Inhibition of EGR1 inhibits glioma proliferation by targeting CCND1 promoter

Abstract

Background: Gliomas are the most common primary tumors in central nervous system. The prognosis of the patients with glioma is poor regardless of the development of therapeutic strategies. Its aggressive behavior mainly depends on the potent ability of proliferation. The transcription factor EGR1 (early growth response 1) is a member of a zinc finger transcription factor family which plays an essential role in cell growth and proliferation.

Methods: EGR1 expression levels in 39 glioma tissues and 10 normal brain tissues were tested by RT-qPCR and Western-blotting. The effects of EGR1 on U251 cells, U251 stem-like cells (GSCs), and U87 cells proliferation were assessed using in vitro and in vivo cell proliferation assays. The specific binding between EGR1 and CCND1 promoter was confirmed by CHIP assay. EGF was used to improve EGR1 expression in this assay.

Results: EGR1 expression levels in human gliomas are decreased compared with normal brain tissues, however, the patients with low EGR1 expression level showed significantly enhanced patient survival in all glioma patients. EGR1 silencing inhibited proliferation and induced G1 phase arrest in glioma cells. EGR1 contributed to proliferation by directly raising CCND1. Meanwhile, EGR1 overexpression induced by EGF was able to promote the proliferation of glioma cells.

Conclusions: Our results show that stable knockdown EGR1 would inhibit glioma proliferation. The results suggest EGR1 showing lower expression in cancer tissues compared with normal tissues maybe still play an important role in tumor proliferation.

Keywords: CCND1; EGR1; Glioma; Proliferation.

Fig. 1

Expression of EGR1 in GBMLGG. a The mRNA levels of EGR1 in giloma tissues and normal brain tissues. P = 0.024. b The mRNA levels of EGR1 in giloma tissues and normal brain tissues, data come from TCGA databases. c Immunoblot analysis of EGR1 total protein levels in glioma tissues and normal brain tissues. d Relative protein levels of EGR1 were determined by Western blotting. The levels of EGR1 were normalized to those of β-ACTIN. P = 0.0447. e. Kaplan-Meier analysis of the GBMLGG RNA-seq data were from the The Cancer Genome Atlas (TCGA) databases. *P < 0.05, (mean ± SEM, Student’s t-test)

Fig. 2

Targeting EGR1 by RNA interference inhibited the proliferation of glioma cells. a Real-time quantitative PCR analysis of EGR1 in U251, U251SLC, and U87 cells transfected with control siRNA vector and siEGR1-vector. b Immunoblot analysis of EGR1 in U251, U251SLC, and U87 cells transduced with control vector and siEGR1-vector. c, d The rates of cell growth were detected by CCK8 assay in U87 and U251 cells transfected with control siRNA or EGR1 siRNA. e Cell proliferation rates as determined by EDU assay in U87, U251 and U251SLC cells transfected with control siRNA or EGR1 siRNA. f Cell cycle analyzed by propidium iodide staining and flow cytometry in U87, U251 or U251SLC cells transfected with control siRNA or siRNA targeting EGR1. (At least three repeated experiments for the all cell types). *P < 0.05, **P < 0.01, and ***P < 0.001 (mean ± SEM)

Fig. 3

CCND1 expression was regulated by EGR1 that directly bound in the promotor of CCND1 in glioma cells. Lysates of U87, U251 and U251SLC cells expressing NT control siRNA, siEGR1 were analyzed by real-time quantitative PCR (a) and by immunoblotting(b). siRNA-mediated knockdown of EGR1 inhibits CCND1 expression. c Chromatin fragments from U251 cells were immunoprecipitated (with antibodies specific to RNA polymerase II (anti- RNA polymerase II; positive control), mouse IgG (IgG, negative control), and EGR1 (anti-EGR1) as indicated. Input, 1% total DNA. After reversal of cross-linking, the immunoprecipitated DNA was amplified by PCR using the specific primers and resolved on 2% agarose gels. ChIP assay demonstrated that EGR1 protein bound to the promoter region of the CCND1 gene. d The immunoprecipitated DNA of U251SLC cells expressing negative control siRNA and siEGR1 was amplified by PCR using the specific primers and resolved on 2% agarose gels. e RT-qPCR analyses the immunoprecipitated DNA of NC-U251SLC and siEGR1-U251SLC. Values were expressed relative to percent input. *P < 0.05, **P < 0.01, and, ***P < 0.001 (mean ± SEM)

Fig. 4

EGR1 RNAi suppressed the growth of glioma xenograft tumor in vivo. a siEGR1-U251 cells (5 × 10⁶) and NC-U251 (5 × 10⁶) cells were inoculated subcutaneously into armpit of BALB/C nude mice, all of the mice examined developed tumors at 50th day. In NC-U251SC (1 × 10⁶) group, all mice developed xenograft tumors at 40th day. In contrast, in siEGR1-U251SCL (1 × 10⁶) group, only 3 mice developed xenograft tumors at 40th day. b Mice were euthanized and implanting tumors were harvested, and examined. siEGR1-U251-derived tumors showed smaller tumor volume compared with the volume of NC-U251-derived tumors, P < 0.05 *. c siEGR1-U251SLC-derived tumors also showed smaller tumor volume compared with the volume of NC-U251SLC-derived tumors, P < 0.01 **. d H&E staining and GFAP immunohistochemistry of siEGR1-U251-derived tumors and NC-U251-derived tumors. e E. Ki-67, EGR1, CCND1 staining of subcutaneous tumors in NC-U251 and siEGR1-U251 groups. Scale bar = 50 μm

Fig. 5

Overexpression of EGR1 induced by EGF improve proliferation of glioma cells. a Real-time quantitative PCR for EGR1 and CCND1 mRNA expression in U251 cells and U251SLCs after adding EGF. β-ACTIN was used as the loading control. b Immunoblots for EGR1 and CCND1 mRNA expression in U251 cells and U251SLCs after adding EGF. β-ACTIN was used as the loading control. c EdU assay for the proliferation of U251 cells group and U251SLCs group with/without EGF. *P < 0.05, **P < 0.01, ***P < 0.001 (mean ± SEM)