CXCL12/CXCR4轴通过调节自噬促进乳腺癌细胞对多柔比星的化疗抗性

CXCL12/CXCR4 axis promotes chemotherapy resistance of breast cancer cells to doxorubicin by regulating autophagy

目的:探究CXCL12/CXCR4轴在乳腺癌细胞多柔比星(Dox)化疗抗性中的作用及相关机制。方法:体外培养乳腺癌细胞系MCF-7,通过MTT实验检测在CXCL12作用下乳腺癌细胞对Dox的敏感性变化,RT-PCR和Western blot检测上述细胞中CXCL12受体CXCR4与CXCR7的mRNA及蛋白的表达水平;采用siRNA干扰技术靶向沉默乳腺癌细胞中CXCR4表达,RT-PCR和Western blot检测转染效率后,依次使用MTT、Annexin V-FITC/PI流式细胞术及Western blot明确在CXCL12作用下沉默CXCR4表达对Dox介导的乳腺癌细胞的抑制率,凋亡、自噬及PI3K/Akt信号通路表达的影响。结果:CXCL12能够通过促进CXCR4的表达,提高乳腺癌细胞对Dox的化疗抗性;转染siRNA能够显著抑制乳腺癌细胞中CXCR4的mRNA及蛋白的表达水平;而沉默CXCR4能显著改善CXCL12作用下的乳腺癌细胞对Dox的敏感性,提高Dox诱导的细胞凋亡率,并通过抑制自噬相关蛋白LC3B与Beclin1表达,下调乳腺癌细胞中的自噬水平;同时,沉默CXCR4能通过抑制PI3K/Akt信号通路中PI3K蛋白表达及Akt蛋白磷酸化水平,下调抗凋亡蛋白BCL-2,促进凋亡相关蛋白Bax与cleaved Caspase-3表达。结论:CXCL12/CXCR4在乳腺癌细胞Dox耐药性中具有重要作用,而沉默CXCR4表达能通过下调MCF-7细胞的自噬水平,提高Dox诱导的细胞凋亡,从而改善肿瘤细胞对Dox的敏感性。

Objective:To explore the role of CXCL12/CXCR4 axis in anti-doxorubicin(Dox)resistance of breast cancer cells and its related mechanism.Methods:Breast cancer cell line MCF-7 was cultured in vitro.The sensitivity of breast cancer cells to Dox was detected by MTT assay.The mRNA and protein expression levels of CXCL12 receptor CXCR4 and CXCR7 were detected by RTPCR and Western blot.siRNA interference was used to silence CXCR4 expression in breast cancer cells.RT-PCR and Western blot were used to detect the transfection efficiency.MTT,Annexin V-FITC/PI flow cytometry and Western blot were used to determine the effects of CXCR4 silencing on DOX mediated proliferation inhibition,apoptosis,autophagy and PI3K/Akt signaling pathway expres-sion in breast cancer cells.Results:CXCL12 could enhance the chemoresistance of breast cancer cells to Dox by promoting the expres-sion of CXCR4.siRNA could significantly inhibit the expression of CXCR4 mRNA and protein in breast cancer cells.Silencing CXCR4 can significantly improve the sensitivity of breast cancer cells to Dox and increase the apoptosis rate mediated by Dox,and down-regu-lation the autophagy level in breast cancer cell by inhibiting the expression of autophagy related proteins LC3B and Beclin1.At the same time,silencing CXCR4 could inhibit the phosphorylation level of PI3K and Akt protein in PI3K/Akt signaling pathway,reduced the anti-apoptotic protein Bcl-2,promoted apoptosis related protein Bax and cleaved caspase-3 expression.Conclusion:CXCL12/CXCR4 played an important role in Dox resistance of breast cancer cells.Silencing CXCR4 expression could improve the sensitivity of tumor cells to Dox by down regulating autophagy level and increasing Dox induced apoptosis.