Med19通过HMGB1介导自噬参与乳腺癌化疗耐药
发布时间：2018-09-07 16:51
【摘要】：目的:耐药是导致乳腺癌化疗失败的主要原因。我们以阿霉素(ariamycin,ADM)耐药的人乳腺癌细胞MCF-7/ADM及其亲本的MCF-7细胞为工具细胞,研究中介体复合物亚基19(mediator complex subunit 19,Med19)对乳腺癌ADM耐药的影响及其机制。方法:构建Med19 RNA干扰(siRNA-Med19)慢病毒载体,分别感染MCF-7/ADM及MCF-7细胞(Med19敲减组,即KD组),并设置空载体感染对照组(NC组)及空白对照组(CON组);高迁移率组蛋白1(high mobility group protein1,HMGB1)RNA干扰慢病毒载体(siRNA-HMGB1),分别感染MCF-7/ADM及MCF-7细胞(HMGB1敲减组,即H-KD组),并设置空载体感染对照组(H-NC组);构建HMGB1过表达慢病毒载体,分别感染KD组和NC组细胞(HMGB1过表达组,KD+H-LV,NC+H-LV),并设置空载体感染对照组(KD+H-LV-NC,NC+H-LV-NC)。CCK8及克隆形成实验检测细胞活力变化;qRT-PCR检测Med19及自噬标志物LC3B、自噬相关基因3(autophagy associated gene 3,Atg3)和自噬相关基因5(autophagy associated gene 5,Atg5)m RNA表达;Western blot检测LC3Ⅰ向LC3Ⅱ的转化、Atg3、Atg5及P62的蛋白表达;免疫荧光检测自噬标志物LC3点的积累量的变化。结果:与亲本MCF-7细胞相比,MCF-7/ADM细胞ADM、紫杉醇(taxinol,TAX)和顺铂(cisplatin,DDP)作用的IC50值显著增高,同时MDR1mRNA及蛋白表达水平显著增高,差异均有统计学意义(P0.05),表明MCF-7/ADM细胞具有多药耐药性。与MCF-7细胞相比,MCF-7/ADM细胞中Med19、LC3B、Atg3和Atg5 mRNA及蛋白水平显著增高,LC3Ⅱ/LC3Ⅰ比例及LC3荧光点的积累显著增高,P62蛋白水平显著降低,差异均有统计学意义(P0.05),表明MCF-7/ADM细胞Med19高表达且呈高自噬水平。与未经新辅助化疗组(non-NACT组)相比,新辅助化疗组(NACT组)中Med19、LC3Ⅱ/LC3Ⅰ比例显著增加,P62表达显著降低。ADM、自噬抑制剂3-甲基嘌呤(3-Methyladenine,3-MA)分别处理MCF-7/ADM和MCF-7细胞,其中ADM组自噬水平显著增加,3-MA处理组呈相反趋势且细胞活力显著降低,差异均有统计学意义(P0.05),表明ADM诱导自噬,抑制自噬增加细胞对ADM的敏感性。雷帕霉素(rapamycin,Rap)为通用自噬诱导剂,在Med19、自噬及化疗药物的关系的探究中发现,与CON及NC组、NC+ADM组、NC+Rap组相比,MCF-7/ADM及MCF-7细胞相应的KD组、KD+ADM组、KD+Rap组自噬水平显著降低,表明抑制Med19表达可抑制自噬;此外,与CON、NC组相比,MCF-7/ADM及MCF-7细胞KD组对ADM、TAX和DDP的细胞活力显著降低,差异均有统计学意义(P0.05);功能回复实验显示,与KD组相比,KD+Rap组自噬水平及细胞活力均显著增加(P0.05),两者均趋于NC组,表明Med19通过诱导自噬介导乳腺癌细胞化疗耐药,抑制Med19表达可抑制自噬,增加乳腺癌细胞化疗敏感性。在Med19调控自噬的机制的探究中发现,与CON、NC组相比,MCF-7/ADM及MCF-7细胞KD组HMGB1 mRNA及蛋白表达水平显著降低,与H-NC组相比,H-KD组自噬水平及细胞活力均显著降低,差异均有统计学意义(P0.05),但并不影响Med19 mRNA及蛋白表达水平(P0.05);功能回复实验显示,与KD+H-LV-NC组相比,KD+H-LV组自噬水平及细胞活力均显著增加(P0.05),两者均趋于NC+H-LV-NC组,这些结果表明,上调HMGB1可以消除Med19敲减引起的化疗增敏作用,即Med19可能通过HMGB1发挥介导自噬和化疗耐药的作用。结论:1、ADM耐药的乳腺癌细胞MCF-7/ADM具有多药耐药性,ADM耐药细胞及NACT人乳腺癌组织高表达Med19且具有高自噬水平;2、Med19敲减抑制乳腺癌细胞MCF-7/ADM、MCF-7的基础自噬水平和ADM诱导的自噬;3、Med19敲减通过抑制自噬增加MCF-7、MCF-7/ADM细胞对ADM的敏感性;4、Med19介导自噬促进乳腺癌细胞的化疗耐药可能通过HMGB1通路发挥作用。
[Abstract]:Objective: Drug resistance is the main cause of chemotherapy failure in breast cancer. Med19 and its mediator complex subunit (Med19) were constructed from adriamycin-resistant human breast cancer cell line MCF-7/ADM and its parent MCF-7 cells. RNA interference (siRNA-Med19) lentiviral vectors were infected with MCF-7/ADM and MCF-7 cells (KD group) respectively, and blank vector infection control group (NC group) and blank control group (CON group); high mobility group protein1 (HMGB1) RNA interference lentiviral vectors (siRNA-HMGB1) were infected with MCF-7/ADM and MCF-7 cells (HMGB1), respectively. MGB1 knockdown group (H-KD group), and set up empty vector infection control group (H-NC group); construct HMGB1 overexpression lentiviral vector, infected KD group and NC group cells (HMGB1 overexpression group, KD+H-LV, NC+H-LV), and set up empty vector infection control group (KD+H-LV-NC, NC+H-LV-NC). CCK8 and cloning formation assay to detect cell viability changes; The expression of autophagy associated gene 3 (Atg3) and autophagy associated gene 5 (Atg5) m RNA was detected by Western blot, and the expression of ATg3, Atg5 and P62 was detected by immunofluorescence. Compared with MCF-7/ADM cells, the IC50 values of ADM, Taxinol (TAX) and cisplatin (DDP) in MCF-7/ADM cells were significantly increased, while the levels of MDR1 mRNA and protein expression were significantly increased (P 0.05), indicating that MCF-7/ADM cells had multidrug resistance. Compared with MCF-7 cells, MCF-7/ADM cells had Med19, LC3B, Atg3 and Atg5 mRN. Compared with the non-NACT group, the neoadjuvant chemotherapy group (NACT group) showed a higher expression of Med19 and a higher level of autophagy. ADM, autophagy