文献类型：期刊文献

中文题名：蒲公英多糖通过下调LncRNA CCAT1表达抑制MDA-MB-231细胞增殖、迁移和侵袭

英文题名：Dandelion polysaccharide inhibits proliferation,migration and invasion of MDAMB-231 cells by down-regulation of LncRNA CCAT1 expression

作者：刘晓燕[1,2];龙凤[1,3,4];黄勇[1,4];赵玉[1];周旋[1];潘靖宇[1];李雪[1];叶海琳[1]

第一作者：刘晓燕

机构：[1]甘肃中医药大学,甘肃兰州730000;[2]西安宝石花长庆医院,陕西西安710000;[3]甘肃省中医药防治慢性疾病重点实验室,甘肃兰州730000;[4]甘肃省高校重大疾病分子医学与中医药防治研究省级重点实验室,甘肃兰州730000

第一机构：甘肃中医药大学

年份：2024

卷号：55

期号：4

起止页码：1145

中文期刊名：中草药

外文期刊名：Chinese Traditional and Herbal Drugs

收录：CSTPCD;;Scopus;北大核心:【北大核心2023】；CSCD:【CSCD2023_2024】；

基金：甘肃省“双一流”科研重点项目(GSSYLXM-05);教育揭榜挂帅项目(2021jyjbgs-03);敦煌医学与转化教育部重点实验室开放课题(DHYX22-10)。

语种：中文

中文关键词：蒲公英多糖;乳腺癌;结肠癌相关转录因子1;蛋白激酶B/糖原合成激酶-3β/细胞周期蛋白D1/周期蛋白依赖性激酶6信号通路;迁移;侵袭

外文关键词：dandelion polysaccharide;breast cancer;colon cancer-associated transcript 1;protein kinase B/glycogen synthase kinase-3β/Cyclin D1/cyclin-dependent kinases 6 signaling pathway;migration;invasion

