FERMT2在肝癌组织中的表达及其对肝癌细胞生长的调控

发布时间：2018-06-08 05:50

本文选题：肝细胞癌 + CRISPR/Cas技术　；参考：《中国病理生理杂志》2017年12期

【摘要】：目的:观察fermitin家族同源蛋白2(FERMT2)在肝细胞癌(HCC)组织中的表达,并以体外实验分析FERMT2基因敲除对肝癌细胞生长及相关调控分子表达的影响。方法:采用real-time PCR及免疫组化实验检测FERMT2在HCC及癌旁正常肝组织中的表达情况;采用CRISPR/Cas9技术构建FERMT2基因敲除的稳定MHCC97H细胞系,通过WST-1法和流式细胞术比较其与野生型MHCC97H细胞活力、细胞周期和凋亡状态的改变,并采用Western blot检测相关调控蛋白的表达变化。结果:FERMT2在HCC组织中的mRNA和蛋白表达水平均显著高于癌旁组织,且FERMT2蛋白的高表达与HCC患者术后复发有关(P0.05);成功构建了FERMT2基因敲除的稳定MHCC97H细胞系;与野生型细胞相比,FERMT2基因敲除后细胞活力明显减弱,S期细胞数量明显减少,细胞凋亡增加,且细胞内增殖细胞核抗原、细胞周期蛋白及细胞周期蛋白依赖性激酶2的表达均显著降低(P0.05);细胞凋亡抑制因子survivin及Bcl2的表达也明显下降(P0.05),并可检测到cleaved caspase-3。结论:FERMT2在肝癌组织中呈高表达,它可能通过调控细胞周期调节蛋白及抗凋亡蛋白的表达影响肝癌细胞的活力、细胞周期及凋亡,进而在肝癌形成及发展过程中发挥促癌作用。
[Abstract]:Aim: to investigate the expression of fermitin family homologous protein 2FERMT2 in human hepatocellular carcinoma (HCC) and to investigate the effect of FERMT2 knockout on the growth and expression of related regulatory molecules in hepatocellular carcinoma (HCC) cells in vitro. Methods: the expression of FERMT2 in HCC and adjacent normal liver tissues was detected by real-time PCR and immunohistochemistry, the stable MHCC97H cell line with FERMT2 gene knockout was constructed by CRISPRR Cas9 technique, and the activity of FERMT2 gene knockout MHCC97H cell line was compared with that of wild type MHCC97H cell line by WST-1 and flow cytometry. The changes of cell cycle and apoptosis were detected by Western blot. Results the expression of FERMT2 mRNA and protein in HCC was significantly higher than that in paracancerous tissues, and the high expression of FERMT2 was related to the recurrence of HCC patients (P 0.05), and a stable MHCC97H cell line with FERMT2 gene knockout was successfully constructed. Compared with wild-type cells, the cell viability of FERMT2 gene knockout decreased significantly, the number of S phase cells decreased, apoptosis increased, and proliferating cell nuclear antigen (PCNA) was found in the cells. The expression of cyclin and cyclin dependent kinase 2 decreased significantly, while the expression of apoptosis inhibitor survivin and Bcl-2 decreased significantly, and cleaved caspase-3 was detected. Conclusion the high expression of FERMT2 may affect the viability, cell cycle and apoptosis of HCC cells by regulating the expression of cell cycle regulatory proteins and antiapoptotic proteins, and thus play a role in the carcinogenesis and development of HCC.
【作者单位】：福建省立医院病理科福建医科大学省立临床医学院;福建医科大学附属肿瘤医院福建省肿瘤医院肿瘤免疫学研究室;福建医科大学附属肿瘤医院福建省转化医学重点实验室;
【基金】：福建省自然科学基金资助项目(No.2016J01510) 福建省卫生计生委青年科研课题(No.2017-1-2)
【分类号】：R735.7

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