摘要
该研究首先通过qRT-PCR和Western blot发现,MITF在人葡萄膜黑色素瘤细胞中的RNA水平及蛋白水平都显著高于葡萄膜黑色素细胞中的水平。通过RNA干扰技术下调葡萄膜黑色素瘤细胞中的MITF的表达,采用MTS实验、细胞平板克隆实验发现,MITF下调后葡萄膜黑色素瘤细胞的增殖能力被显著抑制。利用流式细胞术及Hoechst染色、Caspase 3/7活性检测发现其细胞周期受到阻滞,且凋亡水平增加。通过RTCA xCELLigence DP检测系统定量检测发现,si-MITF能抑制葡萄膜黑色素瘤细胞的迁移及侵袭能力。Western blot检测发现,MITF下调后葡萄膜黑色素瘤细胞中细胞周期相关蛋白p-Rb(retinoblastoma)、CDK2(cyclin-dependent kinase 2)、CDK6、细胞周期蛋白D2(Cyclin D2)以及CyclinE2的表达水平下调,与增殖及迁移侵袭密切相关的FAK(focal adhesion kinase)及ERK(extracellular signal-regulated protein kinases)蛋白的磷酸化水平降低。该研究表明,在葡萄膜黑色素瘤细胞中下调MITF的表达后,细胞内部分周期相关蛋白、细胞增殖及迁移侵袭相关蛋白的表达均有下调,导致细胞发生G1期阻滞,使细胞的增殖、迁移及侵袭能力受到抑制,同时也促使细胞发生凋亡。
In this study,qRT-PCR and Western blot were used to find that the RNA level and protein level of MITF in human uveal melanoma cells were significantly higher than those in uveal melanocytes.The expression of MITF in uveal melanoma cells was down-regulated by RNA interference technique.MTS assay and cell plate cloning experiments showed that the proliferative ability of uveal melanoma cells were significantly inhibited after MITF down-regulation.Flow cytometry,hoechst staining and Caspase 3/7 activity test showed that the cell cycle was blocked and the level of apoptosis increased.Quantitative detection by RTCA xCELLigence DP detection system found that si-MITF could inhibit the migration and invasion of uveal melanoma cells.The expression levels of cell cycle-associated proteins p-Rb (retinoblastoma),CDK2 (cyclin-dependent kinase 2),CDK6,Cyclin D2 and Cyclin E2 in uveal melanoma cells were down-regulated by Western blot.The phosphorylation levels of FAK (focal adhesion kinase) and ERK (extracellular signal-regulated protein kinases) proteins,which are closely related to proliferation and migration decreased.This study shows that si-MITF can lead to G1 phase arrest of uveal melanoma cells,inhibiting cell proliferation,migration and invasion,and also promote cell apoptosis.
作者
徐微微
陈伟伟
王丽花
XU Weiwei;CHEN Weiwei;WANG Lihua(State Key Laboratory of Optometry and Visual Science,Department of Optometry,Wenzhou Medical University,Wenzhou 325027,China)
出处
《中国细胞生物学学报》
CAS
CSCD
2020年第5期858-867,共10页
Chinese Journal of Cell Biology
基金
浙江省自然科学基金(批准号:LQ15H160014、LQ17H120009)资助的课题。
