MicroRNA profiling of diverse endothelial cell types

Abstract

Background: MicroRNAs are ~22-nt long regulatory RNAs that serve as critical modulators of post-transcriptional gene regulation. The diversity of miRNAs in endothelial cells (ECs) and the relationship of this diversity to epithelial and hematologic cells is unknown. We investigated the baseline miRNA signature of human ECs cultured from the aorta (HAEC), coronary artery (HCEC), umbilical vein (HUVEC), pulmonary artery (HPAEC), pulmonary microvasculature (HPMVEC), dermal microvasculature (HDMVEC), and brain microvasculature (HBMVEC) to understand the diversity of miRNA expression in ECs.

Results: We identified 166 expressed miRNAs, of which 3 miRNAs (miR-99b, miR-20b and let-7b) differed significantly between EC types and predicted EC clustering. We confirmed the significance of these miRNAs by RT-PCR analysis and in a second data set by Sylamer analysis. We found wide diversity of miRNAs between endothelial, epithelial and hematologic cells with 99 miRNAs shared across cell types and 31 miRNAs unique to ECs. We show polycistronic miRNA chromosomal clusters have common expression levels within a given cell type.

Conclusions: EC miRNA expression levels are generally consistent across EC types. Three microRNAs were variable within the dataset indicating potential regulatory changes that could impact on EC phenotypic differences. MiRNA expression in endothelial, epithelial and hematologic cells differentiate these cell types. This data establishes a valuable resource characterizing the diverse miRNA signature of ECs.

Figure 1

Endothelial cell miRNA clustering. (A) Hierarchical clustering based on LIMMA pairwise differential analysis of 59 miRNAs that had an unadjusted p value < 0.05. (B) Hierarchical cluster based on the SAM significant miRNAs let-7b, miR-20b and miR-99b. (C) Heat map demonstrating similar expression patterns across miRNAs within the same chromosomal cluster. Light blue represents low expression, dark blue represents high expression.

Figure 2

LNA-ISH of miRNAs miR-126, let-7b and U6 snRNA. PECAM1 (CD31) staining of endothelial cells. (A-D) Human skin including epidermis, (E-H) human lung, (I-L) coronary artery, (M-P) human brain and (Q-T) human umbilical vein. * represents the location of ECs. For LNA-ISH, positive staining is blue. For PECAM1 IHC, positive staining is brown. Original magnification 160× (A-H, M-T) or 100× (I-L).

Figure 3

Sylamer analysis. (A & B) Representative Sylamer enrichment landscape plots for 3' UTR binding sites for miRNAs. (A) Significant peak for miR-99b 7-mer seed sequence in a comparison of HCECs and HAECs. Significant peak for miR-20b 8-mer seed in a comparison between HCECs and HUVECs. Significant peak for let-7b 6-mer seed in a comparison between HUVEC and HCECs. (B) Resampling analysis histogram of Sylamer 3' UTR binding site data. For all EC comparisons, 162 significant miRNA binding sites were identified. Of these 162 sites, 15 were predicted 3' UTR binding sites for miRNAs miR-20b, miR-99b, and let-7b. A histogram of 10, 000 samplings of 3 random miRNA binding sites was generated to determine the likelihood of having a certain number of predicted 3' UTR binding sites. Having miRNAs miR-20b, miR-99b and let-7b represent 15 binding sites was significantly greater than chance (p < 0.0001).

Figure 4

Endothelial, epithelial and hematologic cell comparisons. (A) Cluster analysis of endothelial, epithelial and hematologic cells from the same miRNA expression platform based on LIMMA analysis. (B) Venn diagram indicating the complete unique and shared expression patterns of miRNAs between endothelial, epithelial and heamtologic cell types. (C) Representative heat maps from 6 areas of the Venn diagram identifying specific miRNAs in different colored areas. A complete distribution of all miRNA expression is found in the Additional file 4, Table S2. (D) RT-PCR validation of variable expression between cell lines for miRNAs miR-126, miR-141 and miR-142-3p. The left image is a histogram of the frequency of expression from the GEO data set. Epithelial cell lines are red, ECs are blue and hematologic lines are black. The right image is RT-PCR data of the same miRNAs normalized to U6 snRNA.

Figure 5

Heat maps of 23 polycistronic chromosome miRNA clusters. Each chromosome cluster was selected based on having at least one LIMMA significant pairwise difference. Twenty of 23 chromosome clusters generally display consistent, differential expression between endothelial, epithelial and hematologic cells.