摘要
目的探讨长链非编码RNA(lncRNA)LINC02474在结直肠癌组织及循环外泌体中的表达特征及其对结直肠癌发生发展的影响。方法采用实时荧光定量PCR(qRT-PCR)检测LINC02474在30例结直肠癌患者组织及128例结直肠癌患者和128例健康对照者血清中的相对表达量;使用电子显微镜检测分离所得外泌体的形态特征;使用纳米粒子跟踪分析(NTA)检测分离所得外泌体的粒径大小;Western blotting检测外泌体表面标志物的表达;CCK-8实验、平板克隆形成实验和实时细胞分析系统(RTCA)检测干扰LINC02474后DLD-1细胞的增殖;采用trans基因预测等生物信息学方法分析预测获得LINC02474的潜在靶基因。结果 qRT-PCR检测结果显示,LINC02474在结直肠癌组织中呈高表达,差异有统计学意义(U=213.0,P<0.001)。电子显微镜结果显示,分离所得外泌体呈盘状囊泡结构,大小分布集中于96.9 nm处,且表达表面标志物CD9、CD63和TSG101。CCK-8实验、平板克隆形成实验和RTCA检测结果表明,干扰LINC02474的表达可以抑制结直肠癌细胞的增殖及成瘤能力,差异有统计学意义(CCK-8实验:t=2.640,P=0.046;平板克隆形成实验:t=2.745,P=0.016;RTCA检测:t=28.44,P<0.001)。KEGG和GO富集分析结果显示,LINC02474的潜在靶基因多富集在mTOR及MAPK等信号通路。结论 LINC02474在结直肠癌患者组织和血清外泌体中呈高表达特征,干扰其表达可以抑制结直肠癌细胞的增殖及成瘤能力。
Objective To explore the expression profiles of long non-coding RNA LINC02474 in tissues and circulating exosomes of colorectal cancer(CRC) and the effects of LINC02474 on cell proliferation. Methods The relative expression of LINC02474 in tissues of 30 CRC patients and serum samples from 128 CRC patients and 128 healthy donors by quantitative real-time PCR(qRT-PCR) was obtained. Morphology of the exosomes was detected by electron microscope. The size was analyzed by nanoparticle tracking analysis(NTA). Expression of exosomes markers were shown by Western blotting. The DLD-1 cell proliferation after deletion of LINC02474 was screened by CCK-8 assays, colony formation assays and the real-time cell analysis(RTCA) system. And the potential target genes were acquired by bioinformatics prediction. Results TCGA analysis and qRT-PCR results from tissues of 30 CRC patients and serum samples from 128 CRC patients and 128 healthy donors have shown that LINC02474 was highly expressed in CRC tissues samples(U=213.0, P<0.001). Morphology of the exosomes was disk-like vesicles which detected by electron microscope. The size of the exosomes mainly enriched in 96.9 nm which was analyzed by NTA. CD9, CD63 and TSG101 were expressed in the surface of the exosomes which shown by Western blotting. Deletion of LINC02474 inhibited proliferation of CRC cells, which detected by CCK-8 assays, colony formation assays and the RTCA system(CCK-8 assays: t=2.640, P=0.046;colony formation assays: t=2.745, P=0.016;RTCA system: t=28.44, P<0.001). And there were more target genes enriched in the mTOR and MAPK signaling pathway through KEGG and GO analysis. Conclusion LINC02474 is highly expressed in CRC tissues and serum samples, and its knockdown restrains the cell proliferation.
作者
杜甜甜
李娟
赵颖慧
段伟丽
王景
王允山
杜鲁涛
王传新
DU Tiantian;LI Juan;ZHAO Yinghui;DUAN Weili;WANG Jing;WANG Yunshan;DU Lutao;WANG Chuanxin(Department of Clinical Laboratory,The Second Hospital,Cheeloo College of Medicine,Shandong University,Jinan 250033,Shandong,China;Tumor Marker Detection Engineering Laboratory of Shandong Provine,Jinan 250033,Shandong,China)
出处
《山东大学学报（医学版）》
CAS
北大核心
2021年第10期57-67,共11页
Journal of Shandong University:Health Sciences
基金
国家自然科学基金(2019GHZ003,82002228)
山东省重点研发计划(2020CXGC011304)
山东省自然科学基金(ZR2020QH280)。
关键词
结直肠癌
长链非编码RNA
外泌体
诊断
细胞增殖
Colorectal cancer
Long non-coding RNAs
Exosomes
Diagnosis
Cell proliferation
