三阴乳腺癌细胞中Atg13基因的过表达与化疗耐药的研究

Atg13 gene overexpression induces chemotherapy resistance in triple-negative breast cancer cells

目的探讨自噬相关基因13(Atg13)与三阴性乳腺癌的化疗耐药之间的关系。方法间歇刺激法构建对阿霉素耐受的MDA-MB-231 TNBC细胞株(命名为MDA-MB-231/Dox),并采用药物敏感实验和细胞凋亡实验(TUNEL染色)对耐药细胞株MDA-MB-231/Dox的耐药表型进行鉴定;蛋白免疫印迹实验检测敏感细胞MDA-MB-231和耐药细胞MDA-MB-231/Dox中的自噬相关蛋白LC3A、LC3B的表达情况,免疫荧光法测定细胞内的LC3B荧光斑点数;脂质体转染重组干扰质粒sh-Atg13至耐药细胞MDA-MB-231/Dox中,并经嘌呤霉素筛选得到稳转细胞(命名为MDA-MB-231/Dox+sh-Atg13),蛋白免疫印迹实验检测稳转细胞MDA-MB-231/Dox+sh-Atg13及空载体对照细胞MDA-MB-231/Dox+control plasmid中的Atg13蛋白表达水平,同时检测细胞内的自噬水平(自噬相关蛋白LC3A、LC3B的表达量及LC3B荧光斑点数)及各组细胞对Dox的药物敏感性。结果成功构建对Dox耐药的TNBC细胞株MDA-MB-231/Dox,并且发现耐药细胞中的基础自噬水平显著高于亲本细胞MDA-MB-231(P<0.05)。重组干扰质粒sh-Atg13抑制Atg13基因表达后,使得耐药细胞MDA-MB-231/Dox中的自噬体标志分子LC3B蛋白表达量显著降低(P<0.05),且耐药细胞对Dox的药物敏感性增加(IC50值显著降低,P<0.05)。结论在TNBC细胞MDA-MB-231中,Atg13参与了细胞对Dox获得性耐药的产生。

Objective To explore the impact of gene 13(Atg13)on promoting chemotherapy resistance in triple-negative breast cancer(TNBC)cells.Methods We developed a doxorubicin-resistant MDA-MB-231/Dox cell lines using intermittent application of doxorubicin(Dox).MDA-MB-231/Dox was determined by the drug-sensitive assay and the apoptosis assay(TUNEL staining).The expression of LC3A and LC3B in TNBC cells(MDA-MB-231 and MDA-MB-231/Dox)was detected by Western Blot(WB).The number of LC3B fluorescence spots in the cells was quantified by immunofluorescence.Recombinant plasmid sh-Atg13 was transfected into MDA-MB-231/Dox by liposome,then stable cells(referred to as MDA-MB-231/Dox+sh-Atg13)were obtained by puromycin screening(PM).The Atg13 expression level in MDA-MB-231/Dox+sh-Atg13 and empty plasmid MDA-MB-231/Dox+control plasmid by WB.Changes in autophagy activity were determined by the level of LC3A,LC3B expression and the number of LC3B fluorescent spots.Drug sensitivity of each cell line to Dox was also examined.Results Dox-resistant TNBC cell line MDA-MB-231/Dox were successfully established in vitro.The results showed that the level of basal autophagy in drug-resistant cells was significantly higher than that in sensitive cells MDA-MB-231(P<0.05).After inhibiting the expression of Atg13 gene by recombinant interference plasmid sh-Atg13,the protein expression of autophagy marker LC3B in drug-resistant cells MDA-MB-231/Dox was significantly decreased(P<0.05),the drug sensitivity of MDA-MB-231/Dox cells to Dox was increased(IC50 value was significant,P<0.05).Conclusion In TNBC cell MDA-MB-231,Atg13 increased chemotherapy resistance by inducing autophagy were determined.