Carpi Sara, Fogli Stefano, Polini Beatrice, Montagnani Valentina, Podestà Adriano, Breschi Maria Cristina, Romanini Antonella, Stecca Barbara, Nieri Paola
Department of Pharmacy, University of Pisa, Pisa, Italy.
Department of Pharmacy, University of Pisa, Pisa, Italy.
Toxicol In Vitro. 2017 Apr;40:272-279. doi: 10.1016/j.tiv.2017.01.018. Epub 2017 Jan 26.
The role of endocannabinoid system in melanoma development and progression is actually not fully understood. This study was aimed at clarifying whether cannabinoid-type 1 (CB1) receptor may function as tumor-promoting or -suppressing signal in human cutaneous melanoma. CB1 receptor expression was measured in human melanoma cell lines by real-time PCR. A genetic deletion of CB1 receptors in selected melanoma cells was carried out by using three different short hairpin RNAs (shRNAs). Performance of target gene silencing was verified by real-time PCR and Western blot. The effects of CB1 receptor silencing on cell growth, clonogenicity, migration capability, cell cycle progression, and activation of mitogenic signals was tested. Lentiviral shRNAs vectors targeting different regions of the human CB1 gene led to a significant reduction in CB1 receptor mRNA and a near complete loss of CB1 receptor protein, compared to control vector (LV-c). The number of viable cells, the colony-forming ability and cell migration were significantly reduced in cells transduced with CB1 lentiviral shRNAs compared to LV-c. Cell cycle analyses showed arrest at G1/S phase. p-Akt and p-ERK expression were decreased in transduced versus control cells. Findings of this study suggest that CB1 receptor might function as tumor-promoting signal in human cutaneous melanoma.
内源性大麻素系统在黑色素瘤发生和发展中的作用实际上尚未完全明确。本研究旨在阐明1型大麻素（CB1）受体在人类皮肤黑色素瘤中是作为肿瘤促进信号还是肿瘤抑制信号发挥作用。通过实时PCR检测人类黑色素瘤细胞系中CB1受体的表达。利用三种不同的短发夹RNA（shRNA）对选定的黑色素瘤细胞中的CB1受体进行基因敲除。通过实时PCR和蛋白质免疫印迹法验证靶基因沉默的效果。测试了CB1受体沉默对细胞生长、克隆形成能力、迁移能力、细胞周期进程和有丝分裂信号激活的影响。与对照载体（LV-c）相比，靶向人类CB1基因不同区域的慢病毒shRNA载体导致CB1受体mRNA显著减少，CB1受体蛋白几乎完全丧失。与LV-c相比，用CB1慢病毒shRNA转导的细胞中活细胞数量、集落形成能力和细胞迁移显著降低。细胞周期分析显示在G1/S期停滞。转导细胞与对照细胞相比，p-Akt和p-ERK表达降低。本研究结果表明，CB1受体可能在人类皮肤黑色素瘤中作为肿瘤促进信号发挥作用。
