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, 8 (7), 12003-12012

The Regulator of Calcineurin 1 (RCAN1) Inhibits Nuclear Factor kappaB Signaling Pathway and Suppresses Human Malignant Glioma Cells Growth

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The Regulator of Calcineurin 1 (RCAN1) Inhibits Nuclear Factor kappaB Signaling Pathway and Suppresses Human Malignant Glioma Cells Growth

Xin Chen et al. Oncotarget.

Abstract

Nuclear factor-kappaB (NF-κB) has a vital role in cell survival and inhibition of NF-κB had proven to be an efficient therapeutic pathway for various cancers though little is known about the underlying mechanism. Previously we identified regulator of calcineurin 1 (RCAN1) as an endogenous inhibitor of NF-κB signaling pathway in lymphoma. In the present study, we have solid data to show that RCAN1 can inhibit the nuclear translocation of NF-κB protein then affect the activity of NF-κB signaling pathway in glioma cells. Overexpression of RCAN1 markedly reduced glioma cells viability. We further found that the suppressing glioma cell growth was closely related to the pro-apoptosis effect, not by inhibiting proliferation by the arrest of cell cycle. Our study implicated a novel therapeutic approach for glioma by RCAN1 through inhibition of NF-κB signaling.

Keywords: NF-κB signaling; RCAN1; apoptosis; glioma; suppress.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. NF-κB signaling pathway proteins were elevated in glioma tissue
A. Protein lysates from glioma tissues and controls were separated on a 10% glycine SDS-polyacrylamide gel. The lysates were immunoblotted with anti-NF-κB/p65, anti-IKKα, and anti-IKKβ at a dilution of 1:1000 (lanes 1, 2 and 3). β-actin was used as the internal control. B. Quantification of Western blots showed that NF-κB/p65, IKKα, and IKKβ were increased in glioma tissues. Values represent mean ± S.E. (n = 3), *p <0.05 by Student's t test. C. The expression of NF-κB in glioma was measured using ELISA assay. The values represent the means ± S.E. (n = 3),*p<0.05 by Student's t test.
Figure 2
Figure 2. RCAN1 decreased cell viability in glioma cell lines
U251 and T98G cells were infected with lentivirus expressing RCAN1 (RCAN1) or negative control (Control) for 72 hrs. A. Western blot was performed to assess the RCAN1 protein level. RCAN1 protein levels were normalized to β-actin. The results present the fold-increase relative to control. B. Quantification of (A). Values represent means ± SE (n = 3),*p <0.05 by Student's t test. C. U251 and T98G cells viability were measured by MTT assay. MTT cell viability was measured by the absorbance at 570 nm (ref: 670 nm) at indicated times after infecting 72 hrs. The values represent the means ± S.E. (n = 3), p<0.05 by two-way ANOVA. D. U251 and T98G cells viability were measured by cell colony formation assay. E. Quantification of (D). Values represent means ± SE (n = 3),*p <0.05 by Student's t test.
Figure 3
Figure 3. RCAN1 overexpression had no effect on cell cycle arrest
U251 and T98G cells were infected with lentivirus expressing RCAN1 (RCAN1) or negative control (Control) for 72 hrs. A. Proliferative cells shown by EDU staining (red) and nuclei were counterstained with DAPI (blue). Results were analyzed by a fluorescent microscope with ×100 magnification. Data represents mean ± S.E. (n = 3). B. Quantification of (A). Values represent means ± SE (n = 3),*p <0.05 by Student's t test. C. U251 and T98G cellswere stained with PI and analyzed for their DNA content using FACS Calibur. Values represent means ± SE (n = 3). The right lanes were quantification of (C). Values represent means ± SE (n = 3),*p <0.05 by Student's t test.
Figure 4
Figure 4. RCAN1 overexpression induced glioma cell lines apoptosis
A. U251 and T98G cells were infected with lentivirus expressing RCAN1 (RCAN1) or negative control (Control) for 72 hrs. TUNEL staining was used to indicate cell apoptosis (red color) (lane1 and lane2). Nuclei were counter stained with DAPI (blue color). Then the infected cells were further treated with 10μm H2O2 for 4 h (lane5 and lane6). About 20 ng/ml TNF-α was used to treat glioma cells infected with lentivirus for 3 min (lane 3 and lane4). Apoptotic cells were indicated by a magenta color, which corresponds to an overlap of red TUNEL and blue DAPI staining. The results were analyzed by a fluorescent microscope with ×100 magnification. B. Quantification of (A). Values represent means ± SE (n = 3), *p <0.05 by Student's t test. C. U251and T98G cells transfected with RCAN1expression plasmid, were stained with PI and Annexin V and analyzed by FACS to detect cell apoptosis. D. Quantification of (C). Values represent means ± SE (n = 3), *p <0.05 by Student's t test.
Figure 5
Figure 5. RCAN1 suppressed viability of glioma cells through inhibiting NF-κB signaling pathway
U251 and T98G cells were infected with lentivirus expressing RCAN1 (RCAN1) or negative control (Control) for 72 hrs. A. RCAN1 reduced the luciferase activity controlled by NF-κB. These cells infected by lentivirus expressing RCAN1were transfected with pNF-κBLuc. Renilla luciferase activity was used to normalize transfection efficiency. Dual luciferase assay was performed 24 h after transfection. Data were calculated from 3 experiments. Values represent the means ± S.E. (n = 3), *p <0.05 by Student's t test. B. Cells viability was measured by MTT assay. The values represent the means ± S.E. (n = 3), p<0.05 by two-way ANOVA. C. Nuclear proteins were extracted. NF-κB/p65 protein levels were detected by anti-p65 antibody. RCAN1 protein was detected by anti-RCAN1 antibody. β-actin was used as loading controls for cytosolfractions. TBP detected by anti-TBP was used as loading controls for nuclear proteins. D. Quantification of (C). Values represent the means ± S.E. (n = 3), *p <0.05 by Student's t test.
Figure 6
Figure 6. RCAN1 suppressed viability of glioma cells through inhibiting NF-κB signaling pathway
T98G cells were transfected with pSuper-based shRNA plasmid (siRCAN1) or negative control (siCon) for 48 hrs. A. The knockdown effect of psiRCAN1 was verified in protein level by Western blot. RCAN1 was detected with anti-RCAN1 antibody. β-actin was used as the internal control. The values represented the means ±S.E. (n=3).*p <0.05 by Student's t test. B. Quantification of (A). Values represent the means ± S.E. (n = 3), *p <0.05 by Student's t test. C. Nuclear proteins were extracted. NF-κB/p65 protein levels were detected by anti-p65 antibody. β-actin was used as loading controls for cytosolfractions. TBP detected by anti-TBP was used as loading controls for nuclear proteins. D. Quantification of (C). Values represent the means ± S.E. (n = 3),*p <0.05 by Student's t test. E. RCAN1 knockdown increased the luciferase activity controlled by NF-κB. These cells were cotransfected with siRCAN1 and pNF-κBLuc. Renilla luciferase activity was used to normalize transfection efficiency. Dual luciferase assay was performed 24 h aftertransfection. Data were calculated from 3 experiments. Values represent the means ± S.E. (n = 3),*p <0.05 by Student's t test. F. Cell viability was measured by MTT assay. The values represent the means ± S.E. (n = 3), p<0.05 by two-way ANOVA.

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