Trim72基因在肝癌转移中的功能研究

The function of Trim72 gene in hepatocellular carcinoma metastasis

目的利用CRISPR—Cas9基因编辑技术验证与非小细胞肺癌转移相关的Trim72基因的敲除是否引起肝癌转移能力的变化。方法建立稳定表达Cas9蛋白的小鼠肝癌细胞系Hepa1－6（Cas9），随后利用针对Trim72基因的单导向RNA（sgRNA）进行特异性基因敲除获得Hepa1－6（Trim72-KO）细胞系，再通过体外bTranswell实验和体内皮下瘤肺转移检测评估该细胞系的迁移和侵袭能力。结果利用CRISPR-Cas9系统成功获得了Hepal－6（Trim72－KO）细胞系。Transwell实验结果表明，敲除％m72基因后，Hepa1－6（Cas9）细胞系的体外迁移和侵袭能力增强；体内皮下瘤肺转移病理检查结果显示，Hepa1－6（Trim72－KO）细胞系（实验组）所成皮下瘤的体内转移能力明显强于未转入相应sgRNA的Hepa1－6（Cas9）细胞系（对照组）。结论通过CRISPR-Cas9系统成功获得了敲除Trim72基因的Hepa1-6（Trim72-KO）细胞系，该细胞系表现出较Hepa1-6（Cas9）细胞系更强的侵袭和转移能力，说明Trim72基因可能在肝癌的侵袭和转移中起重要作用，有望成为肝癌基因治疗的潜在靶点。

Objective To investigate the role of non-small cell lung cancer metastasis-related Trim72 gene in hepatocellular carcinoma （HCC） using clustered regularly interspaced short palindromic repeats （CRISPR） Cas9 system. Methods Hepal-6 （Cas9） HCC cells were established with stable expression of Cas9 protein, and then specific gene knockout was performed using sgRNA targeting Trim72 gene. After obtaining the Hepal-6 （Trim72-KO） cells, the metastasis and invasion abilities of the cells were evaluated by in vitro Transwell assay and in vivo subcutaneous lung metastasis examination. Results Hepal-6 （Trim72-KO） cell line was successfully established by the CRISPR-Cas9 system. Transwell assay indicated that the mobility of Hepal-6 （Trim72-KO） cells was increased compared to the control cells. Transwell assay indicated that the metastasis and invasion of Hepal-6 （Cas9） HCC cells were enhanced after the knockout of Trim72 gene. The pathological examination of lung metastasis of subcutaneous tumor in vivo showed that the subcutaneously metastatic ability of Hepal-6 （Trim72-KO） cells （the experimental group） was significantly stronger than Hepal-6 （Cas9） cells （the control troup） that were not transferred to the corresponding sgRNA. Conclusions The trim72 gene knocked-out HCC cells were obtained by CRISPR-Cas9 system, which showed stronger metastasis and invasion abilities than the control ceils. It is suggested that Trim72 gene may play an important role in the invasion and metastasis of HCC, and Tr/m 72 gene is expected to be a potential target for gene therapy of liver cancer.