inhibitor 3-methyladenine (3-MA) respectively treated MCF-7/ADM and MCF-7 cells, ADM group autophagy level increased significantly, 3-MA treatment group showed the opposite trend and cell viability significantly decreased, the difference was statistically significant (P 0.05), indicating that ADM induced autophagy, inhibited autophagy. Rapamycin (Rap) is a universal autophagy inducer. In the study of the relationship between Med19, autophagy and chemotherapeutics, it was found that the autophagy level of MCF-7/ADM and MCF-7 cells in KD group, KD+ADM group, KD+Rap group was significantly lower than that in the control group, NC group, NC+ADM group, NC+Rap group, and the corresponding KD group, KD+ADM group and KD+Rap group. In addition, compared with CON and NC groups, MCF-7/ADM and MCF-7 cell KD groups had significantly decreased cell viability to ADM, TAX and DDP (P 0.05); functional recovery test showed that KD+Rap group had significantly increased autophagy level and cell viability (P 0.05), both tended to NC group, suggesting that Med19 could induce autophagy to mediate mammary gland activity. Inhibiting the expression of Med19 can inhibit autophagy and increase the chemosensitivity of breast cancer cells. In the study of the mechanism of Med19 regulating autophagy, we found that the expression of HMGB1 mRNA and protein in MCF-7/ADM and MCF-7 cell KD groups were significantly lower than those in CN and NC groups, and the autophagy level and cell viability in H-KD group were significantly lower than those in H-NC group. The difference was statistically significant (P 0.05), but did not affect the expression of Med19 mRNA and protein (P 0.05); functional recovery test showed that KD+H-LV-NC group compared with KD+H-LV-NC group, KD+H-LV group autophagy level and cell viability were significantly increased (P 0.05), both tended to NC+H-LV-NC group, these results show that the up-regulation of HMGB1 can eliminate the increase of chemotherapy induced by Med19 knockdown. Conclusion: 1. ADM-resistant breast cancer cells MCF-7/ADM have multidrug resistance, ADM-resistant cells and NACT human breast cancer tissues have high expression of Med19 and high autophagy level; 2, Med19 knockdown inhibits the basal autophagy level of breast cancer cells MCF-7/ADM, MCF-7 and ADF-7. M-induced autophagy; 3, Med19 knockdown increases the sensitivity of MCF-7, MCF-7/ADM cells to ADM by inhibiting autophagy; 4, Med19-mediated autophagy promotes chemotherapeutic resistance of breast cancer cells through the HMGB1 pathway.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
本文编号：2228846
[Abstract]:Objective: Drug resistance is the main cause of chemotherapy failure in breast cancer. Med19 and its mediator complex subunit (Med19) were constructed from adriamycin-resistant human breast cancer cell line MCF-7/ADM and its parent MCF-7 cells. RNA interference (siRNA-Med19) lentiviral vectors were infected with MCF-7/ADM and MCF-7 cells (KD group) respectively, and blank vector infection control group (NC group) and blank control group (CON group); high mobility group protein1 (HMGB1) RNA interference lentiviral vectors (siRNA-HMGB1) were infected with MCF-7/ADM and MCF-7 cells (HMGB1), respectively. MGB1 knockdown group (H-KD group), and set up empty vector infection control group (H-NC group); construct HMGB1 overexpression lentiviral vector, infected KD group and NC group cells (HMGB1 overexpression group, KD+H-LV, NC+H-LV), and set up empty vector infection control group (KD+H-LV-NC, NC+H-LV-NC). CCK8 and cloning formation assay to detect cell viability changes; The expression of autophagy associated gene 3 (Atg3) and autophagy associated gene 5 (Atg5) m RNA was detected by Western