鸟苷三磷酸酶活化蛋白Git2对乳腺癌转移的影响

Effect of GTPase activating protein Git2 on metastasis in breast cancer

目的 探讨鸟苷三磷酸（GTP）酶活化蛋白Git2与乳腺癌转移的关系。方法 以Git2短发夹RNA慢病毒或Git2 cDNA分别对标记荧光素酶的乳腺癌细胞中Git2基因进行敲降或过表达，在雌性小鼠尾静脉或乳腺脂肪垫注射乳腺癌细胞建立乳腺癌转移模型，分析小鼠肿瘤的转移情况。结果 4T1、4TO7、168FARN和67NR乳腺癌细胞中，Git2 mRNA的相对表达水平分别为0.91±0.03、0.125±0.06、0.131±0.04和0.92±0.04，其中内源性低表达Git2蛋白的168FARN细胞和4TO7细胞过表达Git2后，上皮间充质细胞转化分子标记物E-cadherin表达被抑制，N-cadherin及vimentin的表达升高；而内源性高表达Git2蛋白的67NR细胞和4T1细胞敲降Git2后，E-cadherin的表达升高，N-cadherin和vimentin的表达降低。过表达Git2促进了4TO7细胞在肺中从微小转移发展为肿瘤转移灶；抑制Git2的表达使完全没有转移能力的67NR细胞获得向循环系统中内渗的能力，4T1细胞的转移灶克隆形成能力降低。敲降Git2基因的4T1细胞（4T1-luc-KD细胞）肺转移的光子数为（0.4±0.05）×106，低于对照组4T1-luc细胞[（3.0±0.04）×106]，差异有统计学意义（P〈0.05）。结论 Git2参与乳腺癌转移的启动和转移灶的克隆形成。

Objective To investigate the effect of GTPase activating protein Git2 on metastasis in breast cancer. Methods Git2 gene over-expression was induced by Git2 cDNA, and Git2 gene knockdown was induced by Git2 ShRNA lentivirus in four breast cancer cell lines. Six-week old wide type female mice were also used in this study. The cells were tagged with luciferase and injected into wide type female mice by tail vein or 4th mammary fat pad, respectively, to establish a cancer metastasis model. In vivo real time imaging system and immunohistoehemical staining were used to detect the cancer metastasis. Results The relative mRNA expression level of Git2 （normalized by GAPDH） in the 4T1,4TO7, 168FARN and 67NR cells were 0.91 ± 0.03, 0. 125 ± 0.06, 0.131± 0.04 and 0.92±0.04, respectively. The expression of EMT marker E-cadherin was inhibited and N-cadherin and vimentin were enhanced when Git2 was over-expressed in 168FARN cells and 4TO7 cells expressing low level of Git2, whereas the expression of E-cadherin was increased and N-cadherin and vimentin were decreased when Git2 was knocked down in 67NR cells and 4T1 ceils expressing high level of Git2. Furthermore, over-expression of Git2 promoted 4TO7 cells to progress from micro-metastasis to macro-metastasis. The down-regulation of Git2 pushed 67NR ceils to intravasate into blood circulation and suppressed the metastatic ability of 4T1 cells. The number of bloluminescence photos of lung metastatic 4T1-Luc-KD cells was （0.4±0.05） × 10^6, compared with （3.0±0.04） × 10^6 in the control 4T1-Luc cells, showing a significant difference （P〈0.05）. Conclusion Our results indicate that Git2 is involved in breast cancer initiation and metastatic colonization.