二氢杨梅素可逆转SKBR3细胞对赫赛汀的耐药:基于上调miR 98-5p抑制IGF1R/HER2二聚体形成

Dihydromyricetin reverses Herceptin resistance by up-regulating miR-98-5p and inhibit-ing IGF1R/HER2 dimer formation in SKBR3 cells

目的探索二氢杨梅素对SKBR3细胞miR-98-5p表达影响及在赫赛汀耐药发生中的机制。方法通过TargetScan在线数据库对IGF2和miR-98在乳腺癌中的表达及相互作用进行生信分析。细胞实验培养SKBR3细胞并构建耐曲妥珠单抗的SKBR3-R细胞和裸鼠异种移植瘤模型,免疫印迹法和免疫组化法检测细胞和组织内关键信号蛋白表达及其磷酸化水平。shRNA慢病毒载体构建转染细胞系,RT-qPCR检测细胞内RNA表达水平,MTS法检测细胞增殖,Transwell法检测细胞侵袭。荧光素酶实验验证RNA间的相互作用,免疫共沉淀法分析IGF-1R/HER2异源二聚体含量。结果在SKBR3-R细胞中IGF2、p-IGF1R、p-Akt、p-S6K水平高于SKBR3细胞,而PTEN蛋白表达正好相反(P<0.05);SKBR3-R细胞中IGF1R/HER2异源二聚体含量升高(P<0.01);转染miR-98-5p的SKBR3-R细胞内IGF2 mRNA表达水平变化无统计学意义(P>0.05),但IGF2表达水平降低,细胞侵袭能力降低(P<0.05);经二氢杨梅素饲养后耐药裸鼠肿瘤组织中miR-98-5p表达上调,IGF2表达下降,对赫赛汀敏感性升高(P<0.05)。结论二氢杨梅素通过诱导SKBR3-R细胞内miR-98-5p表达,使其结合IGF2 mRNA降低IGF2水平,进一步抑制IGF-1R/HER2异源二聚体形成,从而逆转细胞对赫赛汀耐药。

Objective To explore the effect of dihydromyricetin on the expression of miR-98-5p and its mechanism in the development of Herceptin resistance in SKBR3 cells.Methods The expression of IGF2 and miR-98-5p and their interaction relationship were analyzed by bioinformatics analysis through TargetScan online databases.SKBR3 cells and drug-resistant SKBR3-R cells were cultured in cell experiments.Xenograft tumor mice were constructed by SKBR3 and SKBR3-R cells.Proteins were detected by western blotting and immunohistochemistry.Transfected cells were constructed by shRNA lentivirus vectors.RT-QPCR was used to detect RNA.Cell proliferation was detected by MTS method.Cell jnvasion was detected by Transwell assay.Luciferase reporting assays were used to verify RNA interactions.IGF-1R/HER2 heterodimer was determined by immunocoprecipitation.Results The expression of IGF2,p-IGF1R,p-Akt and p-S6K in SKBR3-R cells were significantly higher than those in SKBR3 cells,while the expression of PTEN protein was lower in SKBR3-R cells(P<0.05).IGF1R/HER2 heterodimer in SKBR3-R cells was significantly increased(P<0.01).The expression of IGF2 and invasion ability were significantly reduced while transfected with miR-98-5p in SKBR3-R cells(P<0.05),but the IGF2 mRNA were no difference in both cells(P>0.05).The expression of miR-98-5p was up-regulated and IGF2 was decreased in drug-resistant xenograft tumor mice after feeding with dihydromyricetin,and the tumor became more sensitivity to Herceptin(P<0.05).Conclusion Dihydromyricetin could induce the expression of miR-98-5p,which binds to IGF2 mRNA to reduce IGF2 expression,inhibit the IGF-1R/HER2 formation,thereby reversing cell resistance to Herceptin in SKBR3-R cells.