blot, and the expression of ATg3, Atg5 and P62 was detected by immunofluorescence. Compared with MCF-7/ADM cells, the IC50 values of ADM, Taxinol (TAX) and cisplatin (DDP) in MCF-7/ADM cells were significantly increased, while the levels of MDR1 mRNA and protein expression were significantly increased (P 0.05), indicating that MCF-7/ADM cells had multidrug resistance. Compared with MCF-7 cells, MCF-7/ADM cells had Med19, LC3B, Atg3 and Atg5 mRN. Compared with the non-NACT group, the neoadjuvant chemotherapy group (NACT group) showed a higher expression of Med19 and a higher level of autophagy. ADM, autophagy inhibitor 3-methyladenine (3-MA) respectively treated MCF-7/ADM and MCF-7 cells, ADM group autophagy level increased significantly, 3-MA treatment group showed the opposite trend and cell viability significantly decreased, the difference was statistically significant (P 0.05), indicating that ADM induced autophagy, inhibited autophagy. Rapamycin (Rap) is a universal autophagy inducer. In the study of the relationship between Med19, autophagy and chemotherapeutics, it was found that the autophagy level of MCF-7/ADM and MCF-7 cells in KD group, KD+ADM group, KD+Rap group was significantly lower than that in the control group, NC group, NC+ADM group, NC+Rap group, and the corresponding KD group, KD+ADM group and KD+Rap group. In addition, compared with CON and NC groups, MCF-7/ADM and MCF-7 cell KD groups had significantly decreased cell viability to ADM, TAX and DDP (P 0.05); functional recovery test showed that KD+Rap group had significantly increased autophagy level and cell viability (P 0.05), both tended to NC group, suggesting that Med19 could induce autophagy to mediate mammary gland activity. Inhibiting the expression of Med19 can inhibit autophagy and increase the chemosensitivity of breast cancer cells. In the study of the mechanism of Med19 regulating autophagy, we found that the expression of HMGB1 mRNA and protein in MCF-7/ADM and MCF-7 cell KD groups were significantly lower than those in CN and NC groups, and the autophagy level and cell viability in H-KD group were significantly lower than those in H-NC group. The difference was statistically significant (P 0.05), but did not affect the expression of Med19 mRNA and protein (P 0.05); functional recovery test showed that KD+H-LV-NC group compared with KD+H-LV-NC group, KD+H-LV group autophagy level and cell viability were significantly increased (P 0.05), both tended to NC+H-LV-NC group, these results show that the up-regulation of HMGB1 can eliminate the increase of chemotherapy induced by Med19 knockdown. Conclusion: 1. ADM-resistant breast cancer cells MCF-7/ADM have multidrug resistance, ADM-resistant cells and NACT human breast cancer tissues have high expression of Med19 and high autophagy level; 2, Med19 knockdown inhibits the basal autophagy level of breast cancer cells MCF-7/ADM, MCF-7 and ADF-7. M-induced autophagy; 3, Med19 knockdown increases the sensitivity of MCF-7, MCF-7/ADM cells to ADM by inhibiting autophagy; 4, Med19-mediated autophagy promotes chemotherapeutic resistance of breast cancer cells through the HMGB1 pathway.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
【参考文献】
相关期刊论文 前6条
1 张进芳;刘晓力;林愈灯;李玉玲;潘建伟;宗飒;周洋;;hnRNP K调控细胞自噬参与急性髓系白血病阿霉素耐药研究[J];中国实验血液学杂志;2016年06期
2 杨晓丹;郭子健;刘贝贝;李莉华;;Med19基因沉默对p53野生型和突变型乳腺癌细胞紫杉醇化疗疗效的影响[J];中国肿瘤生物治疗杂志;2015年06期
3 白玉盘;杨小利;欧周罗;;mTOR信号通路介导产生XCL1可促进乳腺癌耐药细胞株的增殖[J];中国癌症杂志;2014年10期
4 王晓艳;郭子健;吴小红;李莉华;;沉默中介体19对人乳腺癌MCF-7细胞侵袭及迁移的影响[J];实用医学杂志;2014年09期
5 戴桂萍;李莉华;郭子健;王晓艳;;Med19在乳腺癌中的表达及其与临床病理特征的关系[J];江苏医药;2013年01期
6 王晓艳;华东;李莉华;高琪;郭子健;;沉默Med19对人乳腺癌裸鼠成瘤的影响[J];实用医学杂志;2011年17期
，本文编号：2228846
本文链接：https://www.wllwen.com/yixuelunwen/yiyaoxuelunwen/2228846.html
最近更新
教材专著
