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The Effect of Melanoma-associated Antigen A11(MAGE-A11) on the Transcriptional Activity and Stability of P53

Author: XuYingYing
Tutor: DanBaoEn; AiJun
School: Hebei Medical University
Course: Clinical Laboratory Science
Keywords: Melanoma-associated antigen MAGE-A11 p53 MDA-MB-231 Luciferase reporter assay
CLC: R737.9
Type: Master's thesis
Year: 2013
Downloads: 10
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Objective: p53is one of the most studied tumor suppressors in the cancerresearch field. Researches revealed that over50%of human tumors carry p53mutations. p53has an important role in cell cycle-contronlling pathways, suchas apoptosis, cell growth arrest and senescence. Currently, the involvement ofMAGE proteins in the reguation of p53and the apoptotic response wascorroborated by other independent studies. Researches revealed thatMAGE-A2protein was able to bind to p53, and it may recruit histonedeacetylases to p53-binding sites, thereby MAGE-A2repressed p53transcriptional activity. Researches revealed that MAGE-A3repressed p53bybinding to KAP1. However, some researches also revealed that MAGE-A4had some different functions, that MAGE-A4enhanced p53transcriptionalactivity and functions.In the paper, we investigated the effect of MAGE-A11on thetranscriptional activity and stability of p53, and we focus on the mechanismsproposed to explain how MAGE-A11happens in cancer development.Methods:1Gene transfection, reverse transcription-polymerase chain reaction(RT-PCR), and western-blot were used to detect the expression of p21WAF1mRNA and protein in human breast cancer cell line MDA-MB-231.2Gene transfection and luciferase reporter assay were used to evaluatethe effect of MAGE-A11on the transcriptional activity of p53in p53-mutanthuman breast cancer cell line MDA-MB-231.3Gene transfection and colony formation test was used to evaluate theeffect of MAGE-A11on the transcriptional activity of p53in p53-mutanthuman breast cancer cell line MDA-MB-231.4RT-PCR, and western-blot were used to used to detect the expression of p53, and to research the effect of MAGE-A11on p53stability.5Gene transfection and western-blot protein decay analysis bycycloheximide were used to research the effect of MAGE-A11on p53stabilityin p53-mutant human breast cancer cell line MDA-MB-231.Results:1In human breast cancer cell line MDA-MB-231, RT-PCR showed thatp21WAF1mRNA expressions in the group with p53transfection alone wereincreased compared with control group (p<0.05), the result showed that p53plasmid was transfected well; p21WAF1mRNA expressions in the groups withp53transfection alone and p53and MAGE-A11co-transfection were notsignificantly different (p>0.05); p21WAF1mRNA expressions in the group withMAGE-A11transfection alone were not significantly different compared withthe control group (p>0.05). These results showed that MAGE-A11had noeffect on p21WAF1mRNA expressions.Western-blot showed that p21WAF1protein expressions in the group withp53transfection alone were increased compared with control group (p<0.05),the result showed that p53plasmid was transfected well; p21WAF1proteinexpressions in the groups with p53transfection alone and p53andMAGE-A11co-transfection were not significantly different (p>0.05);p21WAF1protein expressions in the group with MAGE-A11transfection alonewere not significantly different compared with the control group (p>0.05).These results showed that MAGE-A11had no effect on p21WAF1proteinexpressions.2Luciferase reporter assay showed, the luciferase activities induced byp21WAF1promotor were1.00±0.00after transfection of empty plasmids; theluciferase activities induced by p21WAF1promotor were27.72±1.03aftertransfection of25ng p53, significantly higher than in control MDA-MB-231cells (p<0.05); the luciferase activities induced by p21WAF1promotorrespectively were48.90±4.77,60.11±2.66,51.47±4.52after co-transfectionof25ng p53and MAGE-A11(100,200,400ng), significantly higher than incontrol MDA-MB-231and p53-transfected MDA-MB-231cells (p<0.05); the luciferase activities induced by p21WAF1promotor respectively was1.86±0.71after transfection of400ng MAGE-A11, not significantly different comparedwith the empty plasmids control group (p>0.05). These results showed thatexogenous MAGE-A11enhanced p53transcriptional activity.3In human breast cancer cell line MDA-MB-231, the number of coloniesin the groups with empty plasmids transfection, p53transfection alone, p53and MAGE-A11co-transfection and MAGE-A11transfection alone were121.67±2.08,60.33±1.53,26.33±1.53and127.00±2.00, respectively.Colony formation test showed that the colony numbers were decreased inMDA-MB-231cells after transfection with p53compared with the control(p<0.05), that the colony numbers were further decreased in MDA-MB-231cells after co-transfection of p53and MAGE-A11compared with the groupthat tranfected with p53alone (p<0.05). These results showed that exogenousMAGE-A11enhanced p53transcriptional activity and funtions.4In human breast cancer cell line MDA-MB-231, p53mRNAexpressions in the group with1μg p53transfection were increased comparedwith the control group (p<0.05), the result showed that p53plasmid wastransfected well; p53mRNA expressions in the groups with1μg p53transfection,1μg p53and1μg MAGE-A11transfection,1μg p53and2μgMAGE-A11transfection were not significantly different (p>0.05).Western-blot showed that p53protein expressions in the p53-transfectedMDA-MB-231cells were increased compared with the control (p<0.05), theresult showed that p53plasmid was transfected well; p53protein expressionswere increased in MDA-MB-231cells after co-transfection with1μg p53and1μg MAGE-A11compared with p53-transfected MDA-MB-231cells (p<0.05)and p53protein expressions were increased in MDA-MB-231cells afterco-transfection of1μg p53and2μg MAGE-A11compared with the group thattranfected with1μg p53and1μg MAGE-A11(p<0.05). These results showedthat MAGE-A11had no effect on p53mRNA expressions, but thatMAGE-A11had the increased effect on p53protein expressions.5In human breast cancer cell line MDA-MB-231, after transfection of p53and cycloheximide treatment for0h,1h,2h,4h,6h, the ratios of p53andGAPDH were0.978±0.002,0.787±0.002,0.477±0.001,0.345±0.003and0.285±0.002, respectively; while after transfection of p53and MAGE-A11and then cycloheximide treatment for0h,1h,2h,4h,6h, the ratios of p53andGAPDH were1.019±0.001,0.976±0.001,0.894±0.004,0.678±0.001and0.499±0.003, respectively (p<0.05). These results revealed that MAGE-A11can increase the stability of p53.Conclusion:1Exogenous MAGE-A11enhanced p53transcriptional activity.2MAGE-A11can increase the stability of p53on protein level.

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