Gene expression analysis uncovers novel hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

Abstract

Hedgehog interacting protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis.

Trial registration: ClinicalTrials.gov NCT00292552.

Conflict of interest statement

Competing Interests Statement:

E.K.S. has received grant support and consulting and speaker’s fees from GlaxoSmithKline, consulting and speaker’s fees from Astra-Zeneca, and consulting fees from Merck. X.Z., W.Q., M.H.C., J.F.S., J.D.M., T.L., D.M.T, G.L., P.S.B., A.G., D.A.L., T.H.B., C.P.H., C.A., U.G., B.A.R., S.I.R., M.A.P., A.M.K.C. and J.Q. do not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Copyright © 2013 Elsevier Inc. All rights reserved.

Figures

Figure 1

Gene expression profiling analysis in Beas-2B cells stably infected with HHIP shRNAs. A. Venn diagram showing the number of differentially expressed genes based on iSVA or SVA. B. Expression heat map of 296 differentially expressed genes (columns) (p adjusted <0.05). Each row represents a separate sample (array). Bio: biological replicate; tech: technical replicate; NT shRNA: non-targeting shRNA control; shRNA: HHIP-targeting shRNA

Figure 2

Significantly increased (A) and decreased (B) expression (**, pHHIP silencing in Beas-2B cells, as measured by TaqMan assay-based RT-PCR. Three different HHIP-targeted shRNAs were used for knock-down. Relative expression values were calculated as described in Materials and Methods and represent means ± standard errors from two independent shRNA infections.

Figure 3

Genes associated with cell growth that demonstrated significant differential expression (pHHIP-knock-down in Beas-2B (A) or normal human bronchial epithelial cells (NHBE) (B), as measured by TaqMan assay-based RT-PCR. Three different HHIP-targeted shRNAs were used for knock-down. Relative expression values were calculated as described in Materials and Methods and represent means ± standard errors from two (A) or three (B) replicates for each hairpin. P values in figure 3B are calculated based on two biological replicates and one is represented here. C. Expression of CCND1, TGM2 and BIRC5 in Beas-2B cells infected with HHIP-targeted or non-target (NT) control shRNA, as detected by western blot. α-actin was used as loading control. Band density was quantified using Image J and then normalized to α-actin control.

Figure 4

Expression of HHIP target genes associated with ECM (A) or cell growth (B) were measured in human COPD and control lung tissues by RT-PCR. Relative expression values were calculated as described in Materials and Methods and represent means± standard errors for COPD (n=15) and Controls (n=18). **p

Figure 5

Network analysis of validated HHIP targets related to ECM and cell proliferation (highlighted by pink circles). Edge colors represent sources of the interaction (brown: Pathway Commons, purple: functional interaction, blue: PubMed abstract, teal: Medline abstract, red: manual curation based on recently published literature) and node colors represent the level of differential expression (red: highly differential, gray: no expression change.) Thickened edges connect pairs of differentially expressed genes.