Roles of E2F transcription factor 1 in regulating cellular senescence of neuroblastoma and its mech-anisms
Objective To investigate regulatory effect and mechanisms of E2F transcription factor 1(E2F1)on cellular senescence in neuroblastoma(NB).Methods The human neuroblastoma cell line SK-N-BE(2)(CRL-2271)was purchased from the American Type Culture Collection(Rockville,MD).NB cells were divided into empty vector(mock),E2F1 over-expression,scramble short hairpin RNA(shRNA),sh-E2F1 #1,and sh-E2F1 #2 groups,while stable cells were screened.The E2F1 expression was detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)and Western blotting.Kaplan-Meier Plot,ChEA3 and other databases were used to search possible target genes.Senescence-associated β-galactosidase(SA-β-Gal)staining was performed for 12 h to detect β-gal-actosidase-positive senescent cells.5-ethynyl-2'-deoxyuridine(EdU)incorporation for 2 h was applied for assessing cellular proliferation.Incubation within soft agar for 3 weeks was undertaken to assess cell growth.Matrigel transwell assay for 24 h was applied for observing cellular invasion capability.Student's t-test was applied for statistical analysis.Results In subclonal lines over-expressing E2F1,the transcript and protein levels increased compared with mock group(3.24±0.12 vs.1.00±0.04,t=32.06,P＜0.05).Meanwhile,its transcript levels were reduced in E2F1 silencing groups as compared with sh-Scb group(0.32±0.08 vs.1.00±0.03,t=14.24,P＜0.05;0.33±0.08 vs.1.00±0.03,t=13.80,P＜0.05).Similarly,its protein levels were reduced.As compared with the mock group,insulin like growth factor binding protein 2(IGFBP2)transcript and protein levels increased in E2F1 over-expression group(2.17±0.15 vs.1.02±0.00,t=13.00,P＜0.05).DNA polymerase gamma catalytic subunit(POLG)transcript and protein levels increased in E2F1 over-expression group(2.38±0.11 vs.1.02± 0.01,t=15.20,P＜0.05).Senescent cell positivity decreased(6.66±1.52 vs.23.33±2.51,t=9.80,P＜0.05),and proliferation rate of tumor cells increased(51.66±1.52 vs.23.00±4.00,t=11.60,P＜0.05).The number of clone formation and infiltrating cells increased,respectively(334.66± 6.11 vs.157.66±4.16,t=41.46,P＜0.05;318.33±8.02 vs.126.33±5.50,t=34.18,P＜0.05).In contrast,IGFBP2 transcript and protein expression levels were reduced(0.38±0.06 vs.1.02±0.01,t=17.05,P＜0.05;0.33±0.05 vs.1.02±0.01,t=19.76,P＜0.05)in sh-E2F1 group when compared with scramble shRNA group.POLG transcript levels were reduced(0.50±0.02 vs.1.02±0.01,t=30.35,P＜0.05;0.35±0.05 vs.1.02±0.01,t=20.72,P＜0.05)in sh-E2Fl group,as well as protein levels.Senescent cell positivity increased(49.66±3.05 vs.18.66±1.52,t=15.72,P＜0.05;40.33±3.05 vs.18.66±1.52,t=10.99,P＜0.05),and cellular proliferation slowed down(3.33±2.31 vs.19.33±1.52,t=10.01,P＜0.05;3.33±1.52 vs.19.33±1.52,t=12.83,P＜0.05).There was a decrease in the number of clone formation(52.00±6.08 vs.127.00± 3.60,t=18.37,P＜0.05;51.33±5.03 vs.127.00±3.60,t=51.33,P＜0.05)and a decrease in the number of infiltrating cells(44.33±2.08 vs.109.66±7.37,t=14.77,P＜0.05;52.00±5.56 vs.109.66±7.37,t=10.81,P＜0.05).Conclusion Transcription factor E2F1 regulates the expression of senescence-related genes IGFBP2 and POLG,thus inhibiting senescence of NB cells.
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