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(P<0.05);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(P<0.05);细胞凋亡抑制因子survivin及Bcl2的表达也明显下降(P<0.05),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。
AIM ： To identify the expression of fermitin family homolog 2 （ FERMT2 ） in hepatocellular carci-noma （HCC） tissues and the effect of FERMT2 on the cell growth and related protein expression. METHODS： Real-time PCR and immunohistochemistry were used to detect FERMT2 expression in the HCC tissues. The technique of CRISPR/ Cas9 was applied to construct stable FERMT2 knockout MHCC97H cell line. WST-1 assay and flow cytometry were used to measure the cell viability, cell-cycle distribution and cell apoptosis. Western blot was used to determine the expression of related proteins in the MHCC97H cells. RESULTS： In HCC tissues, the expression level of FERMT2 was higher than that in adjacent liver tissues （P 〈 0. 05 ） . High expression of FERMT2 was significantly correlated with postoperative recurrence of tumor. Knockout of FERMT2 gene evidently inhibited MHCC97H cell viability and accelerated cell apoptosis. Mean-while, the expression levels of proliferating cell nuclear antigen, cyclins, cyclin-dependent kinases 2 and anti-apoptotic fac-tors were significantly downregulated in MHCC97H cells with FERMT2 knockout （P 〈 0 . 05）. CONCLUSION： FERMT2 may function as a promoter of hepatocarcinogenesis and progression via regulating the cell viability, cell-cycle distribution and cell apoptosis, which is related with the expression of cell cycle regulators and anti-apoptotic factors.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2017年第12期2157-2164,共8页
Chinese Journal of Pathophysiology
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)
