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. 2014 May;2(3):354-358.
doi: 10.3892/br.2014.250. Epub 2014 Mar 12.

RelB regulates Bcl-xl expression and the irradiation-induced apoptosis of murine prostate cancer cells

Affiliations

RelB regulates Bcl-xl expression and the irradiation-induced apoptosis of murine prostate cancer cells

Liang Zhu et al. Biomed Rep. 2014 May.

Abstract

Apoptosis in prostate cancer (PCa) induced by ionizing radiation (IR) is believed to play a critical role in radioresistance. Bcl-xl, an important member of the anti-apoptotic Bcl-2 family, has critical roles in tumor progression and development. The aim of the present study was to investigate the association of Bcl-xl expression and radiosensitivity from murine PCa RM-1 cells. An adenovirus-mediated RNA interference technique was employed to inhibit the expression of the RelB gene. RelB proteins were detected upon irradiation following transfection with small interfering (si)RelB, as shown by western blot analysis. The radiosensitivity of the RM-1 cells was determined by clonogenic assays. The apoptosis of the RM-1 cells were detected by flow cytometry assay, then quantitative polymerase chain reaction assays were performed to determine the expression level of Bcl-xl mRNA in the RM-1 cells. Radiation treatment increased the RelB protein levels from the cytosol and nucleus in the RM-1 cells. The protein expression levels of RelB in the pLentilox-sh-RelB-transfected RM-1 cells were significantly lower than in the negative interference group following radiation treatment. The percentage of cells undergoing apoptosis in the siRelB-RM-1 group was significantly higher than that in the control group following radiation treatment. Finally, a positive link between Bcl-xl expression and RelB activity was established in the RM-1 cells. Inhibition of RelB correlates with a decrease in expression of Bcl-xl. In conclusion, adenovirus-mediated siRNA targeting RelB inhibits Bcl-xl expression, enhances radiosensitivity and regulates the irradiation-induced apoptosis of the murine PCa RM-1 cell line.

Keywords: Bcl-xl; RNA interference; RelB; prostate cancer; radiosensitivity.

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Figures

Figure 1
Figure 1
RelB-siRNA decreases RelB protein levels in RM-1 cells following radiation treatment. (A) N-fold changes of RelB mRNA level. Non-trans-RM-1, siVector-RM-1 and siRelB-RM-1 cells were treated by 6 Gy irradiation. (*P<0.05 and **P<0.01 vs. control). (B) RelB protein cytosol levels, observed by western blot analysis in response to 6 Gy radiation. (C) RelB protein nuclear levels, observed by western blot analysis, in response to 6 Gy radiation. si, small interfering; siRelB-RM-1, pLentilox-sh-RelB-transfected RM-1 cells; non-trans-RM-1, non-transfected RM-1 cells; siVector-RM-1, RM-1 cells transfected with empty vector; control-RM-1, non-irradiated RM-1 cells.
Figure 2
Figure 2
Dose-survival curves of siRelB-RM-1, siVector-RM-1 and non-trans-RM-1 cells following 2, 4, 6 and 8 Gy radiation. Compared with the controls, the siRelB-RM-1 cells were more sensitive to irradiation. siRelB-RM-1, pLentilox-sh-RelB-transfected RM-1 cells; siVector-RM-1, RM-1 cells transfected with empty vector; non-trans-RM-1, non-transfected RM-1 cells; IR, ionizing radiation; small interfering.
Figure 3
Figure 3
(A) Apoptosis induction with radiation in siRelB-RM-1, siVector-RM-1 and non-trans-RM-1 cells. The apoptosis rate was measured with Annexin V/PI flow cytometry in the various RM-1 cells that were treated with 6 Gy radiation, along with the untreated control RM-1 cells. Radiation treatment induced apoptosis in all the treated cells compared with the control cells. (B) Irradiation activates Bcl-xl in RM-1 cells. The cells were pretreated with siRelB prior to irradiation. The RM-1 cells were subjected to qPCR assay. Total RNA isolated from irradiated cells and untreated control RM-1 cells. (C) Bcl-xl mRNA levels were measured by qPCR assay normalized by the level of β-actin. Significant differences were observed, as indicated, when compared with the untreated groups (*P<0.05 and**P<0.01 vs. control). si, small interfering; control-RM-1, non-irradiated RM-1 cells; non-trans-RM-1, non-transfected RM-1 cells; siVector-RM-1, RM-1 cells transfected with empty vector; siRelB-RM-1, pLentilox-sh-RelB-transfected RM-1 cells; qPCR, quantitative polymerase chain reaction.

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