DKC1影响黏膜型黑色素瘤细胞的增殖、凋亡和细胞周期且与MEKERK通路相关  

DKC1 affects proliferation,apoptosis and cell cycle of mucosal melanoma cells relating to MEK-ERK pathway

作　　者：殷婷 孔燕[1] YIN Ting;KONG Yan(Key Laboratory of Carcinogenesis and Translational Research,Department of Renal Cancer and Melanoma,Peking University Cancer Hospital and Institute,Beijing 100142,China)

机构地区：[1]恶性肿瘤发病机制及转化研究教育部重点实验室,北京大学肿瘤医院暨北京市肿瘤防治研究所肾癌黑色素瘤内科,北京100142

出　　处：《中国肿瘤生物治疗杂志》2020年第8期860-866,共7页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.81772912,81972557)。

摘　　要：目的:研究角化不良素假尿苷合成酶1(dyskerin pseudouridine synthase 1,DKC1)对黏膜型黑色素瘤细胞的增殖、细胞周期和凋亡的影响及其可能的机制。方法:qPCR检测DKC1 mRNA在黏膜型黑色素瘤细胞系HMVⅡ、GAK和正常皮肤细胞株BJ中的表达水平;用DKC1 siRNA(si-DKC1组)和对照siRNA(si-Ctrl组)转染HMVⅡ和GAK细胞,干扰48 h后用qPCR和Western blotting验证敲减效率,CCK-8法检测敲减DKC1对黏膜型黑色素瘤细胞增殖的影响,流式细胞技术检测对细胞凋亡和周期的影响,Western blotting和qPCR检测MEK-ERK通路相关分子表达变化。结果:HMVⅡ、GAK细胞的DKC1 mRNA和蛋白表达水平均显著高于BJ细胞(均P<0.01)。转染siRNA 48 h后,与si-Ctrl组相比,si-DKC1组HMVⅡ和GAK细胞中DKC1 mRNA和蛋白水平均显著降低(均P<0.01),细胞的增殖水平显著下降(P<0.05或P<0.01),细胞的凋亡率显著升高(均P<0.01)且促凋亡分子caspase 9、BAK和PUMA mRNA的表达显著升高(P<0.05或P<0.01),发生细胞周期阻滞(P<0.05或P<0.01),MEK和ERK1/2的磷酸化水平显著下调(P<0.05)。结论:敲减DKC1可抑制黏膜型黑色素瘤细胞的增殖,促进细胞周期阻滞并诱导细胞凋亡,其机制可能与MEK/ERK信号通路有关。Objective: To investigate the effects of dyskerin pseudouridine synthase 1(DKC1) on the proliferation, cell cycle and apoptosis of mucosal melanoma cells and its potential mechanisms. Methods: qPCR was used to detect the mRNA expression of DKC1 in mucosal melanoma cell lines HMV Ⅱ, GAK and normal skin cell line BJ. HMV Ⅱ and GAK cells were interfered with DKC1 siRNA(si-DKC1 group) and control siRNA(si-Ctrl group) respectively;48 h later, qPCR and Western blotting were used to verify the interference efficiency. CCK-8 assay was used to detect the effect of DKC1 knockdown on the proliferation of mucousal melanoma cells. Flow cytometry was used to detect the apoptosis and cell cycles. Western blotting and qPCR were used to detect the molecule expressions of related pathways. Results: The mRNA and protein expression levels of DKC1 in HMV Ⅱ and GAK cells were significantly higher than those in BJ cells(all P<0.01). After 48 h of siRNA transfection, compared with the si-Ctrl group, the m RNA and protein levels of DKC1 in HMV Ⅱ and GAK cells of the si-DKC1 group significantly reduced(all P<0.01), the cell proliferation level significantly reduced(P<0.05 or P<0.01), and the apoptosis rate of cells significantly increased(all P<0.01);in addition, the mRNA expressions of proapoptotic molecules caspase 9, BAK and PUMA increased significantly(P<0.05 or P<0.01) and the cell cycle was blocked(P<0.05 or P<0.01);moreover, the phosphorylation levels of MEK and ERK1/2 were significantly reduced(P<0.05). Conclusion: Knockdown of DKC1 can inhibit the proliferation of mucousal melanoma cells, promote cell cycle arrest and induce apoptosis, and its mechanism may be related to MEK/ERK signal pathway.

关 键 词：角化不良素假尿苷合成酶1 黏膜型黑色素瘤 增殖 凋亡 细胞周期 

分 类 号：R739.5[医药卫生—肿瘤] R730.2[医药卫生—临床医学]