中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
9期
1926-1928
,共3页
卢建华%罗琨%冯梦宇%杨娟%张波
盧建華%囉琨%馮夢宇%楊娟%張波
로건화%라곤%풍몽우%양연%장파
BRIT1%p53%DNA损伤%乳腺癌
BRIT1%p53%DNA損傷%乳腺癌
BRIT1%p53%DNA손상%유선암
BRIT1%p53%DNA damage%Breast cancer
目的 探讨BRIT1基因在乳腺癌细胞中的功能,并研究其可能通过调控p53蛋白的活性发挥肿瘤抑制作用.方法 构建BRIT1真核表达载体p3×FLAG-CMV-BRIT1,实现BRIT1基因的过表达,检测p53蛋白的表达;以及利用小干扰RNA(siRNA)技术敲低了MCF10A细胞中BRIT1基因的表达,建立DNA损伤模型后检测p53蛋白在DNA损伤时的反应.利用体外细胞增殖实验观察BRIT1的肿瘤抑制功能是否依赖p53蛋白的活性.结果 BRIT1过表达可以上调p53蛋白的活性,敲低BRIT1的功能使p53蛋白的表达下降;当面临DNA损伤时,BRIT1的功能缺陷能够阻碍p53的表达水平.当细胞具备正常p53功能时,BRIT1基因的过表达可以显著抑制细胞的增殖(10.55±0.12比15.83±0.11,P＜0.05);而当p53蛋白表达被降低时,BRIT1基因的肿瘤抑制功能降低(13.84 ±0.09比10.55±0.12,P＜0.05).结论 BRIT1基因不仅调控p53蛋白的活性,而且依赖野生型p53基因的功能来发挥肿瘤抑制作用.
目的 探討BRIT1基因在乳腺癌細胞中的功能,併研究其可能通過調控p53蛋白的活性髮揮腫瘤抑製作用.方法 構建BRIT1真覈錶達載體p3×FLAG-CMV-BRIT1,實現BRIT1基因的過錶達,檢測p53蛋白的錶達;以及利用小榦擾RNA(siRNA)技術敲低瞭MCF10A細胞中BRIT1基因的錶達,建立DNA損傷模型後檢測p53蛋白在DNA損傷時的反應.利用體外細胞增殖實驗觀察BRIT1的腫瘤抑製功能是否依賴p53蛋白的活性.結果 BRIT1過錶達可以上調p53蛋白的活性,敲低BRIT1的功能使p53蛋白的錶達下降;噹麵臨DNA損傷時,BRIT1的功能缺陷能夠阻礙p53的錶達水平.噹細胞具備正常p53功能時,BRIT1基因的過錶達可以顯著抑製細胞的增殖(10.55±0.12比15.83±0.11,P＜0.05);而噹p53蛋白錶達被降低時,BRIT1基因的腫瘤抑製功能降低(13.84 ±0.09比10.55±0.12,P＜0.05).結論 BRIT1基因不僅調控p53蛋白的活性,而且依賴野生型p53基因的功能來髮揮腫瘤抑製作用.
목적 탐토BRIT1기인재유선암세포중적공능,병연구기가능통과조공p53단백적활성발휘종류억제작용.방법 구건BRIT1진핵표체재체p3×FLAG-CMV-BRIT1,실현BRIT1기인적과표체,검측p53단백적표체;이급이용소간우RNA(siRNA)기술고저료MCF10A세포중BRIT1기인적표체,건립DNA손상모형후검측p53단백재DNA손상시적반응.이용체외세포증식실험관찰BRIT1적종류억제공능시부의뢰p53단백적활성.결과 BRIT1과표체가이상조p53단백적활성,고저BRIT1적공능사p53단백적표체하강;당면림DNA손상시,BRIT1적공능결함능구조애p53적표체수평.당세포구비정상p53공능시,BRIT1기인적과표체가이현저억제세포적증식(10.55±0.12비15.83±0.11,P＜0.05);이당p53단백표체피강저시,BRIT1기인적종류억제공능강저(13.84 ±0.09비10.55±0.12,P＜0.05).결론 BRIT1기인불부조공p53단백적활성,이차의뢰야생형p53기인적공능래발휘종류억제작용.
Objective To investigate the role of BRIT1 as a tumor suppressor in breast cancer and to discover that BRIT1 functions as a regulator of p53 expression.Methods BRIT1 over-expressing U-2OS cell line was established by FLAG-tagged BRIT1 expression vector transfection.BRIT1 knockdown was established by BRIT1-specific siRNA in non-tumorigenic breast epithelial MCF10A cells,and p53 level was detected by Western blotting.Methyl thiazol tetrazolium (MTF) assay was used to measure the cell proliferation and the optical density was measured spectrophotometrically at 570 nm.Results p53 protein expression was significantly increased in the BRIT1-overexpressing U-2OS cells and reduced in the BRIT1 knockdown MCF10A cells.Transient BRIT1 knockdown MCF10A cells were treated with UV radiation and cell lysates were harvested at indicated time points.It was found that UV DNA damage signals induced p53 expression in control cells.However,this induction of p53 was markedly reduced in BRIT1-deficient cells.Moreover,in cells with wild-type p53,BRIT1 had a much stronger tumor suppression effect than in cells with p53 depletion (10.55 ±0.12 vs.15.83 ±0.11) (P＜0.05).Conclusion BRIT1 could not only regulate the activity of p53 protein,but also could function as a tumor suppressor in p53-dependent pathways.
