MicroRNA-19a mediates the suppressive effect of laminar flow on cyclin D1 expression in human umbilical vein endothelial cells - PubMed (original) (raw)

MicroRNA-19a mediates the suppressive effect of laminar flow on cyclin D1 expression in human umbilical vein endothelial cells

Xiaomei Qin et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Endothelial cells (ECs) respond to changes in mechanical forces, leading to the modulation of signaling networks and cell function; an example is the inhibition of EC proliferation by steady laminar flow. MicroRNAs (miRs) are short noncoding 20-22 nucleotide RNAs that negatively regulate the expression of target genes at the posttranscriptional level. This study demonstrates that miRs are involved in the flow regulation of gene expression in ECs. With the use of microRNA chip array, we found that laminar shear stress (12 dyn/cm(2), 12 h) regulated the EC expression of many miRs, including miR-19a. We further showed that stable transfection of miR-19a significantly decreased the expression of a reporter gene controlled by a conserved 3'-untranslated region of the cyclinD1 gene and also the protein level of cyclin D1, leading to an arrest of cell cycle at G1/S transition. Laminar flow suppressed cyclin D1 protein level, and this suppressive effect was diminished when the endogenous miR-19a was inhibited. In conclusion, we demonstrated that miR-19a plays an important role in the flow regulation of cyclin D1 expression. These results revealed a mechanism by which mechanical forces modulate endothelial gene expression.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

miR-19a is rapidly induced by shear stress in ECs. (A) Scatter plot of miR expression profiles in ECs in response to laminar shear stress (12 dyn/cm2 for 12 h, y axis) and control ECs kept under static condition (x axis). The miRs differentially expressed with statistical significance are marked in red. (B) qRT-PCR shows that miR-19a was induced in ECs after being exposed to laminar shear stress for various time durations. U6 snRNA level was used for normalization. Data are presented as the mean ± SEM (n = 3). *P < 0.05 compared with static control).

Fig. 2.

Fig. 2.

miR-19a represses cyclin D1 expression through a conserved 3′-UTR binding site. (A) Sequence alignment of the miR-19a base-pairing sites in the 3′-UTR of cyclinD1 mRNAs showed that the regions complementary to the 9 nt of miR-19a are highly conserved among human, mouse, rat, cow, dog, and chicken. The “seed” sequences of miR-19a complementary to cyclin D1 are shown in red and boxed. (B) Endothelial cell stable lines were generated by transfecting EA.hy.926 cells with pEF1-RF-miR-19 or pEF-RF and by the puromycin selection. Photomicrographs show ubiquitous expression of red fluorescence protein in both transfected cells. (C) Overexpression of miR-19a was confirmed in the RF-miR-19a stable cell line with the use of qRT-PCR. U6 was used as the internal control. Data are presented as mean ± SEM (n = 3). **P < 0.01.

Fig. 3.

Fig. 3.

Cyclin D1 is a target of miR-19a in ECs. (A) Western blot analysis showed that protein level of cyclinD1 was decreased in the miR-19a–transfected cell line. (B) Cyclin D1 mRNA was not different between miR-19a–transfected cells and control cells as detected by qRT-PCR. (C) Luciferase activity of the reporter of the cyclin D1 3′-UTR containing miR-19a site (3′-UTR-CCND1-miR19a), but not the pMIR-REPORT control, was decreased in the miR19a-expressing cells. Data are presented as mean ± SEM of three experiments. *P < 0.05 compared with that in the control cell line.

Fig. 4.

Fig. 4.

Overexpression of miR-19a arrests cell cycle at the G1 phase in ECs. Flow-cytometric analysis indicated that the population of cells in G0/G1 phase was increased and that in S phase decreased in miR-19a–transfected cells. Data are presented as mean ± SEM of three repeated experiments. *P < 0.05 compared with control cells.

Fig. 5.

Fig. 5.

miR-19a mediates the suppressive effect of laminar flow on cyclin D1 expression in HUVECs. (A) Effects of laminar flow on the expression of cyclin D1 and p21, another cell cycle–related genes. Confluent HUVECs were exposed to 12 dyn/cm2 shear stress or static control for 12 h and 24 h. Results shown are representative of three independent experiments. (B) Pretreatment with anti–miR-19a prevented the inhibitory effect of laminar flow on cyclin D1 expression. HUVECs were pretreated with anti–miR-19a or control oligonucleotides (negative anti-miR) for 48 h followed by laminar flow for 24 h, and cyclinD1 expression was examined with Western blotting. (C) miR-19a inhibitor partially attenuated the laminar flow-induced cell cycle arrest. HUVECs were transfected with anti–miR-19a or anti-miR negative control for 48 h, followed by 12 dyn/cm2 shear stress or static control for 24 h, and cell cycle distribution of ECs was analyzed with flow cytometry. *P < 0.05 compared with static plus anti-miR negative control. (D) Shear stress–induced suppression of ECs entry S and G2/M phases were reduced by anti–miR-19a. *P < 0.05 compared with anti-miR negative control. Results shown are representative of three independent experiments.

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