Bit1 knockdown contributes to growth suppression as well as the decreases of migration and invasion abilities in esophageal squamous cell carcinoma via suppressing FAK-paxillin pathway.
Bit1 knockdown contributes to growth suppression as well as the decreases of migration and invasion abilities in esophageal squamous cell carcinoma via suppressing FAK-paxillin pathway.
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DOI：
10.1186/s12943-016-0507-5
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发表时间：
2016-03-08
期刊：
影响因子：
37.3
通讯作者：
Liu H
中科院分区：
文献类型：
--
作者：
Fan T;Chen J;Zhang L;Gao P;Hui Y;Xu P;Zhang X;Liu H
越来越多的证据表明，Bit1在人类癌症的发生和发展中发挥着不同的作用。虽然在我们先前的研究中，Bit1在食管癌组织中高度表达，但其在食管癌发生发展中的作用和分子机制仍不清楚。Western blotting检测Bit1蛋白在ESCC细胞系和正常食道上皮细胞中的表达。Western blotting检测Bit1 shRNA介导的Bit1蛋白表达。采用四甲基偶氮唑盐比色法、迁移实验、侵袭实验、ELISA法和流式细胞术分别检测Bit1基因敲除对细胞增殖、迁移、侵袭和凋亡的影响。采用异种移植模型检测体内致瘤性，免疫组织化学和原位末端标记法检测相关蛋白表达和细胞凋亡情况。采用Agilent SurePrint G3人GE8 × 60K芯片检测基因芯片，免疫沉淀法检测Bit1与FAK蛋白的相互作用，Western blotting检测FAK-PXLING通路关键蛋白的表达。我们发现在所有的人鳞状细胞癌细胞系中，Bit1的表达均显著高于正常食道上皮细胞Het-1a(P <； 0.05)，其中EC9706Bit1的表达水平最高。在第1天，针对Bit1的特异性shRNA显著下调了Bit1的蛋白水平(P <； 0.05)。在第2、3天，Bit1 shRNA表达水平达到最低值。此外，Bit1缺失在体外和体内均可抑制EC9706和TE1细胞的生长，降低细胞迁移和侵袭能力，并诱导细胞凋亡。更重要的是，不同剂量的Bit1 shRNA处理EC9706裸鼠移植瘤组织后，Bit1表达下调显著降低了移植瘤组织中Bcl2和MMP2的表达，同时也引发了肿瘤细胞的凋亡。进一步的基因芯片显示，Bit1-RNAi组有23个基因明显下调，16个基因明显上调。值得注意的是，Bit1与EC9706细胞中的FAK蛋白存在内在的相互作用。此外，Bit1 shRNA组在mRNA和蛋白水平下调了paxlin，并降低了FAK的mRNA和蛋白表达。Bit1可能通过靶向FAK-paxlin通路对ESCC的生长、凋亡、迁移和侵袭起重要调节作用，因此联合操作Bit1和FAK-paxlin通路可能成为ESCC治疗的新靶点。
There is growing evidence that Bit1 exerts different roles in the development and progression of human cancers. Although Bit1 was highly exhibited in ESCC tissues in our previous study, its roles and molecular mechanisms implicated in development and progression of ESCC remain unknown. Bit1 protein expression in ESCC cell lines and normal esophageal epithelial cell was detected by Western blotting. Bit1 protein expression mediated by Bit1 shRNA was investigated by Western blotting. MTT, migration assay, invasion experiment, ELISA and Flow cytometry were utilized to determine the effects of Bit1 knockdown on cell proliferation, migration, invasion and apoptosis, respectively. A xenograft model was used to examine in vivo tumourigenicity, and immunohistochemistry and TUNEL were utilized to evaluate the related protein expression and apoptosis. Gene microarray was determined by Agilent SurePrint G3 Human GE 8 × 60 K Microarray, the interaction of Bit1 and FAK proteins were detected by Immunoprecipitation and the key protein expressions of FAK-paxillin pathway were detected by Western blotting. We found Bit1 expression in all human ESCC cell lines tested was significantly higher than that in normal esophageal epithelial cell Het-1A (P < 0.05), in which EC9706 presented the highest Bit1 level. Bit1 protein level was significantly downregulated at day 1 after transfection with specific shRNA against Bit1 (P < 0.05). At days 2 and 3, Bit1 level reached the lowest value after transfection with Bit1 shRNA. Moreover, Bit1 depletion contributed to growth inhibition in vitro and in vivo, reduced cell migration and invasion abilities, and induced cell apoptosis in EC9706 and TE1 cells. More importantly, Bit1 downregulation significantly lowered Bcl-2 and MMP-2 levels in EC9706 xenografted tumor tissues, meanwhile triggered apoptosis after treatment with different doses of Bit1 shRNA. Further gene microarray revealed that 23 genes in Bit1-RNAi group were markedly downregulated, whereas 16 genes were obviously upregulated. Notably, Bit1 intrinsically interacted with FAK protein in EC9706 cells. Moreover, paxillin was downregulated at mRNA and protein levels in Bit1 shRNA group, coupled with the decreases of FAK mRNA and protein expressions. Bit1 may be an important regulator in cell growth, apoptosis, migration and invasion of ESCC via targeting FAK-paxillin pathway, and thereby combinative manipulation of Bit1 and FAK-paxillin pathway may be the novel and promising therapeutic targets for the patients with ESCC.