Role of microRNA-23b in flow-regulation of Rb phosphorylation and endothelial cell growth

Abstract

MicroRNAs (miRs) can regulate many cellular functions, but their roles in regulating responses of vascular endothelial cells (ECs) to mechanical stimuli remain unexplored. We hypothesize that the physiological responses of ECs are regulated by not only mRNA and protein signaling networks, but also expression of the corresponding miRs. EC growth arrest induced by pulsatile shear (PS) flow is an important feature for flow regulation of ECs. miR profiling showed that 21 miRs are differentially expressed (8 up- and 13 downregulated) in response to 24-h PS as compared to static condition (ST). The mRNA expression profile indicates EC growth arrest under 24-h PS. Analysis of differentially expressed miRs yielded 68 predicted mRNA targets that overlapped with results of microarray mRNA profiling. Functional analysis of miR profile indicates that the cell cycle network is highly regulated. The upregulation of miR-23b and miR-27b was found to correlate with the PS-induced EC growth arrest. Inhibition of miR-23b using antagomir-23b oligonucleotide (AM23b) reversed the PS-induced E2F1 reduction and retinoblastoma (Rb) hypophosphorylation and attenuated the PS-induced G1/G0 arrest. Antagomir AM27b regulated E2F1 expression, but did not affect Rb and growth arrest. Our findings indicate that PS suppresses EC proliferation through the regulation of miR-23b and provide insights into the role of miRs in mechanotransduction.

Fig. 1.

Twenty-four-hour PS reduces cell proliferation and regulates cell cycle-related gene expression. (A) HUVECs were subjected to 24-h PS, with BrdU added during the last 4 h. Cells were fixed and stained (Upper) with anti-BrdU and 7-AAD (for DNA staining). Flow cytometry (Lower) showed a significant increase of cell number in G0/G1 phase and a significant decrease in S phase. The number of cells in G2 + M was not significantly changed. (B) Hierarchical clustering of differentially expressed genes in cell cycle under PS vs. ST. Biological repeats are shown at the Upper. Right column lists the selected gene symbols. Color of each band reflects fold change of mRNA in PS vs. ST. Red bands represent increase, and green bands decrease. Exemplar mRNAs representing cell cycle components are shown in this heat map.

Fig. 2.

miRNA expression profile under PS vs. ST. Expression levels of 856 human miRs (Sanger miRBase 12.0) in ECs under PS and ST were measured with miR microarray (n = 3). (A) Unsupervised hierarchical clustering analysis of miR expression profile. Heat map shows PS vs. ST miR log2 ratios with a threshold of 1.25-fold and P < 0.05. Red bands represent upregulated miRs and green bands, downregulated miRs. (B) Differential expression of miR clusters. miRs are considered to belong to the same cluster if they are located in close proximity to each other in the genome. PS upregulates the miR-23b cluster in chromosome 9 and downregulates miR-17–92, -16, and -221 clusters in chromosomes 13, 3, and X, respectively. The images of chromosomes are copied from http://genomics.energy.gov.

Fig. 3.

Association matrix of miR levels with functional gene sets under PS vs. ST. (A) Columns represent 394 functional gene sets, and rows represent flow-regulated miRs. Functional categories are positively (red bands), negatively (blue bands), or not (white bands) associated with miR expression profiles under PS vs. ST. The boxed region shows high associations between functional categories and miRs. The genes extracted from the functional gene sets in the box were used to perform the gene ontology analysis. (B) The −log (P values) of top 10 GO terms are plotted.

Fig. 4.

Antagomir against miR-23b attenuates the flow-induced EC growth arrest. (A) Flow cytometry for cells transfected with anti-miR negative control (Right), AM23b (Center), and AM27b (Left) in ST (Upper) vs. 24-h PS (Lower). R1, R2, and R3 region gates denote cells in G1/G0, G2 + M, and S phases, respectively. (B) The bar graph summarized the percentage of cells in G1/G0, G2/M, and S phases under PS and ST with antagomir transfection. *,P < 0.05 in comparison with the corresponding data for negative AM transfection under static condition. #, P < 0.05 in comparison with the corresponding data for negative AM transfection under PS condition.

Fig. 5.

Effects of miR-23b and 27b on cell cycle proteins. HUVECs were transfected with Neg. AM, AM23b, and AM27b. Twenty-four-hour posttransfection, cells were subjected to PS or ST for 24 h. Western blot analysis of cell cycle regulatory proteins, including (A) phosphorylation levels of Rb protein and (B) E2F1 expressions on total cell extracts, were determined with antibodies against phospho-Rb (BD Biosciences), Rb, and E2F1 (Santa Cruz). Blots shown here are representative of three independent experiments with similar results. *, P < 0.05 in comparison with the corresponding data for neg. AM transfection under ST.

Fig. 6.

Schematic representation of the role of miR-23b in flow regulation of EC growth. PS increases the expression of miR-23b (bold), which alters Rb into a hypophosphorylated state to complex with E2F family proteins and suppress EC growth. The upregulation of miR-27b (gray letters) also decreased the expression of E2F1, which is not sufficient for cell growth arrest without modulating Rb status. Qin et al. (44) show that miR-19 (italics) regulates cyclin D1 expression to cause EC growth arrest in response to steady laminar flow, and this effect may also be mediated via Rb hypophosphorylation.