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、200 μg/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（200 μg/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（200 μg/mL）和siCCAT1均能抑制MDA-MB-231细胞活力（P＜0.01），且DP（200 μg/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（200 μg/mL）和siCCAT1均能抑制MDA-MB-231细胞的迁移和侵袭（P＜0.01），且DP（200 μg/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1 929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（200 μg/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（200 μg/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 MDA-MB-231 cells. Methods Online data tools were used to analyze the differential expression of CCAT1 in breast cancer tissues. qRT-PCR was used to detect the differential expression of CCAT1 in breast cancer cell lines, the effect of DP (200 μg/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 (200 μg/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 (200 μg/mL) and siCCAT1 could inhibit MDA-MB-231 cell viability(P < 0.01), and the inhibitory effect of DP (200 μg/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 (200 μg/mL) and siCCAT1 inhibited the migration and invasion of MDA-MB-231 cells (P < 0.01), DP (200 μg/mL) combined with siCCAT1 inhibited the migration and invasion ability of MDA-MB-231 cells more significantly (P < 0.01); EMT process-related protein E-cadherin 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 1 929 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 (200 μg/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 (200 μg/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 MDA-MB-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.

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）