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. 2022 Feb 20;42(2):207-214.
doi: 10.12122/j.issn.1673-4254.2022.02.06.

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

[Article in Chinese]
M Zhang  1 , C Guo  1 , Y Chu  1 , R Xu  1 , F Yin  1 , J Qian  1
Affiliations

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

[Article in Chinese]
M Zhang et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

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.

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。

Keywords: Herceptin; breast cancer; dihydromyricetin; drug resistance; heterodimer; miR-98-5p.

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Figures

图 1
图 1
SKBR3及SKBR3-R细胞中IGF2表达 IGF2 expression in SKBR3 and SKBR3-R cells. A: SKBR3- R cells were significantly resistant to Herceptin; B: Compared with SKBR3 cells, there was no significant difference in IGF2 mRNA level between SKBR3-R cells and SKBR3 cells, but the expression of IGF2 protein was significantly increased (****P < 0.001).
图 2
图 2
SKBR3-R, SKBR3细胞内信号蛋白磷酸化水平及PTEN蛋白表达量 The phosphorylation level of signal protein and the expression of PTEN protein in SKBR3- R and SKBR3 cells. *P < 0.05, **P < 0.01.
图 3
图 3
SKBR3-R、SKBR3细胞内IGF1-R/HER2异源二聚体含量 The IGF1-R/HER2 heterodimer in SKBR3-R and SKBR3 cells. ***P < 0.01.
图 4
图 4
miR-98-5p与IGF2 mRNA 3’UTR端存在相互作用 The interaction between miR- 98- 5p and IGF2 mRNA 3'UTR.
图 5
图 5
miR-98-5p的抑制剂和模拟对野生型、突变型荧光素酶报告基因活性的影响 Affection of inhibitors and mimics of miR-98-5p in Wilder type and mutant luciferase reporter gene activity. *P < 0.05.
图 6
图 6
miR-98-5p调控IGF2蛋白表达 The miR-98-5p regulates the IGF2 protein expression. IGF2 protein expression was significantly up-regulated by miR-98-5p inhibitors (***P < 0.01), miR-98-5p mimics significantly down-regulated IGF2 protein expression (**P < 0.01).
图 7
图 7
miR-98-5p对SKBR3细胞侵袭性影响 The invasive effect of miR-98-5p on SKBR3 cells. miR-98-5p inhibitor significantly promoted SKBR3 invasion (*P < 0.05), and miR-98-5p mimic significantly inhibited SKBR3 invasion (***P < 0.01).
图 8
图 8
SKBR3-R细胞异种移植肿瘤对赫赛汀的敏感性显著降低 The sensitivity of SKBR3-R cell xenografts to Herceptin was significantly decreased (P < 0.05).
图 9
图 9
相较于SKBR3，SKBR3-R细胞异种移植肿瘤组织内IGF2、p-IGF1R、p-Akt、p-S6K水平升高 The levels of IGF2, P-IGF1R, P-Akt and P-S6K in SKBR3-R cell xenograft tumor tissue were increased, compared with SKBR3. Scate bar: 50 μm.
图 10
图 10
二氢杨梅素在体内增加SKBR3-R细胞异种移植瘤对赫赛汀敏感性 Dihydromyricetin increases the sensitivity of SkBR3- R xenografts to Herceptin in vivo (*P < 0.05).
图 11
图 11
二氢杨梅素在体内通过miR-98-5p调节IGF2转录后表达水平 Dihydroarxin regulates post-transcriptional IGF2 expression levels in vivo through miR-98-5p. A: Dihydromyristine significantly up-regulated miR- 98- 5p levels in tumor tissues (****P < 0.01); B: Dihydromyricetin had no significant effect on IGF2 mRNA level in tumor tissue (P > 0.05); C: Dihydromyricetin significantly reduced the level of IGF2 protein in tumor tissue (***P < 0.01).
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