癌症进展
癌癥進展
암증진전
ONCOLOGY PROGRESS
2014年
4期
394-398
,共5页
刘健%王海娟%刘群%李春晓%刘欢%黄骁舾%孟希亭%赵枚%林晨%黄常志%钱海利
劉健%王海娟%劉群%李春曉%劉歡%黃驍舾%孟希亭%趙枚%林晨%黃常誌%錢海利
류건%왕해연%류군%리춘효%류환%황효서%맹희정%조매%림신%황상지%전해리
MTA1%NuRD复合体%相互作用%In Situ PLA技术
MTA1%NuRD複閤體%相互作用%In Situ PLA技術
MTA1%NuRD복합체%상호작용%In Situ PLA기술
MTA1%NuRD complex%interaction%in situ PLA technology
目的：原位检测肿瘤转移相关蛋白MTA1与NuRD复合体其他组分-Mi2、HDAC2及MBD3的相互作用并分析MTA1/NuRD复合体在HCT116细胞中的分布情况。方法首先利用co-IP技术体外验证MTA1与NuRD复合体其他组分Mi2、HDAC2及MBD3在HCT116细胞裂解液中的相互作用。然后利用In Situ PLA技术，体内原位检测MTA1与三者的相互作用，并根据镜下阳性荧光信号数目，比较MTA1与Mi2、HDAC2及MBD3之间的作用强弱；根据相互作用位点的亚细胞分布情况，分析间期及M期MTA1/NuRD复合体的核质分布情况。结果首次从体内原位水平证实MTA1与NuRD复合体其他组分-Mi2、HDAC2及MBD3的相互作用；其作用强度HDAC2最高，Mi2次之，MBD3最弱；首次发现MTA1/NuRD复合体存在很明显的胞质分布（约占总量的1/4）；另外，我们还首次发现在M期MTA1/NuRD复合体不再位于核区，而是位于染色体外周。结论In Situ PLA技术是一项有效的原位检测蛋白相互作用的新技术；MTA1/NuRD复合体可能参与MTA1胞质中功能的发挥；并且其胞质分布可能参与M期进程调控。
目的：原位檢測腫瘤轉移相關蛋白MTA1與NuRD複閤體其他組分-Mi2、HDAC2及MBD3的相互作用併分析MTA1/NuRD複閤體在HCT116細胞中的分佈情況。方法首先利用co-IP技術體外驗證MTA1與NuRD複閤體其他組分Mi2、HDAC2及MBD3在HCT116細胞裂解液中的相互作用。然後利用In Situ PLA技術，體內原位檢測MTA1與三者的相互作用，併根據鏡下暘性熒光信號數目，比較MTA1與Mi2、HDAC2及MBD3之間的作用彊弱；根據相互作用位點的亞細胞分佈情況，分析間期及M期MTA1/NuRD複閤體的覈質分佈情況。結果首次從體內原位水平證實MTA1與NuRD複閤體其他組分-Mi2、HDAC2及MBD3的相互作用；其作用彊度HDAC2最高，Mi2次之，MBD3最弱；首次髮現MTA1/NuRD複閤體存在很明顯的胞質分佈（約佔總量的1/4）；另外，我們還首次髮現在M期MTA1/NuRD複閤體不再位于覈區，而是位于染色體外週。結論In Situ PLA技術是一項有效的原位檢測蛋白相互作用的新技術；MTA1/NuRD複閤體可能參與MTA1胞質中功能的髮揮；併且其胞質分佈可能參與M期進程調控。
목적：원위검측종류전이상관단백MTA1여NuRD복합체기타조분-Mi2、HDAC2급MBD3적상호작용병분석MTA1/NuRD복합체재HCT116세포중적분포정황。방법수선이용co-IP기술체외험증MTA1여NuRD복합체기타조분Mi2、HDAC2급MBD3재HCT116세포렬해액중적상호작용。연후이용In Situ PLA기술，체내원위검측MTA1여삼자적상호작용，병근거경하양성형광신호수목，비교MTA1여Mi2、HDAC2급MBD3지간적작용강약；근거상호작용위점적아세포분포정황，분석간기급M기MTA1/NuRD복합체적핵질분포정황。결과수차종체내원위수평증실MTA1여NuRD복합체기타조분-Mi2、HDAC2급MBD3적상호작용；기작용강도HDAC2최고，Mi2차지，MBD3최약；수차발현MTA1/NuRD복합체존재흔명현적포질분포（약점총량적1/4）；령외，아문환수차발현재M기MTA1/NuRD복합체불재위우핵구，이시위우염색체외주。결론In Situ PLA기술시일항유효적원위검측단백상호작용적신기술；MTA1/NuRD복합체가능삼여MTA1포질중공능적발휘；병차기포질분포가능삼여M기진정조공。
Objective To detect the in situ interactions of tumor metastasis associated protein MTA1 with other NuRD complex components: -Mi2, HDAC2 and MBD3 and analyze the subcellular distribution of MTA1/NuRD in HCT116 cells. Method The interaction of MTA1 with Mi2, HDAC2 and MBD3 in HCT116 cell lysate was first vali-dated by in vitro co-IP analysis and then further detected by in vivo visualization using the PLA technology. The strength of interactions were compared by number analysis of the positive signals; moreover, the subcellular distribu-tion of MTA1/NuRD in interphase and mitosis was analyzed by localization analysis of the positive signals. Result We, for the first time, validated the interaction of MTA1 with NuRD components at in vivo level; For the interaction strength, MTA1-HDAC2 was the highest (363 positive signals / 50 cells), followed by MTA1-Mi2 (280 positive sig-nals / 50 cells), and the weakest was MTA1-MBD3 (208 positive signals / 50 cells); Also it was the first time that MTA1/NuRD was found with obvious cytoplasm distribution which accounted for about 1/4 of the total; In addition, we also first noticed that during mitosis, MTA1/NuRD was no longer located in the nuclear area, but at the periph-ery of the chromosome which was different from interphase. Conclusion The in situ PLA is an effective new tech-nology to detect the in vivo interaction of two proteins. MTA1 may also possess cytoplasmic functions through NuRD complex; And MTA1/NuRD may play a role in controlling mitosis process in the cytoplasm.
