冬凌草甲素通过下调lncRNA AFAP1-AS1表达对人乳腺癌细胞增殖、迁移及侵袭的抑制作用

Inhibitory effects of oridonin on proliferation, migration and invasion of human breast cancer cells by down regulating lncRNA AFAP1-AS1 expression

目的 通过观察siRNA靶向沉默长链非编码RNA AFAP1-AS1并联合冬凌草甲素对人乳腺癌MDA-MB-231细胞增殖、迁移、侵袭及上皮间质转化的影响,探究冬凌草甲素抗乳腺癌可能的分子机制。方法 在线数据库分析lncRNA AFAP1-AS1在三阴性乳腺癌组织中的差异表达,并确定其差异表达与乳腺癌患者生存期的关系。RT-qPCR法检测冬凌草甲素和siAFAP1-AS1对MDA-MB-231细胞AFAP1-AS1表达的影响,CCK-8法、划痕实验、Transwell侵袭实验分别检测冬凌草甲素和siAFAP1-AS1对MDA-MB-231细胞活力、细胞迁移和侵袭能力的影响,生物信息学技术分析预测lncRNA AFAP1-AS1的靶基因,Western blot法检测冬凌草甲素和siAFAP1-AS1对MDA-MB-231细胞EMT相关蛋白及MAPK通路中Cdc42、MAP3K10蛋白表达的影响。结果 lncRNA AFAP1-AS1在三阴性乳腺癌组织中表达上调且与乳腺癌患者生存不良相关(P<0.05);冬凌草甲素下调MDA-MB-231细胞中AFAP1-AS1表达(P<0.05);siAFAP1-AS1和冬凌草甲素联合处理MDA-MB-231细胞后,细胞活力、细胞迁移和侵袭能力减弱,N-cadherin和Vimentin蛋白表达降低,E-cadherin蛋白表达升高(P<0.01)。在线数据库筛选出AFAP1-AS1靶基因显著性富集的信号通路有MAPK、PI3K-Akt、催乳素信号等。冬凌草甲素联合siAFAP1-AS1下调MDA-MB-231细胞中Cdc42、MAP3K10蛋白表达(P<0.01)。结论 冬凌草甲素联合siAFAP1-AS1能抑制人乳腺癌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lncRNA AFAP1-AS1的表达进而阻断Cdc42/MAP3K10通路有关。

AIM To explore the possible molecular mechanism of oridonin against breast cancer by observing the effects of siRNA targeted silencing long chain non-coding RNA AFAP1-AS1 and their associations with the proliferation, migration, invasion and epithelial-mesenchymal transformation of human breast cancer MDA-MB-231 cells.METHODS The differential expressions of lncRNA AFAP1-AS1 in triple negative breast cancer tissues were analyzed by online database, and the relationship between its differential expressions and the survival time of breast cancer patients was determined. The effects of oridonin and siAFAP1-AS1 on the expression of AFAP1-AS1 in MDA-MB-231 cells were detected by RT-qPCR;and their effects on the viability, migration and invasion of MDA-MB-231 cells were detected by CCK-8 assay, wound healing assay and Transwell invasion assay, respectively. The target genes of lncRNA AFAP1-AS1 were analyzed and predicted by bioinformatics technology. The effects of oridonin and siAFAP1-AS1 on the expressions of EMT-related proteins and Cdc42 and MAP3 K10 proteins in MAPK pathway of MDA-MB-231 cells were detected by Western blot.RESULTS The up-regulated expression of lncRNA AFAP1-AS1 in triple negative breast cancer tissue associated with the poor survival of breast cancer patients(P<0.05). Oridonin down-regulated the expression of AFAP1-AS1 in MDA-MB-231 cells(P<0.05);MDA-MB-231 cells co-treated with siAFAP1-AS1 and oridonin were observed with decreased cell viability, cell migration and invasion ability, and decreased protein expressions of N-cadherin and Vimentin, but increased protein expression of E-cadherin(P<0.01). As a result of online database screening, MAPK, PI3 K-Akt and prolactin signals were the signal pathways of significant enrichment of AFAP1-AS1 target genes. The co-treatment of oridonin and siAFAP1-AS1 down-regulated the protein expressions of Cdc42 and MAP3 K10 in MDA-MB-231 cells(P<0.05).CONCLUSION The co-treatment of oridonin and siAFAP1-AS1 inhibits the proliferation, migration, invasion and EMT progression of human breast cancer MDA-MB-231 cells, and the mechanism may be associated with its effects in down-regulating the expression of lncRNA AFAP1-AS1 and thus blocking the Cdc42/MAP3 K10 pathway.