摘要
目的 探讨沉默CD147基因对人甲状腺髓样癌TT细胞ANXA2、基质金属蛋白酶（MMP）-2和组织金属蛋白酶抑制因子（TIMP）-2蛋白表达和生物学特性的影响.方法 首先用免疫细胞化学技术检测CD147、ANXA2、MMP-2、TIMP-2等蛋白在TT细胞中的表达情况；应用RNA干扰（RNAi）技术筛选出能有效沉默TT细胞CD147基因的小干扰RNA（siRNA）序列和最佳时间点；在最佳时间点,用逆转录-聚合酶链反应（RT-PCR）、Western blot分别检测ANXA2、MMP-2、TIMP-2等在mRNA和蛋白水平的表达情况；四甲基偶氮唑盐法检测CD147基因沉默后TT细胞的增殖情况；流式细胞技术检测CD147基因被沉默后各实验组TT细胞的周期和凋亡的改变；Transwell小室实验检测沉默CD147基因对TT细胞迁移和侵袭的影响.结果 CD147、ANXA2、MMP-2、TIMP-2等蛋白在TF细胞中均有不同程度的表达；筛选出2条能够在72 h时有效沉默TT细胞CD147基因表达的siRNA序列；MMP-2 mRNA和蛋白的相对表达量也同时下降；而ANXA2 mRNA和蛋白的相对表达量均无明显变化：TIMP-2 mRNA相对表达量无变化,但其蛋白的表达水平明显下降；沉默CD147基因后TT细胞的增殖受到抑制,Go/G1期细胞明显增加,G2/M期细胞比例明显下降,但各实验组凋亡细胞的比例没有变化；沉默TT细胞CD147基因后,Transwell小室迁移和侵袭实验中,各实验组的穿膜细胞数差异均无统计学意义（P＞0.05）.结论 通过RNAi技术,2条CD147 siRNA可以有效的沉默TT细胞CD147基因在mRNA和蛋白水平上的表达,同时下调MMP-2 mRNA和蛋白的表达,对TIMP-2 mRNA无明显影响,但是导致了TIMP-2蛋白表达下降,对ANXA2 mRNA和蛋白的表达均无明显影响；沉默CD147基因可以抑制TT细胞的生长,改变其细胞周期,但是对TT细胞的凋亡、迁移和侵袭影响不大.
Objective To study the influence of CD147 gene silencing on the expression of ANXA2,MMP-2 and TIMP-2 of thyroid medullary carcinoma TT cells and related biological characteristics.Methods Protein expression of CD147,ANXA2,MMP-2 and TIMP-2 was detected by immunocytochemistry.RNAi technology was used to identify the specific siRNA sequences and the optimal time point of effective inhibition of CD147 gene.The expression of ANXA2,MMP-2 and TIMP-2 at mRNA and protein levels was detected with RT-PCR and Western blot,respectively.MTT method was used to detect the proliferation of the TT cells,flow cytometry （FCM） to detect the cell cycle and apoptosis changes of TT cells and transwell chamber assays to document the influence of CD147 gene silencing on migration and invasion of the TT cells.Results The protein expression of CD147,ANXA2,MMP-2 and TIMP-2 proteins was variable in the TT cells.Two siRNA sequences were identified to effectively silence CD147 gene in the TT cells,in which relative expression of MMP-2 was reduced at both mRNA and protein levels; although the expression of ANXA2 mRNA and protein did not change significantly.TIMP-2 protein expression markedly decreased in an absence of its mRNA expression.The proliferation of the TT cells was inhibited upon the CD147 gene silencing along with a significant increase of G0/G1 phase cells and a decrease of G2/M phase cells.However,the proportion of the apoptotic cells in all experimental groups did not change.The number of the penetrating cells through the membrane filters did not show significant changes in all experimental groups in the Transwell chamber assays.Conclusions Through RNAi technology,two CD147 siRNA sequences are identified and shown to effectively inhibit CD147 gene expression of the TT cells.CD147 gene silencing leads to growth inhibition of the TT cells and alteration of the cell cycle.However,silencing CD147 does not significantly affect the apoptosis,migration and invasion of the TT cells.
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2014年第2期103-108,共6页
Chinese Journal of Pathology
基金
河南省基础与前沿技术研究项目（112300410155）
关键词
甲状腺肿瘤
癌
髓样
细胞系
肿瘤
基质金属蛋白酶类
Thyroid neoplasms
Carcinoma, medullary
Cell line, tumor
Matrix metalloproteinases
