| 慢病毒介导Ras-GRF1调控肝癌干细胞干性维持与自我更新能力的机制 |
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| 引用本文： | 宫东伟,邢贻雷,王开琼,黄 涛,郑进方.慢病毒介导Ras-GRF1调控肝癌干细胞干性维持与自我更新能力的机制[J].现代肿瘤医学,2022,0(17):3076-3082. |
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| 作者姓名： | 宫东伟 邢贻雷 王开琼 黄 涛 郑进方 |
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| 作者单位： | 1.海南省人民医院肝胆胰外科，海南 海口 570311;
2.河南省肿瘤医院肝胆胰外科，河南 郑州 450004 |
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| 基金项目： | 海南省基础与应用基础研究计划项目（编号:2019RC373） |
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| 摘 要： | 目的:探究Ras蛋白特异性的鸟苷酸释放因子1（Ras protein specific guanylate releasing factor 1,Ras-GRF1）在肝癌干细胞（cancer stem cells,CSC）生物学特性及自我更新中的作用，并初步探究其作用机制。方法:实时荧光定量PCR（quantitative real-time PCR，qRT-PCR)检测人正常肝细胞系（HL-7702）和不同肝癌细胞系（Huh7、HepG2、Hep3B、CLC4、CLCl3、SMMC-7721）中Ras-GRF1表达水平;流式细胞术分选出肝癌干细胞，利用Ras-GRF1-pCMV6-GFP重组慢病毒感染肝癌干细胞，MTT法、EdU染色和平板克隆形成实验检测过表达Ras-GRF1对肝癌干细胞增殖能力的影响；Transwell小室实验检测过表达Ras-GRF1后肝癌干细胞迁移与侵袭变化；成球实验检测过表达Ras-GRF1后肝癌干细胞的自我更新能力；蛋白免疫印迹（Western blot）检测肿瘤干细胞中Hippo信号通路关键蛋白大肿瘤抑制因子（large tumor suppressor，LATS）、Yes协同蛋白（Yes cooperative protein，YAP）和哺乳动物STE20样蛋白激酶（mammalian STE20-like protein kinase，MST）的蛋白及磷酸化表达水平。结果:不同肝癌细胞中Ras-GRF1 mRNA表达量较人正常肝细胞中显著降低（P＜0.05）；分离到的肝癌干细胞在感染Ras-GRF1-pCMV6-GFP重组慢病毒后，细胞中Ras-GRF1 mRNA表达量显著升高，差异具有统计学意义（P＜0.05）；在两种肝癌干细胞 HepG2-CSC和SMMC-7721-CSC中，相较于空白对照组，Ras-GRF1-pCMV6组细胞增殖活性显著降低，EdU阳性染色数目和细胞克隆形成数目均显著减少，细胞迁移与侵袭数目也均显著减少，细胞成球能力明显降低，p-LATS/LATS、p-YAP/YAP及p-MST/MST蛋白表达显著增加，差异均具有统计学意义（P＜0.05）。结论:通过慢病毒介导Ras-GRF1在肝癌干细胞中过表达后，能够抑制肝癌干细胞的增殖、迁移与侵袭，降低自我更新能力，其机制可能与调控Hippo信号通路相关。
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| 关 键 词： | Ras蛋白特异性的鸟苷酸释放因子1 肝癌干细胞 细胞干性 自我更新 |
To explore the mechanism of lentivirus-mediated Ras-GRF1 to regulate the stemness maintenance and self-renewal ability of liver cancer stem cells |
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| GONG Dongwei,XING Yilei,WANG Kaiqiong,HUANG Tao,ZHENG Jinfang.To explore the mechanism of lentivirus-mediated Ras-GRF1 to regulate the stemness maintenance and self-renewal ability of liver cancer stem cells[J].Journal of Modern Oncology,2022,0(17):3076-3082. |
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| Authors: | GONG Dongwei XING Yilei WANG Kaiqiong HUANG Tao ZHENG Jinfang |
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| Affiliation: | 1.Department of Hepatobiliary and Pancreatic Surgery,Hainan Provincial People's Hospital，Hainan Haikou 570311，China;2.Department of Hepatobiliary and Pancreatic Surgery,Henan Cancer Hospital,Henan Zhengzhou 450004，China. |
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| Abstract: | Objective:To explore the role of Ras protein specific guanylate releasing factor 1（Ras-GRF1) in the biological characteristics and self-renewal of liver cancer stem cells（CSC),and to explore its mechanism of action.Methods:Real-time fluorescent quantitative PCR（qRT-PCR) was used to detect the expression level of Ras-GRF1 in human normal liver cell lines（HL-7702) and different liver cancer cell lines（Huh7,HepG2,Hep3B,CLC4,CLCl3,SMMC-7721).Flow cytometry sorts out liver cancer stem cells,infect liver cancer stem cells with Ras-GRF1-pCMV6-GFP recombinant lentivirus,MTT method,EdU staining and plate clone formation experiment were used to detect the effect of overexpression of Ras-GRF1 on the proliferation of liver cancer stem cells.Transwell chamber experiment detects the migration and invasion of liver cancer stem cells after overexpression of Ras-GRF1.The spheroidization experiment detects the self-renewal ability of liver cancer stem cells after overexpression of Ras-GRF1.Western blot was used to detect the protein and phosphorylation expression levels of Hippo signaling pathway key proteins large tumor suppressor（LATS),Yes cooperative protein（YAP) and mammalian STE20-like protein kinase（MST) in cancer stem cells.Results:The expression of Ras-GRF1 mRNA in different liver cancer cells was significantly lower than that in normal human liver cells（P＜0.05).After the isolated liver cancer stem cells were infected with Ras-GRF1-pCMV6-GFP recombinant lentivirus,the expression of Ras-GRF1 mRNA in the cells was significantly increased,and the difference was statistically significant（P＜0.05).In the two liver cancer stem cells HepG2-CSC and SMMC-7721-CSC,compared with the blank control group,the cell proliferation activity of the Ras-GRF1-pCMV6 group was significantly reduced.The number of EdU positive staining and the number of cell clone formation were significantly reduced,and the cells migrated and the number of invasions were also significantly reduced.The ability of cells to form spheroids was significantly reduced.At the same time,the expression of p-LATS/LATS,p-YAP/YAP and p-MST/MST protein increased significantly,the difference were statistically significant（P＜0.05).Conclusion:The overexpression of Ras-GRF1 in liver cancer stem cells mediated by lentivirus can inhibit the proliferation,migration and invasion of liver cancer stem cells,and reduce the ability of self-renewal,the mechanism may be related to the regulation of Hippo signaling pathway. |
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| Keywords: | Ras protein specific guanylate releasing factor 1 liver cancer stem cells cell stemness self-renewal |
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