转移消失蛋白B与微囊蛋白-1在肝细胞癌中的相互作用

Interaction between missing in metastasis B and caveolin - 1 in hepatocellular carcinoma

目的探讨转移消失蛋白B（MIM—B）与微囊蛋白-1（caveolin-1）在肝细胞癌细胞中的相互作用。方法构建携带MIM—B与FLAG融合基因的pcDNA3．1载体，脂质体法转染Hep3B、SMMC7721细胞。通过细胞免疫荧光以及共聚焦显微镜检测MIM—B蛋白与caveolin-1蛋白共定位；应用内、外源免疫共沉淀确认相互作用；以Hep3B、SMMC7721以及MHCC97H细胞为研究模型，分成空载体组、MIM—B与FLAG融合基因组，进行免疫共沉淀实验。结果成功构建携带MIM—B与FLAG融合基因的pcDNA3．1载体，pcDNA3．1-MIM—B—FLAG转染效率为90．0％以上，显著表达MIM-B。共聚焦显微镜结果显示MIM—B能沉淀caveolin-1；MIM-B与caveolin-1共定位；内、外源免疫共沉淀结果显示MIM-B与caveolin-1相互作用。结论MIM-B与caveolin-1在肝癌细胞中共定位并相互作用。

Objective To investigate the protein interaction between missing in metastasis B （MIM -B） and caveolin - 1 in hepatocellular carcinoma （HCC）. Methods After the construction of pcDNA3. 1 vector carrying the fusion gene of FLAG- MIM -B, we transfected the vectors into Hep3B and SMMC7721 cells respectively. The immunofluorescence and confocal microscopy were applied in detecting distribution of MIM - B and caveolin - 1 in MHCC97H cells. The immunoprecipitation in vitro and in vivo was used to confirm the protein interaction between MIM -B and caveolin - 1 in Hep3B, SMMC7721 and MHCC97H cells. The cells were divided into empty vector group and FLAG - MIM - B fusion protein group. Results The pcDNA3.1 vector with FLAG - MIM - B fusion gene was successfully constructed and the transfection efficiency of pcDNA3. 1 - MIM - B - FLAG was over 90%. The results of Western blotting analysis demonstrated that the expression of MIM - B was significantly increased. The results of mass spec- trometry showed that MIM - B could precipitate caveolin - 1. The confocal microscopy analysis showed that MIM - B was co - localized with caveolin - 1. The immunoprecipitation in endo and in exo illustrated the interaction between MIM- B and caveolin- 1. Conclusion Our research found that MIM - B and caveolin - 1 co - localized and interacted in HCC cell lines, and it is possible to provide a new molecu- lar target therapy for HCC.