复方斑蝥通过miR-520d/Beclin1信号轴逆转三阴乳腺癌化疗耐药的机制研究

Compound Cantharis Reverses Chemotherapy Resistance of TNBC via MiR-520d/Beclin1 Signal Axis

目的探讨复方斑蝥注射液(Compound Cantharis Injection,CCI)通过miR-520d/Beclin1信号轴逆转三阴乳腺癌(Triple-negative breast cancer,TNBC)化疗耐药的机制研究。方法构建人三阴性乳腺癌细胞MDA-MB-231,MDA-MB-468的耐药模型MDA-MB-231/Doc,MDA-MB-468/Doc。MTT法检测亲本株和耐药株细胞活性;Western blot检测自噬相关蛋白表达;miRNA芯片检测复方斑蝥处理前后的TNBC耐药细胞;实时定量PCR(RealtimePCR)检验miRNA表达以验证芯片检测结果;荧光素酶实验验证miRNA和BECN1/Beclin1的结合位点。结果MTT结果显示,CCI能够显著提高TNBC耐药细胞株化疗敏感性。WB结果表明,CCI能够浓度依赖性的抑制TNBC耐药细胞的自噬水平;miRNA芯片检测发现复方斑蝥能够显著上调miR-520d水平,RealtimePCR验证了这一结果。荧光素酶实验表明miR-520d通过作用于Beclin1/BECN13′UTR区域抑制其表达;最后,WB检测证明miR-520d mimics能够明显抑制TNBC耐药细胞株中Beclin1/BECN1的蛋白表达,MTT法结果显示miR-520d mimics与TNBC耐药性细胞对多西他赛的敏感性成正相关。结论CCI通过调控miR-520d/BECN1/Beclin1信号轴逆转三阴性乳腺癌化疗耐药。

Objective To explore the mechanism of compound cantharidin injection reversing chemoresistance of triple-negative breast cancer through microRNA-520 d/Beclin1 signal axis.Methods The human TNBC cell MDAMB-231,MDA-MB-468 resistance model MDA-MB-231/Doc,MDA-MB-468/Doc was constructed.MTT assay was used to detect the activity of parental and drug-resistant strains;Western blot was used to detect the expression of autophagy-related proteins;miRNA microarray was used to detect TNBC-resistant cells before and after treatment with compound cantharidin injection;Realtime PCR was used to detect the expression of miRNA to verify the detection results;luciferase The experiment verified the binding sites of miRNA and BECN1/Beclin1.Results MTT results showed that Compound Cantharidin injection can significantly improve the chemosensitivity of TNBC resistant cell lines.The WB results showed that the compound cantharidin injection could inhibit the autophagy level of TNBC-resistant cells in a concentration-dependent manner.The miRNA microarray showed that the compound cantharidin injection can significantly up-regulate the level of miR-520 d,and RealtimePCR verified the result.Luciferase assay showed that MiR-520 d inhibited its expression by acting on the Beclin1/BECN13′UTR region.Finally,WB assay demonstrated that miR-520 d mimics can significantly inhibit Beclin1/BECN1 protein expression in TNBC-resistant cell lines.MTT assay results It is shown that miR-520 d mimics is positively correlated with the sensitivity of TNBC-resistant cells to docetaxel.Conclusion Compound Cantharidin injection reverses TNBC chemotherapy resistance by regulating miR-520 d/BECN1/Beclin1 signal axis.