SAHA靶定组织蛋白酶V诱导乳腺癌MDA-MB-231细胞自噬  

SAHA targeted CTSV for autophagy induction in MDA-MB-231 breast cancer cells

作　　者：周慧[1] 韩翰[2] 周伟强[3] ZHOU Hui;HAN Han;ZHOU Wei-qiang(Dept of Histology and Embryology,Shenyang Medical College,Shenyang 110034,China;Dept of Biochemistry and Molecular Biology,Shenyang Medical College,Shenyang 110034,China;Dept of Pathogen Biology,Shenyang Medical College,Shenyang 110034,China)

机构地区：[1]沈阳医学院组织学与胚胎学教研室,辽宁沈阳110034 [2]沈阳医学院生物化学与分子生物学教研室,辽宁沈阳110034 [3]沈阳医学院病原生物学教研室,辽宁沈阳110034

出　　处：《中国药理学通报》2020年第11期1563-1568,共6页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81172509);辽宁省教育厅辽宁大学创新人才支持基金(No LR2017040);辽宁省科技厅研究基金(No 2018010621-301,2017012046-301);沈阳医学院科技基金项目(No 20172035,20187074)。

摘　　要：目的探讨组织蛋白酶V(cathepsin V,CTSV)在辛二酰苯胺异羟肟酸(suberoylanilide hydroxamic acid,SAHA)诱导乳腺癌MDA-MB-231细胞自噬中的作用机制。方法以人乳腺癌MDA-MB-231细胞为研究对象,Muse细胞分析仪检测SAHA对细胞活力的影响;Real-time PCR和Western blot检测SAHA对CTSV的mRNA和蛋白的作用;CTSV-siRNA转染细胞,Real-time PCR和Western blot检测CTSV的mRNA和蛋白的情况;细胞免疫荧光法检测SAHA对LC3B表达的影响;Real-time PCR和Western blot检测SAHA诱导自噬相关ATG分子和LC3的mRNA和蛋白的变化;Bafilomycin A1抑制自噬,随后Western blot测定p62蛋白水平,Muse细胞分析仪检测SAHA对细胞活力的作用。结果SAHA在抑制MDA-MB-231细胞增殖的同时促进CTSV的表达。CTSV-siRNA转染细胞后,CTSV的mRNA和蛋白水平均明显降低。SAHA促进LC3免疫荧光点的增加,与CTSV-siRNA联合作用后可以恢复由CTSV-siRNA干扰引起的细胞自噬减弱。SAHA增强了Beclin1的表达,并伴有LC3B升高和mTOR降低。CTSV-siRNA的加入逆转了SAHA的效应。Bafilomycin A1阻断自噬后,被SAHA抑制的p62蛋白的表达和细胞活力明显增加。结论SAHA靶定CTSV诱导乳腺癌MDA-MB-231细胞自噬。Aim To detect the effect of cathepsin V(CTSV)on autophagy induced by SAHA in breast cancer MDA-MB-231 cells.Methods Muse cell analyzer was used to screen the optional treatment doses of drug for cells,and real-time PCR and Western blot were to detect the mRNA and protein expressions of CTSV.Then,specific siRNA was transfected in the cells for silence the CTSV function.Immunofluorescent assay was employed to detect the distribution of LC3B,and related ATG molecule expressions was assessed.Bafilomycin A1 was used to inhibit autophagy flux,then p62 protein was detected.Results 2μmol·L^-1 SAHA inhibited the proliferation of MDA-MB-231 cells accompanied with high expression of CTSV.CTSV-siRNA transfected MDA-MB-231 cells significantly reduced the function of CTSV.With SAHA treatment,the distribution of LC3 increased in cells.While knock-off the function of CTSV by siRNA,the autophagy effect was recovered.Finally,SAHA enhanced the expression of Beclin1,with the increase of LC3B and the decrease of mTOR.The addition of CTSV-siRNA reversed the effect of SAHA.After Bafilomycin A1 blocked autophagy,the p62 protein expression and cell viability inhibited by SAHA increased significantly.Conclusion SAHA-CTSV participates in the induction of autophagy in MDA-MB-231 breast cancer cells.

关 键 词：三阴乳腺癌 组织蛋白酶V SAHA MDA-MB-231 自噬 LC3 

分 类 号：R345.69[医药卫生—基础医学] R394.2