Yonsei Med J.  2016 Jan;57(1):247-253. 10.3349/ymj.2016.57.1.247.

Galpha12 Protects Vascular Endothelial Cells from Serum Withdrawal-Induced Apoptosis through Regulation of miR-155

• Lee HJ
• Lee EJ
• Seo M

• KMID: 2466377
• DOI: http://doi.org/10.3349/ymj.2016.57.1.247

Abstract

PURPOSE
Apoptosis of vascular endothelial cells is a type of endothelial damage that is associated with the pathogenesis of cardiovascular diseases such as atherosclerosis. Heterotrimeric GTP-binding proteins (G proteins), including the alpha 12 subunit of G protein (Galpha12), have been found to modulate cellular proliferation, differentiation, and apoptosis of numerous cell types. However, the role of Galpha12 in the regulation of apoptosis of vascular cells has not been elucidated. We investigated the role of Galpha12 in serum withdrawal-induced apoptosis of human umbilical vein endothelial cells (HUVECs) and its underlying mechanisms.
MATERIALS AND METHODS
HUVECs were transfected with Galpha12 small-interfering RNA (siRNA) to knockdown the endogenous Galpha12 expression and were serum-deprived for 6 h to induce apoptosis. The apoptosis of HUVECs were assessed by Western blotting and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expressions of microRNAs were analyzed by quantitative real-time PCR.
RESULTS
Knockdown of Galpha12 with siRNA augmented the serum withdrawal-induced apoptosis of HUVECs and markedly repressed the expression of microRNA-155 (miR-155). Serum withdrawal-induced apoptosis of HUVECs was inhibited by the overexpression of miR-155 and increased significantly due to the inhibition of miR-155. Notably, the elevation of miR-155 expression prevented increased apoptosis of Galpha12-deficient HUVECs.
CONCLUSION
From these results, we conclude that Galpha12 protects HUVECs from serum withdrawal-induced apoptosis by retaining miR-155 expression. This suggests that Galpha12 might play a protective role in vascular endothelial cells by regulating the expression of microRNAs.

Keyword

Galpha12 protein; apoptosis; microRNAs; endothelial cells

MeSH Terms

*Apoptosis
Atherosclerosis/*blood/genetics/immunology
Cell Proliferation
Endothelial Cells/*metabolism
GTP-Binding Protein alpha Subunits, G12-G13/*genetics
Gene Expression Profiling
Gene Expression Regulation
Human Umbilical Vein Endothelial Cells/cytology
Humans
MicroRNAs/*metabolism
Protective Agents
*RNA, Small Interfering
Real-Time Polymerase Chain Reaction
*Transfection
GTP-Binding Protein alpha Subunits, G12-G13
MicroRNAs
Protective Agents
RNA, Small Interfering

Figure

• 

  Fig. 1 Gα12 inhibits serum withdrawal-induced apoptosis of HUVECs. (A and B) Effect of serum deprivation on HUVEC apoptosis. HUVECs were deprived of serum for the indicated times, and apoptosis was assessed via immunoblot analysis with antibody to cleaved caspase-3 and a TUNEL assay. (C and D) Effect of Gα12 siRNA on serum withdrawal-induced apoptosis. HUVECs were transfected with Gα12 siRNA. These cells were cultured 48 h after transfection in the serum-free condition for 6 h and were analyzed for apoptosis via immunoblotting with antibodies to cleaved caspase-3 and phosphorylated p38 and the TUNEL assay. The blots are representative of at least three independent experiments, and the histograms show the average and standard deviation. *p<0.05 compared with control cells. Gα12, alpha 12 subunit of G protein; HUVEC, human umbilical vein endothelial cell; siRNA, small-interfering RNA; PI, propidium iodide.

• 

  Fig. 2 Gα12 alters the expression of miRNAs in HUVECs. HUVECs were transfected with Gα12 siRNA, and the expression of miRNAs was assessed via quantitative RT-PCR. The RNA was isolated, and the levels of miR-17-3, miR-31, miR-155, and miR-191 were measured. The histograms show the average and standard deviation of three independent experiments. *p<0.05, **p<0.005 compared with control cells. Gα12, alpha 12 subunit of G protein; HUVEC, human umbilical vein endothelial cell; siRNA, small-interfering RNA; miRNA, microRNA; RT-PCR, realtime PCR.

• 

  Fig. 3 Gα12 modulates the apoptosis of HUVECs by regulating the level of miR-155. (A) Effect of serum deprivation on the expression of miR-155. HUVECs were deprived of serum for 6 h, and the expression of miR-155 was determined by quantitative RT-PCR. (B) Regulation of miR-155 level by a mimic or inhibitor of miR-155. HUVECs were transfected with a mimic or inhibitor of miR-155, and the expression of miR-155 was determined by quantitative RT-PCR. (C and D) Effects of a mimic and inhibitor of miR-155 on serum withdrawal-induced apoptosis. HUVECs were transfected with the mimic or inhibitor for miR-155 and then deprived of serum for 6 h, and the expression of cleaved caspase-3 was measured. (E) Effect of miR-155 mimic on apoptosis in Gα12-deficient HUVECs. HUVECs were cotransfected with Gα12 siRNA and miR-155 mimic and cultured in the serum-free condition for 6 h, and cleaved caspase-3 and phosphorylated p38 were assessed. The blots are representative of at least three independent experiments, and the histograms show the average and standard deviation. *p<0.05 compared with control cells. Gα12, alpha 12 subunit of G protein; HUVEC, human umbilical vein endothelial cell; RT-PCR, real-time PCR; siRNA, small-interfering RNA.

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