干扰hsa_circ 0103232对葡萄膜黑色素瘤细胞生物学行为的影响
Effect of interfering hsa_circ_0103232 on the biological behavior of uveal melanoma cells
杨萱 1魏文斌1
作者信息
- 1. 首都医科大学附属北京同仁医院 北京同仁眼科中心 眼内肿瘤诊治研究北京市重点实验室北京市眼科学与视觉科学重点实验室 医学人工智能研究与验证工信部重点实验室,北京 100730
- 折叠
摘要
目的 探讨干扰hsa_circ_0103232对葡萄膜黑色素瘤C918和MUM2B细胞增殖、迁移、细胞周期及凋亡的影响.方法 培养C918和MUM2B细胞株,通过实时荧光定量PCR分别检测3个靶向hsa_circ_0103232的小干扰RNA(siRNA)的干扰效果,并选择干扰效果最好的siRNA进行后续实验.将C918和MUM2B细胞均分为阴性对照转染(siCtrl)组和干扰(si-hsa_circ_0103232)组,采用细胞计数试剂盒8(CCK-8)和细胞克隆形成实验检测细胞增殖情况;采用Transwell实验检测细胞迁移情况;采用流式细胞术检测细胞周期分布和细胞凋亡情况;采用荧光原位杂交实验检测hsa_circ_0103232在细胞中的定位.结果 实时荧光定量PCR结果显示,3个靶向hsa_circ_0103232的siRNA中,以si-hsa_circ_0103232#1的靶点效果最好,在C918和MUM2B细胞中的干扰后表达水平分别为0.263±0.016和0.469±0.028,显著低于对照组的1.013±0.008 和 1.004±0.108(均P＜0.001).CCK-8 结果显示,与 siCtrl 组相比,si-hsa_circ_0103232 组 C918和MUM2B细胞增殖活力在转染后不同时间均显著降低,差异均有统计学意义(均P＜0.05);细胞克隆形成实验结果显示,si-hsa_circ_0103232组C918和MUM2B细胞形成克隆数分别为(12±1)和(45±7)个,分别少于siCtrl组的(28±4)和(83±3)个,差异均有统计学意义(t=4.93、7.42,均P＜0.05);Transwell实验结果显示,si-hsa_circ_0103232组C918和MUM2B细胞迁移数量分别为(4±1)和(24±2)个,分别少于siCtrl组的(37±12)和(57±3)个,差异均有统计学意义(t=3.91、10.80,均P＜0.05);流式细胞术检测结果显示,与siCtrl组相比,si-hsa_circ_0103232组C918和MUM2B细胞中G1期细胞的比例均明显升高、G2/M期比例均显著下降,细胞凋亡率均显著升高,差异均有统计学意义(均P＜0.05);荧光原位杂交实验结果显示,hsa_circ_0103232在C918和MUM2B细胞中定位在细胞核.结论 干扰hsa_circ_0103232可抑制C918和MUM2B细胞增殖、迁移和周期进程,并促进细胞凋亡.hsa_circ_0103232可能成为葡萄膜黑色素瘤新的治疗靶点.
Abstract
Objective To investigate the effects of interference with hsa_circ_0103232 on the proliferation,metastasis,cell cycle and apoptosis of melanoma cells C918 and MUM2B.Methods C918 and MUM2B cells were cultured,and the interference efficiency of three small interfering RNA(siRNA)targeting hsa_circ_0103232 were detected by real-time fluorescent quantitative polymerase chain reaction(PCR).The siRNA with the highest interference efficiency was used for the following experiment.Both C918 and MUM2B cells were divided into negative control transfection(siCtrl)groups and interference(si-hsa_circ_0103232)groups.The proliferation of C918 and MUM2B cells was examined by cell counting kit-8(CCK-8)assay and cell colony formation assay.The migration of C918 and MUM2B cells was determined by transwell assay.The cell cycle distribution and apoptosis rate of C918 and MUM2B cells were detected by flow cytometry.The localization of hsa_circ_0103232 in C918 and MUM2B cells was tested by the fluorescence in situ hybridization experiment(FISH).Results The results of real-time quantitative PCR showed that among the three siRNAs targeting hsa_circ_0103232,si-hsa_circ_0103232#1 had the best effect,which reduced the expression level of gene in C918 and MUM2B cells to 0.263±0.016 and 0.469±0.028,significanthy lower than 1.013±0.008 and 1.004±0.108 of control groups(both at P＜0.001).CCK-8 results showed that the proliferation activity of C918 and MUM2B cells was significantly lower in si-hsa_circ_0103232 group than in siCtrl group after transfection(all at P＜0.05).The results of cell clone formation showed that the clone number of C918 and MUM2B cells in si-hsa_circ_0103232 group were 12±1 and 45±7,which were significantly lower than 28±4 and 83±3 in siCtrl group,and the differences were statistically significant(t=4.93,7.42;both at P＜0.05).Transwell assay results showed that the number of migrating C918 and UM2B cells in si-hsa_circ_0103232 group were 4±1 and 24±2,respectively,which were significantly lower than 37±12 and 57±3 in siCtrl group,and the differences were statistically significant(t=3.91,10.80;both at P＜0.05).The results of flow cytometry showed that compared with siCtrl group,the proportion of G1 phase cells in C918 and MUM2B cells in si-hsa_circ_0103232 group increased significantly,the proportion of G2/M phase cells decreased significantly,and the number of apoptotic cells increased significantly(all at P＜0.05).FISH experiment showed that hsa_circ_0103232 was located in the nuclei of C918 and MUM2B cells.Conclusions Interference with hsa_circ_0103232 can inhibit the proliferation,migration and cycle progression of C918 and MUM2B cells,and promote their apoptosis.hsa_circ_0103232 may be a new therapeutic target for uveal melanoma.
关键词
葡萄膜黑色素瘤/环状RNA/小干扰RNA/治疗靶点Key words
Uveal melanoma/RNA,circular/RNA,small interfering/Therapeutic target引用本文复制引用
基金项目
国家自然科学基金(82002883)
国家自然科学基金(82220108017)
国家自然科学基金(82141128)
首都卫生发展科研专项(首发2020-1-2052)
北京市科委科技项目(Z201100005520045)
北京市科委科技项目(Z181100001818003)
首都医科大学附属北京同仁医院青年人才培养计划种子基金(2020)(2020-YJJ-ZZL-017)
出版年
2024
NETL
NSTL
万方数据