Silencing effect of RNA interference on suppressor of cytokine signaling-1 in human umbilical vein endothelial cells*,⋆

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Abstract

BACKGROUND:

Suppressor of cytokine signaling-1 (SOCS1) silencing by RNAi can induce apoptosis of neuroendocrine tumor cells. However, it is not clear whether apoptosis of endothelial cells can be enhanced by silencing of SOCS1 similarly.

OBJECTIVE:

To determine whether SOCS1 is expressed in endothelial cell, in addition, to select the most extremely silences SOCS1 from the siRNAs designed.

DESIGN, TIME AND SETTING:

The in vitro control cell experiment was performed at the Jiangxi Province Key Laboratory of Molecular Medicine, the Second Affiliated Hospital of Nanchang University Medical College from December 2007 to June 2008.

MATERIALS:

Human umbilical vein endothelial cell (HUVEC) lines were provided by NanJing KeyGen Biotech Co., Ltd.

METHODS:

HUVEC was cultured. RT-PCR was used to determine whether SOCS1 was expressed in HUVEC. HUVEC were divided into the positive control (GAPDH-siRNA-PC), the negative control (siRNA-NC), the mock control (Mock) and the blank control groups. Four different siRNAs were designed and synthesized; they were siRNA-1, siRNA-2, siRNA-3, siRNA-4, and then transfected with liposome. The negative control siRNAs labelled with fluorescence were transfected with different concentrations (25, 50, 75, 100 nmol/L). Then fluorescent microscope was employed to determine the concentration at which siRNAs were transfected most successfully. RNAs were extracted from siRNAs groups, which had optimum transfection efficiency after 48 hours.

MAIN OUTCOME MEASURES:

RT-PCR was employed to assess the expression of SOCS1 at gene level.

RESULTS:

SOCS1 were highly expressed in HUVEC. It was in the 50 nmol/L group that the siRNAs were transfected most successfully. The expressions of SOCS1 in the siRNA-1, siRNA-2, siRNA-3 and siRNA-4 group were reduced compared to the siRNA-NC, Mock and the blank group (P < 0.05). Meanwhile, they were significantly down-regulated compared to the GAPDH-siRNA-PC (P < 0.01). In addition, siRNA-3 had the best silencing efficiency in the four different siRNAs designed group (P < 0.05). The expressions of SOCS1 in the siRNA-NC, Mock and blank group were reduced compared to the GAPDH-siRNA-PC group (P < 0.05). There was no statistical significance among the siRNA-NC, Mock and the blank group (P > 0.05).

CONCLUSIONS:

SOCS1 is expressed in endothelial cells. The expression of SOCS1 in the endothelial cell can be inhibited by RNA interference. One siRNA with a maximal inhibition rate was selected.

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