干扰hsa_circ_0103232对葡萄膜黑色素瘤细胞生物学行为的影响

杨萱; 魏文斌

doi:10.3760/cma.j.cn115989-20231122-00179

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中华实验眼科杂志

• 实验研究 •

ENGLISH ABSTRACT

干扰hsa_circ_0103232对葡萄膜黑色素瘤细胞生物学行为的影响

作者及单位信息

出版日期： 2024 -03 -10 ·

DOI： 10.3760/cma.j.cn115989-20231122-00179

Effect of interfering hsa_circ_0103232 on the biological behavior of uveal melanoma cells

Authors Info & Affiliations

Yang Xuan

Beijing Tongren Eye Center, Beijing Key Laboratory of Intraocular Tumor Diagnosis and Treatment, Beijing Key Laboratory of Ophthalmology and Visual Science, Medical Artificial Intelligence Research and Validation Key Laboratory of the Ministry of Industry and Information Technology, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, China

Wei Wenbin

Published： 2024 -03 -10 ·

DOI： 10.3760/cma.j.cn115989-20231122-00179

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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。

关键词：葡萄膜黑色素瘤;环状RNA;小干扰RNA;治疗靶点

ABSTRACT

ObjectiveTo investigate the effects of interference with hsa_circ_0103232 on the proliferation, metastasis, cell cycle and apoptosis of melanoma cells C918 and MUM2B.

MethodsC918 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).

ResultsThe 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.

ConclusionsInterference 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.

KEYWORDS： Uveal melanoma;RNA, circular;RNA, small interfering;Therapeutic target

Corresponding author: Wei Wenbin, Email: mocdef.3ab61rtnibnewiew

FUNDING:

National Natural Science Foundation of China（82002883, 82220108017, 82141128）

The Special Project of Capital Health Research and Development（2020-1-2052）

Science & Technology Project of Beijing Municipal Science & Technology Commission（Z201100005520045, Z181100001818003）

The Priming Scientific Research Foundation for the Junior Researcher in Beijing Tongren Hospital, Capital Medical University（2020-YJJ-ZZL-017）

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All articles published represent the opinions of the authors, and do not reflect the official policy of the Chinese Medical Association or the Editorial Board, unless this is clearly specified.

引用本文

杨萱,魏文斌. 干扰hsa_circ_0103232对葡萄膜黑色素瘤细胞生物学行为的影响[J]. 中华实验眼科杂志,2024,42(03)：224-231.

DOI:10.3760/cma.j.cn115989-20231122-00179

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葡萄膜黑色素瘤(uveal melanoma，UM)是成年人常见的原发性眼内恶性肿瘤，具有恶性程度高、发病隐匿的特点，严重威胁患者的视力乃至生命安全。UM起源于眼球壁的中间葡萄膜层，包括虹膜、睫状体及脉络膜，具有高度侵袭性，约50%的患者会出现转移性UM ^{[
1
,

2
,

3
,

4
]}。目前，对于UM缺乏有效的治疗手段，患者的预后和生存率较差，UM发生转移后整体的生存时间为10～12个月 ^{[
5
,

6
]}。因此，深入了解UM的分子机制，进而确定更有效的生物标志物和治疗靶点，对其早期临床诊断、制定预防和治疗策略具有重要意义。环状RNA(circular RNA，circRNA)是一种新型内源性非编码RNA，被认为是哺乳动物中一类稳定的调节转录物 ^{[
7
,

8
]}。circRNA由"反向剪接"反应产生，具有特征性的共价闭环结构，并且在特定细胞类型或发育阶段中特异性表达 ^{[
9
,

10
,

11
]}。circRNA具有选择性保守的微小RNA(microRNA，miRNA)靶位点，可以通过miRNA海绵调节下游基因的表达 ^{[
12
]}。近年来研究表明，circRNA具有保守性、稳定性、丰富性以及动态性的特点，使其成为新型临床诊断与治疗的重要靶点及标志物；同时，circRNA广泛存在于胶质瘤、乳腺癌、肝癌等多种肿瘤中，是肿瘤发生和发展的重要调节因子 ^{[
13
]}。目前，已有部分circRNA被发现可作为皮肤黑色素瘤的潜在靶标，如circ_0062270 ^{[
14
]}等，但是circRNA在UM中的研究较匮乏。本课题组先前研究比较circRNA在UM组织和正常葡萄膜组织中的表达 ^{[
15
]}，探讨了多个circRNA作为UM诊断与预后标志物的可能，发现与正常葡萄膜组织相比，hsa_circ_0103232在UM组织中呈高表达，表明hsa_circ_0103232有望成为UM治疗新的潜在靶点。此外，Lin等 ^{[
16
]}研究发现hsa_circ_0103232可能通过miR-661增强RAB3D的表达来促进皮肤黑色素瘤(SKMel1、A375和A875)的进展，进一步提示了hsa_circ_0103232在黑色素瘤中的作用。但尚未见hsa_circ_0103232对UM细胞作用的研究报道。本研究旨在探讨干扰hsa_circ_0103232对UM细胞C918和MUM2B增殖、迁移、周期和凋亡的影响。

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摘要
参考文献

参考文献

[1]

Toro MD , Gozzo L , Tracia L ,et al. New therapeutic perspectives in the treatment of uveal melanoma：a systematic review[J/OL]. Biomedicines， 2021，9(10)∶1311[2023-11-10]. http://www.ncbi.nlm.nih.gov/pubmed/34680428. DOI： 10.3390/biomedicines9101311 .