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[1]高帆,田爽,王琬瑶,等.VCY通过液-液相分离参与mRNA剪接增强NCCIT细胞迁移和侵袭能力的研究[J].天津医科大学学报,2025,31(04):349-357.[doi:10.20135/j.issn.1006-8147.2025.04.0349]
　GAO Fan,TIAN Shuang,WANG Wanyao,et al.VCY enhances the migration and invasion capabilities of NCCIT cells by participating in mRNA splicing through liquid-liquid phase separation[J].Journal of Tianjin Medical University,2025,31(04):349-357.[doi:10.20135/j.issn.1006-8147.2025.04.0349]
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VCY通过液-液相分离参与mRNA剪接增强NCCIT细胞迁移和侵袭能力的研究(PDF)

《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:
31卷
期数:
2025年04期
页码:
349-357
栏目:
基础医学
出版日期:
2025-07-10

文章信息/Info

Title:
VCY enhances the migration and invasion capabilities of NCCIT cells by participating in mRNA splicing through liquid-liquid phase separation
文章编号:
1006-8147(2025)04-0349-09
作者:
高帆; 田爽; 王琬瑶; 熊昌欢; 刘小萌; 周一平; 李鲲鹏; 时文涛
（天津医科大学基础医学院遗传学系，天津 300070）
Author(s):
GAO Fan;  TIAN Shuang;  WANG Wanyao;  XIONG Changhuan;  LIU Xiaomeng;  ZHOU Yiping;  LI Kunpeng;  SHI Wentao
（Department of Genetics，School of Basic Medical Sciences，Tianjin Medical University，Tianjin 300070，China）
关键词:
癌-睾丸抗原; VCY; 液-液相分离; 畸胎瘤; 选择性剪接
Keywords:
cancer-testis antigen;  VCY;  liquid-liquid separation;  embryonal carcinoma;  alternative splicing
分类号:
R394
DOI:
10.20135/j.issn.1006-8147.2025.04.0349
文献标志码:
A
摘要:
目的：探究可变电荷Y（VCY）的液-液相分离特性及其对畸胎瘤细胞系NCCIT细胞增殖、迁移和侵袭能力的影响，并探索其发挥作用的分子机制。方法：应用原核表达系统表达VCY蛋白，蛋白纯化后在缓冲液中检测其相分离特性。在NCCIT细胞中外源性表达VCY（记作NCCIT VCY组），并设NCCIT Vector组和NCCIT组为对照组。通过免疫荧光技术检测细胞内VCY定位及相分离能力。应用基因本体（GO）功能分析、京都基因与基因组百科全书（KEGG）信号通路分析VCY对NCCIT细胞转录组的影响；通过CCK8实验和Transwell实验检测VCY对细胞增殖、迁移和侵袭能力的影响。应用rMATS软件分析转录组测序数据，探索VCY对选择性剪接的调控；设计外显子特异性引物，通过逆转录-聚合酶链反应（RT-PCR）验证VCY对STMN2 pre-mRNA选择性剪接的具体影响。结果：VCY的氨基酸序列中包含固有无序区域，在缓冲液和NCCIT细胞中均可发生液-液相分离；外源性VCY表达在转录组水平引起NCCIT细胞的38个基因表达上调（|log2FC|＞1，q＜0.05），50个基因表达下调（|log2FC|＞1，q＜0.05）；GO和KEGG富集分析发现VCY主要参与调控细胞增殖、细胞黏附等信号通路（q＜0.01）；与NCCIT组和NCCIT Vector组细胞相比，NCCIT VCY组细胞增殖（F=92.79，P<0.000 1）、迁移（F=42.57，P<0.001）和侵袭（F=206.10，P<0.000 1）能力显著提高。VCY参与STMN2基因的选择性剪接过程，使细胞中STMN2-001转录本水平提高，对STMN2-007和STMN2-008转录本无影响。结论：VCY作为一种癌-睾丸抗原，通过液-液相分离参与STMN2等基因的选择性剪接，促进NCCIT细胞的增殖、迁移和侵袭。
Abstract:
Objective： To investigate the liquid-liquid phase separation characteristics of variable charge Y-lined（VCY） and its effects on the proliferation， migration， and invasion capabilities of NCCIT cells， as well as to explore the underlying molecular mecha-nisms. Methods： The VCY protein was expressed using a prokaryotic expression system， and its phase separation properties were assessed in a buffered solution following purification.Exogenous expression of VCY was introduced in NCCIT cells（denoted as the NCCIT VCY group）， with the NCCIT Vector group and NCCIT group serving as control groups. The localization and phase separation ability of intracellular VCY were detected using immunofluorescence technology. Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） analyses were conducted to evaluate the impact of VCY on the transcriptome of NCCIT cells； the effe-cts of VCY on cell proliferation， migration， and invasion were assessed through CCK8 assays and Transwell experiments. The rMATS software was utilized to analyze transcriptome sequencing data to explore the regulation of alternative splicing by VCY； exon-specific primers were designed to validate the specific effects of VCY on the alternative splicing of STMN2 pre-mRNA via reverse transcription polymerase chain reaction（RT-PCR）. Results： The amino acid sequence of VCY contained an intrinsically disordered region， which was capable of undergoing liquid-liquid phase separation in both buffered solutions and NCCIT cells. Exogenous expression of VCY resulted in the upregulation of 38 genes（|log2FC|＞1， q<0.05） and downregulation of 50 genes（|log2FC|＞1， q<0.05） at the transcriptome level in NCCIT cells. GO and KEGG enrichment analyses revealed that VCY primarily participated in the regulation of signaling pathways related to cell proliferation and adhesion（q<0.01）. Compared with the NCCIT group and NCCIT Vector group， cells proliferation （F=92.79， P<0.000 1）， migration （F=42.57， P<0.001）， and invasion （F=206.10， P<0.000 1） capabilities significantly enhanced in the NCCIT VCY group. VCY was involved in the alternative splicing process of the STMN2 gene， leading to an increase in the STMN2-001 transcript level， with no effect on the STMN2-007 and STMN2-008 transcripts. Conclusion： VCY， as a cancer-testis antigen， participates in the alternative splicing of genes such as STMN2 through liquid-liquid phase separation， thereby promoting the proliferation， migration， and invasion of NCCIT cells.

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备注/Memo

备注/Memo:
基金项目:国家自然科学基金项目（32170649）
作者简介:高帆（1998-），女，硕士在读，研究方向：医学遗传学；通信作者：时文涛，E-mail：shiwentao@tmu.edu.cn。
更新日期/Last Update: 2025-07-10