (club cell secretory proteins) and CYP2B6 (a member of the cytochrome p450 family) in the differentiated NHBEs when compared to the undifferentiated ones-we conducted further investigations seeking club cells in the differentiated NHBEs (Hukkanen et al 2002;Martinez-Anton et al 2013;Mori et al 1996;). Our immunofluorescence for anti-CC10 was consistently negative on our FFPE differentiated NHBE cells and human distal trachea samples.…”

“…To ascertain whether neuroendocrine and professional immune cells were present in fully differentiated NHBE cells, we mined data from a previously completed microarray analysis of a day 28, fully differentiated, NHBE cell sample from a non-smoker (Martinez-Anton et al 2013). Briefly, sample preparation was completed according to standard Affymetrix procedures using the Genechip 3'IVT Express Kit and the Human Genome U133 plus 2.0 chip (Affymetrix; Santa Clara, CA).…”

MicroRNA: Endotyping United Airways?

“…The use of miR microarrays makes it possible to perform profiling studies that evaluate the differences between healthy and pathological tissues, treated and untreated samples and undifferentiated and differentiated cells [12]. Moreover, this systematic screening approach provides us with a starting point for the identification of new miR functions.…”

A primate-specific microRNA enters the lung cancer landscape

“…However, knockdown of miR-4423 did not greatly affect ciliated differentiation of bronchial epithelial cells, implying its functional redundancy with other factors, possibly other miRNAs, like miR-449/miR-34 family members with whom miR-4423 shares partial seed sequences and predicted targets. miR-449 accumulates when bronchial epithelial cells are differentiated into mucociliary epithelia at an air-liquid interface (15) and controls ciliated differentiation and cell fate (16,17). Expression of miR-34 is frequently reduced in cancers, and its reexpression has pleiotropic effects on cancer cell phenotypes, including inhibition of cancer stemness (18).…”