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. 2014 Mar 15;7(3):607-15.
eCollection 2014.

RNAi targeting GPR4 influences HMEC-1 gene expression by microarray analysis

Juan Ren  1 , Yuelang Zhang  2 , Hui Cai  3 , Hongbing Ma  4 , Dongli Zhao  1 , Xiaozhi Zhang  1 , Zongfang Li  4 , Shufeng Wang  1 , Jiangsheng Wang  1 , Rui Liu  1 , Yi Li  1 , Jiansheng Qian  1 , Hongxia Wei  1 , Liying Niu  1 , Yan Liu  1 , Lisha Xiao  3 , Muyang Ding  3 , Shiwen Jiang  5
Affiliations

RNAi targeting GPR4 influences HMEC-1 gene expression by microarray analysis

Juan Ren et al. Int J Clin Exp Med. .

Abstract

G-protein coupled receptor 4 (GPR4) belongs to a protein family comprised of 3 closely related G protein-coupled receptors. Recent studies have shown that GPR4 plays important roles in angiogenesis, proton sensing, and regulating tumor cells as an oncogenic gene. How GPR4 conducts its functions? Rare has been known. In order to detect the genes related to GPR4, microarray technology was employed. GPR4 is highly expressed in human vascular endothelial cell HMEC-1. Small interfering RNA against GPR4 was used to knockdown GPR4 expression in HMEC-1. Then RNA from the GPR4 knockdown cells and control cells were analyzed through genome microarray. Microarray results shown that among the whole genes and expressed sequence tags, 447 differentially expressed genes were identified, containing 318 up-regulated genes and 129 down-regulated genes. These genes whose expression dramatically changed may be involved in the GPR4 functions. These genes were related to cell apoptosis, cytoskeleton and signal transduction, cell proliferation, differentiation and cell-cycle regulation, gene transcription and translation and cell material and energy metabolism.

Keywords: GPR4; RNAi; microarray.

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Figures

Figure 1
Figure 1
Verification of GPR4 knockdown in HMEC-1 cells by lentiviral-mediated RNA interference. Images of GFP expression showing shRNA delivery efficiency. Bar=50 μM. A: LV-CON-infected HMEC-1 cells for 24 h; B: LV-CON-infected HMEC-1 cells for 48 h; C: LV-shGPR4-infected HMEC-1 cells for 24 h; D: LV-shGPR4-infected HMEC-1 cells for 48 h.
Figure 2
Figure 2
Lentiviral vector-mediated delivery of siRNA targeting GPR4 results in specific knockdown of expression. The indicated lentiviral siRNA expression constructs were cotransfected into HMEC-1 cells with an expression construct for the GPR4 target. A: Real-time PCR analyzed the expression of target gene expression in the RNA level. B: Immunoblotting of whole cell extracts was performed with anti-GPR4 antibody to detect GPR4 (upper panel). An anti-GAPDH antibody was used to confirm equal protein loading (bottom panel).
Figure 3
Figure 3
Cluster analysis of the gene expression. Clustering of differentially expressed genes in the HMEC-1 cells infected with LV-Con or LV-shGPR4.
Figure 4
Figure 4
Comparison of 4 differential expression genes between the microarray and RT-PCR analyses.
See this image and copyright information in PMC

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