摘要：目的通过观察蒲公英多糖(dandelion polysaccharide,DP)联合小干扰RNA(small interfering RNA,siRNA)靶向沉默长链非编码RNA(LncRNA)结肠癌相关转录因子1(colon cancer-associated transcript 1,CCAT1)对三阴性乳腺癌MDA-MB-231细胞增殖、迁移、侵袭和上皮间质转化(epithelial-mesenchymal transition,EMT)的影响,探究DP抗乳腺癌可能的分子机制。方法在线数据工具分析CCAT1在乳腺癌组织中的差异表达,qRT-PCR分别检测CCAT1在乳腺癌细胞系中的差异表达、DP(100、200μg/mL)对MCF-10A、MCF-7、MDA-MB-231细胞中CCAT1表达的影响以及siCCAT1对MDA-MB-231细胞中CCAT1表达的影响;CCK-8实验、划痕实验和Transwell实验分别检测DP联合siCCAT1对MDA-MB-231细胞活力、迁移和侵袭能力的影响;在线数据工具预测CCAT1下游靶基因及靶基因富集的信号通路;Western blotting检测DP联合siCCAT1对MDA-MB-231细胞EMT相关蛋白及蛋白激酶B(protein kinase B,Akt)/糖原合成激酶-3β(glycogen synthase kinase-3β,GSK-3β)/细胞周期蛋白D1(Cyclin D1)/周期蛋白依赖性激酶6(cyclin-dependent kinases 6,CDK6)信号通路中关键蛋白表达的影响。结果与癌旁正常组织及正常乳腺上皮细胞MCF-10A相比,CCAT1在人乳腺癌组织和细胞中高表达(P<0.01);与对照组比较,DP呈剂量和时间相关性地抑制MDA-MB-231及MCF-7细胞活力(P<0.05、0.01),但对MCF-10A细胞活力无明显影响;与MCF-10A及MCF-7细胞相比,DP可显著下调MDA-MB-231细胞中CCAT1表达水平(P<0.01);与siNC组比较,siRNA可显著降低CCAT1在MDA-MB-231细胞中的表达(P<0.01),且DP(200μg/mL)联合siCCAT1更加显著降低了MDA-MB-231细胞中CCAT1的表达水平(P<0.01);与对照组及siNC组比较,DP(200μg/mL)和siCCAT1均能抑制MDA-MB-231细胞活力(P<0.01),且DP(200μg/mL)联合siCCAT1对MDA-MB-231细胞活力的抑制作用更为明显(P<0.01);与对照组及siNC组比较,DP(200μg/mL)和siCCAT1均能抑制MDA-MB-231细胞的迁移和侵袭(P<0.01),且DP(200μg/mL)联合siCCAT1抑制MDA-MB-231细胞迁移和侵袭能力的作用更为明显(P<0.01);与单独用DP或siCCAT1相比,DP联合siCCAT1处理MDA-MB-231细胞中EMT进程相关蛋白E-cadherin表达上调更为显著(P<0.01),N-cadherin和Vimentin蛋白表达下调更为显著(P<0.01);在线生物工具预测出CCAT1靶基因共1929个,这些靶基因主要富集在RNA聚合酶II启动子转录调控、信号转导、转录调控等关键生物过程;富集的通路主要有癌症、磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/Akt信号通路;与对照组及siNC组比较,DP(200μg/mL)和siCCAT1均能下调MDA-MB-231细胞中Akt/GSK-3β/Cyclin D1/CDK6信号通路关键蛋白p-PI3K、p-Akt、p-GSK-3β、Cyclin D1、CDK6蛋白表达水平(P<0.05、0.01),且DP(200μg/mL)联合siCCAT1应用时对其关键蛋白下调作用更为显著(P<0.01)。结论DP联合siCCAT1显著抑制MDA-MB-231细胞增殖、迁移、侵袭和EMT进程,其机制可能与下调MDA-MB-231细胞中CCAT1表达进而抑制Akt/GSK-3β/Cyclin D1/CDK6信号通路有关。
Objective To investigate the possible molecular mechanism of dandelion polysaccharide(DP)against breast cancer by observing the effect of combined siRNA-targeted silencing of long-stranded non-coding RNA colon cancer-associated transcript 1(CCAT1)on the proliferation,migration,invasion and epithelial-mesenchymal transition(EMT)of triple-negative breast cancer MDAMB-231 cells.Methods Online data tools were used to analyze the differential expression of CCAT1 in breast cancer tissues.qRTPCR was used to detect the differential expression of CCAT1 in breast cancer cell lines,the effect of DP(200μg/mL)on the expression of CCAT1 in MCF-10A,MCF-7 and MDA-MB-231 cells and the effect of siCCAT1 on CCAT1 expression in MDA-MB-231 cells;CCK-8 assay,scratch assay and Transwell assay were used to detect the effects of DP combined with siCCAT1 on the viability,migration and invasion ability of MDA-MB-231 cells,respectively;Online data tools was used to predict CCAT1 downstream target genes and target gene enrichment signaling pathways;Western blotting was used to detect the effects of DP combined with siCCAT1 on the expression levels of EMT-related proteins and key proteins in protein kinase B(Akt)/glycogen synthase kinase-3β(GSK-3β)/Cyclin D1/cyclin-dependent kinases 6(CDK6)signaling pathway in MDA-MB-231 cells.Results CCAT1 was highly expressed in human breast cancer tissues and cells compared with normal tissues adjacent to cancer and normal breast epithelial cells MCF-10A(P<0.01);Compared with control group,DP inhibited MDA-MB-231 and MCF-7 cell viability in a dose-and time-dependent manner(P<0.05,0.01),but had no significant effect on cytosolic MCF-10A cell viability;DP significantly downregulated CCAT1 expression levels in MDA-MB-231 cells compared with MCF-10A and MCF-7 cells(P<0.01);Compared with siNC group,siRNA significantly reduced CCAT1 expression in MDA-MB-231 cells(P<0.01),DP(200μg/mL)combined with siCCAT1 more significantly reduced the expression level of CCAT1 in MDA-MB-231 cells(P<0.01);Compared with control group and siNC group,both DP(200μg/mL)and siCCAT1 could inhibit MDA-MB-231 cell viability(P<0.01),and the inhibitory effect of DP(200μg/mL)combined with siCCAT1 on MDA-MB-231 cell viability was more significant(P<0.01);Compared with control group and siNC group,both DP(200μg/mL)and siCCAT1 inhibited the migration and invasion of MDA-MB-231 cells(P<0.01),DP(200μg/mL)combined with siCCAT1 inhibited the migration and invasion ability of MDA-MB-231 cells more significantly(P<0.01);EMT process-related protein Ecadherin expression was more significantly upregulated and N-cadherin and Vimentin protein expression were more significantly downregulated in MDA-MB-231 cells treated with DP combined with siCCAT1 than with DP or siCCAT1 alone(P<0.01);Online biological tools predicted a total of 1929 CCAT1 target genes,which are mainly enriched in RNA polymerase II promoter transcriptional regulation,signal transduction,transcriptional regulation and other key biological processes;Enriched pathways were mainly involved cancer,phosphoinositide 3-kinase(PI3K)/Akt signaling pathway;Compared with control group and siNC group,both DP(200μg/mL)and siCCAT1 down-regulated the expression levels of p-PI3K,p-Akt,p-GSK-3β,Cyclin D1 and CDK6 proteins in MDA-MB-231 cells(P<0.05,0.01),which were key proteins of the Akt/GSK-3β/Cyclin D1/CDK6 signaling pathway,DP(200μg/mL)combined with siCCAT1 application had a more significant effect on the downregulation of above proteins(P<0.01).Conclusion DP combined with siCCAT1 significantly inhibited the proliferation,migration,invasion and EMT process of MDAMB-231 cells,and the mechanism may be related to the downregulation of CCAT1 expression in MDA-MB-231 cells and thus the inhibition of Akt/GSK-3β/Cyclin D1/CDK6 signaling pathway.