Med19通過HMGB1介導自噬參與乳腺癌化療耐藥
發(fā)布時間：2018-09-07 16:51
【摘要】：目的:耐藥是導致乳腺癌化療失敗的主要原因。我們以阿霉素(ariamycin,ADM)耐藥的人乳腺癌細胞MCF-7/ADM及其親本的MCF-7細胞為工具細胞,研究中介體復合物亞基19(mediator complex subunit 19,Med19)對乳腺癌ADM耐藥的影響及其機制。方法:構(gòu)建Med19 RNA干擾(siRNA-Med19)慢病毒載體,分別感染MCF-7/ADM及MCF-7細胞(Med19敲減組,即KD組),并設置空載體感染對照組(NC組)及空白對照組(CON組);高遷移率組蛋白1(high mobility group protein1,HMGB1)RNA干擾慢病毒載體(siRNA-HMGB1),分別感染MCF-7/ADM及MCF-7細胞(HMGB1敲減組,即H-KD組),并設置空載體感染對照組(H-NC組);構(gòu)建HMGB1過表達慢病毒載體,分別感染KD組和NC組細胞(HMGB1過表達組,KD+H-LV,NC+H-LV),并設置空載體感染對照組(KD+H-LV-NC,NC+H-LV-NC)。CCK8及克隆形成實驗檢測細胞活力變化;qRT-PCR檢測Med19及自噬標志物LC3B、自噬相關(guān)基因3(autophagy associated gene 3,Atg3)和自噬相關(guān)基因5(autophagy associated gene 5,Atg5)m RNA表達;Western blot檢測LC3Ⅰ向LC3Ⅱ的轉(zhuǎn)化、Atg3、Atg5及P62的蛋白表達;免疫熒光檢測自噬標志物LC3點的積累量的變化。結(jié)果:與親本MCF-7細胞相比,MCF-7/ADM細胞ADM、紫杉醇(taxinol,TAX)和順鉑(cisplatin,DDP)作用的IC50值顯著增高,同時MDR1mRNA及蛋白表達水平顯著增高,差異均有統(tǒng)計學意義(P0.05),表明MCF-7/ADM細胞具有多藥耐藥性。與MCF-7細胞相比,MCF-7/ADM細胞中Med19、LC3B、Atg3和Atg5 mRNA及蛋白水平顯著增高,LC3Ⅱ/LC3Ⅰ比例及LC3熒光點的積累顯著增高,P62蛋白水平顯著降低,差異均有統(tǒng)計學意義(P0.05),表明MCF-7/ADM細胞Med19高表達且呈高自噬水平。與未經(jīng)新輔助化療組(non-NACT組)相比,新輔助化療組(NACT組)中Med19、LC3Ⅱ/LC3Ⅰ比例顯著增加,P62表達顯著降低。ADM、自噬抑制劑3-甲基嘌呤(3-Methyladenine,3-MA)分別處理MCF-7/ADM和MCF-7細胞,其中ADM組自噬水平顯著增加,3-MA處理組呈相反趨勢且細胞活力顯著降低,差異均有統(tǒng)計學意義(P0.05),表明ADM誘導自噬,抑制自噬增加細胞對ADM的敏感性。雷帕霉素(rapamycin,Rap)為通用自噬誘導劑,在Med19、自噬及化療藥物的關(guān)系的探究中發(fā)現(xiàn),與CON及NC組、NC+ADM組、NC+Rap組相比,MCF-7/ADM及MCF-7細胞相應的KD組、KD+ADM組、KD+Rap組自噬水平顯著降低,表明抑制Med19表達可抑制自噬;此外,與CON、NC組相比,MCF-7/ADM及MCF-7細胞KD組對ADM、TAX和DDP的細胞活力顯著降低,差異均有統(tǒng)計學意義(P0.05);功能回復實驗顯示,與KD組相比,KD+Rap組自噬水平及細胞活力均顯著增加(P0.05),兩者均趨于NC組,表明Med19通過誘導自噬介導乳腺癌細胞化療耐藥,抑制Med19表達可抑制自噬,增加乳腺癌細胞化療敏感性。在Med19調(diào)控自噬的機制的探究中發(fā)現(xiàn),與CON、NC組相比,MCF-7/ADM及MCF-7細胞KD組HMGB1 mRNA及蛋白表達水平顯著降低,與H-NC組相比,H-KD組自噬水平及細胞活力均顯著降低,差異均有統(tǒng)計學意義(P0.05),但并不影響Med19 mRNA及蛋白表達水平(P0.05);功能回復實驗顯示,與KD+H-LV-NC組相比,KD+H-LV組自噬水平及細胞活力均顯著增加(P0.05),兩者均趨于NC+H-LV-NC組,這些結(jié)果表明,上調(diào)HMGB1可以消除Med19敲減引起的化療增敏作用,即Med19可能通過HMGB1發(fā)揮介導自噬和化療耐藥的作用。結(jié)論:1、ADM耐藥的乳腺癌細胞MCF-7/ADM具有多藥耐藥性,ADM耐藥細胞及NACT人乳腺癌組織高表達Med19且具有高自噬水平;2、Med19敲減抑制乳腺癌細胞MCF-7/ADM、MCF-7的基礎自噬水平和ADM誘導的自噬;3、Med19敲減通過抑制自噬增加MCF-7、MCF-7/ADM細胞對ADM的敏感性;4、Med19介導自噬促進乳腺癌細胞的化療耐藥可能通過HMGB1通路發(fā)揮作用。
[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.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96
本文編號：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.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96
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