摘要
目的 观察新型抑癌基因Arid2对人肝癌细胞HepG2和Huh7细胞中CD44的表达调控,初步探讨Arid2调控肝癌细胞侵袭和迁移的机制. 方法 构建pGL3-CD44报告质粒并转染至人肝癌细胞HepG2及Huh7细胞中,感染腺病毒Ad-Arid2后,双荧光素酶实验检测报告质粒相对荧光素酶活性的变化;Western blot检测过表达Arid2对跨膜糖蛋白CD44表达的影响.细胞迁移与侵袭实验检测过表达Arid2对细胞迁移和侵袭能力的影响,并在裸鼠荷瘤实验中观察体内移植瘤的生长情况.两组数据之间的比较采用独立样本t检验,多组间比较采用单因素方差分析.结果 荧光素酶活性实验发现,转染不同长度CD44报告质粒后其相对荧光素酶活性均有不同程度的提高,而变化明显的pGL3-CD44-791 ~ +225 bp报告质粒相对荧光素酶在给予过表达Arid2处理后明显下调,抑制率分别为73.83%±0.92%(P<0.05,HepG2)和48.99%±1.37%(P<0.05,Huh7).外源性过表达Arid2能够明显抑制CD44蛋白表达.Transwell实验证实过表达Arid2可以明显抑制人肝癌细胞的迁移能力,在HepG2、Huh7细胞中其抑制率分别为66.95%±0.59%(P<0.05)、73.86%±0.49%(P< 0.05).动物实验结果表明,Arid2明显延缓或抑制荷瘤小鼠皮下移植瘤的生长,其抑制率为98.57%±0.34%(P<0.05).结论 外源过表达Arid2明显下调CD44启动子活性和CD44蛋白表达,并在肝癌细胞和荷瘤裸鼠模型中也明显抑制肿瘤细胞的生长和迁移,提示CD44在Arid2调控肝癌细胞的侵袭和迁移过程中可能发挥了重要作用.该研究为阐明Arid2与CD44分子的关系以及其在肝癌发生、发展中的作用提供了新的研究方向.
Objective To investigate the effect of a new tumor-suppressor gene Arid2 on the expression level of CD44 and the mechanism that Arid2 regulates the invasion and metastasis in human hepatocellular carcinoma cells HepG2 and Huh7.Methods Recombinant pGL3-CD44 reporter plasmids were transfected into hepatoma cell lines HepG2 and Huh7 cells infected with adenovirus Arid2(Ad-Arid2).Dual luciferase assays were used to determine the relative luciferase activities of reporter plasmids.Western blot technique was used to detect the influence of Arid2 on the expression of transmembrane glycoprotein CD44.Cell migration assays of tumor cells were employed to observe the impact of overexpression of Arid2 on the invasion and metastasis abilities of tumor cells.The sizes of transplanted tumors were recorded to observe the growth of subcutaneous transplanted tumors in nude mice.Statistical significance was analyzed by one-way ANOVA for multiple comparisons,and independent-samples t-test was utilized to compare two groups.Results Luciferase assay showed cells were transfected with different length of CD44 reporter plasmids,and their relative luciferase activities were improved to different degrees,compared with pGL3-Basic control.Meanwhile,the mean luciferase activities ofpGL3-CD44 -791~+224bp reporter plasmids were significantly repressed by the overexpression of Arid2 which inhibition rates were up to 73.83%±0.92%(P 〈 0.05,HepG2) or 48.99%±1.37% (P 〈0.05,Huh7),compared with Ad-GFP control.Western blot results showed that CD44 protein expression was obviously decreased by overexpression of Arid2.Cell migration assays confirmed that the invasion and metastasis abilities were inhibited by increasing Arid2 expression in Human HepG2 or Huh7 cells,which inhibition rates were 66.95%±0.59%(P 〈 0.05)in HepG2 cells and 73.86%±0.49%(P 〈 0.05) in Huh7 ceils respectively.The animal experiment results indicated that Arid2 could obviously delay or restrict the subcutaneous transplanted tumors growth in nude mice,which was declined by 98.57%±0.34%(P 〈 0.05).Conclusion CD44 promoter activities and protein expressions were significantly down-regulated by Arid2 in vitro.The growth and metastasis of tumors were obviously restrained in the hepatocellular carcinoma cells and nude models.In brief,these researches indicate CD44 may play important roles in the process where the invasion and metastasis of hepatocellular carcinoma cells are under the control of Arid2.The studies introduce and evaluate the relationships between Arid2 and CD44,and further provide a new research direction with the occurrence and development of hepatocellular carcinoma.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2016年第3期196-201,共6页
Chinese Journal of Hepatology
基金
国家自然科学基金(81572683,81371827,31171307)
“十二五”传染病国家科技重大专项(2012ZXl0002005-003-002,2013ZXl0002002-005-003)
重庆医科大学附属第二医院“优秀青年人才”项目
关键词
癌
肝细胞
Arid2
细胞迁移
基因
CD44
转录活性
Carcinoma, hepatocellular
Arid2
Cell metastasis
Gene, CD44
Transcription activity