miRNA-139-5p靶向Notch1/Jagged1调控胃癌干细胞增殖转移及益气补肾方的干预作用

miRNA-139-5p targets Notch1/Jagged1 to regulate the proliferation and metastasis of gastric cancer stem cells and the intervention effect of Yiqi Bushen Formula

目的:明确miRNA-139-5p对Notch1/Jagged1信号通路的靶向调控关系,探讨益气补肾方抗胃癌干细胞增殖转移的作用机制。方法:通过免疫磁珠分选及鉴定CD44+EpCAM+SGC-7901 CSC;双荧光素酶报告基因检测miRNA-139-5p与Notch1的结合作用;qRT-PCR检测不同细胞株中miRNA-139-5p、Notch1与Jagged1 mRNA表达;Western Blot检测不同细胞株中Notch1、Jagged1蛋白表达;qRT-PCR检测益气补肾方含药血清干预后,胃癌细胞株中miRNA-139-5p、Notch1与Jagged1 mRNA表达以及Notch1、Jagged1蛋白表达。结果:免疫磁珠能较好地分选出CD44+EpCAM+SGC-7901 CSC细胞株。转染克隆有Notch13’-UTR野生型载体质粒实验中,miRNA-139-5p组荧光素酶活性明显受到抑制,与阴性对照组比较,差异有统计学意义(P<0.01)。胃癌细胞中miRNA-139-5p的表达量显著减少,Notch1、Jagged1的mRNA与蛋白表达量均显著增加(P<0.05,P<0.01)。益气补肾含药血清干预后,胃癌细胞中miRNA-139-5p表达水平显著升高,Notch1、Jagged1 mRNA与蛋白表达水平均显著降低(P<0.01)。结论:Notch1基因是miRNA-139-5p直接作用的靶基因,益气补肾方可能是通过调控miRNA-139-5p/Notch1/Jagged1信号通路而发挥抗胃癌增殖转移的作用。

Objective:To clarify the targeted regulation of miRNA-139-5 p on Notch1/Jagged1 signaling pathway,and explore the mechanism of Yiqi Bushen Formula against gastric cancer stem cell proliferation and metastasis.Methods:By immunomagnetic bead sorting and identification CD44+EpCAM+SGC-7901 CSC;dual luciferase reporter gene to detect the binding effect of miRNA-139-5 p and Notch1;qRT-PCR detection of miRNA-139-5 p,Notch1 and Jagged1 mRNA in different cell lines expression;Western Blot detection of Notch1,Jagged1 protein expression in different cell lines;qRT-PCR detection of Yiqi Bushen Formula-containing serum intervention,miRNA-139-5 p,Notch1 and Jagged1 mRNA expression and Notch1,Jagged1 in gastric cancer cell lines Protein.Results:Immunomagnetic beads can sort CD44+EpCAM+SGC-7901 cell lines well.Transfected with Notch13’-UTR wild-type vector plasmid experiment,miRNA-139-5 p group luciferase activity was significantly inhibited,compared with the negative control group,the difference in luciferase activity was significant(P<0.01).The expression of miRNA-139-5 p in gastric cancer cells was significantly reduced,and the mRNA and protein expression of Notch1 and Jagged1 were significantly increased(P<0.05,P<0.01).After the intervention of Yiqi Bushen Formula-containing serum,the expression level of miRNA-139-5 p in gastric cancer cells increased significantly,and the expression levels of Notch1,Jagged1 mRNA and protein decreased(P<0.01).Conclusion:Notch1 gene is the direct target gene of miRNA-139-5 p.Yiqi Bushen Formula may play the anti-proliferative and metastatic role in gastric cancer by regulating the miRNA-139-5 p/Notch1/Jagged1 signaling pathway.