Hint1在体外通过调控Girdin活性抑制人肝癌细胞迁移侵袭
Hint1在体外通过调控Girdin活性抑制人肝癌细胞迁移侵袭
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DOI：
10.1007/s13277-016-5336-z
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发表时间：
2016
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影响因子：
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通讯作者：
Lin Wang
中科院分区：
文献类型：
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作者：
Xue-Song Wu;Tian-Hao Bao;Yang Ke;De-Yun Sun;Zhi-Tian Shi;Hao-Ran Tang;Lin Wang
组氨酸三联体核苷酸结合蛋白1 （Hint1）是一种单倍不足的肿瘤抑制基因。它在癌细胞迁移中的作用以前没有被推测过。在本研究中，我们检测了Hint1在肝细胞癌（HCC）患者转移性和非转移性淋巴结中的表达，进一步阐明了Hint1表达对girdin表达、AKT和ERK1/2磷酸化以及肝癌细胞体外迁移的影响。RT-PCR检测原发性HCC组织、转移性和非转移性淋巴结中Hint1和Girdin的表达。用过表达Hint1或靶向Hint1、girdin、Hint1 + girdin或打乱RNA的小干扰RNA （siRNA）质粒载体转染HepG2细胞。采用创面法和Transwell法检测肝癌细胞的迁移和侵袭。免疫荧光法和免疫印迹法检测蛋白表达。RT-PCR检测显示，与原发性肝癌组织和非转移性淋巴结相比，Hint1 mRNA转录水平明显降低（P<0.01）。相比之下，girdin mRNA转录物水平显著高于非转移淋巴结（P<0.05）。此外，siRNA敲低HINT1导致HepG2细胞中girdin mRNA转录水平显著升高（P<0.05）。伤口实验和Transwell实验显示，与转染混乱siRNA的HepG2细胞相比，siRNA敲低Hint1显著增强了HepG2细胞的迁移和侵袭能力。Hint1敲除后，HepG2细胞中girdin和AKT的磷酸化水平也显著升高（P<0.05），而siRNA敲除girdin可有效抑制其磷酸化水平（P<0.05）。Hint1在转移性淋巴结中下调，并通过调节Girdin和AKT的表达和磷酸化参与肝癌细胞的迁移和侵袭。Hint1-girdin-AKT信号轴在HCC迁移和侵袭中的作用有待进一步研究，并可能成为抑制肿瘤生长和转移的治疗靶点。
Histidine triad nucleotide-binding protein 1 (Hint1) is a haploinsufficient tumor suppressor gene. Its role in cancer cell migration has not been previously speculated. In the current study, we examined the expression of Hint1 in metastatic and non-metastatic lymph nodes of hepatocellular carcinoma (HCC) patients and further elucidated the effect of Hint1 expression on girdin expression and phosphorylation of AKT and ERK1/2 and on the migration of HCC cells in vitro. Expression of Hint1 and Girdin in primary HCC tissues and metastatic and non-metastatic lymph nodes was determined by RT-PCR assays. HepG2 cells were transfected with plasmid vectors overexpressing Hint1 or small interfering RNA (siRNA) targeting hint1, girdin, hint1 plus girdin, or the scrambled RNA. Migration and invasion of HCC cells were examined by wound and Transwell assays. Protein expression was detected by immunofluorescence and immunoblotting assays. RT-PCR assays revealed that the mRNA transcript levels of Hint1 were markedly lower than those of primary HCC tissues and non-metastatic lymph nodes (P<0.01). By contrast, the mRNA transcript levels of girdin were significantly higher than non-metastatic lymph nodes (P<0.05). Furthermore, siRNA knockdown of HINT1 resulted in a significant increase in the mRNA transcript levels of girdin in HepG2 cells (P<0.05). Wound assays and Transwell assays showed that Hint1 knockdown by siRNA significantly enhanced the migration and invasion of HepG2 cells compared to HepG2 cells transfected with scrambled siRNA. Hint1 knockdown also led to significantly increased phosphorylation of girdin and AKT in HepG2 cells (P<0.05), which, however, was effectively aborted by Girdin knockdown by siRNA (P<0.05). Hint1 is downregulated in metastatic lymph nodes and is implicated in migration and invasion of HCC cells in vitro by modulating Girdin and AKT expression and phosphorylation. The Hint1-girdin-AKT signaling axis should be further dissected for its role in HCC migration and invasion and may be therapeutically targeted to suppress tumor growth and metastasis.