Med19 Is Involved In Chemoresistance By Mediating Autophagy Through HMGB1 In Breast Cancer

Purpose Drug resistance is the main cause of chemotherapy failure in breast cancer.Here we investigated the effects and mechanisms of mediator complex subunit 19(Med19)in ariamycin(ADM)resistance of breast cancer.Methods Lentivirus-mediated Med19 RNA interference vectors(siRNA-Med19)were constructed to infect ADM-resistant MCF-7 human breast cancer cell line(MCF-7/ADM)and the parental MCF-7 cell line respectively(KD groups),and the empty vector-infected groups(NC groups),non-infected groups(CON groups)were used as control.Lentivirus-mediated high mobility group protein1(HMGB1)RNA interference vectors(siRNA-HMGB1)were constructed to infect MCF-7/ADM cells and the parental MCF-7 cells(H-KD groups),respectively,and the empty vectorinfected groups(H-NC groups)were served as control.Lentivirus-mediated overexpression vectors of HMGB1(H-LV)were constructed to infect KD groups or NC groups(KD+H-LV groups or NC+H-LV groups),respectively,and the empty vector-infected groups(KD+H-LV-NC groups or NC+H-LV-NC groups)were served as control.CCK-8 assay and clone formation assay were performed to determine the cell viability.Quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR)was applied to detect the mRNA expression of Med19,LC3 B,autophagy-related gene 3(Atg3),autophagy-related gene 5(Atg5)and HMGB1.Western blot was applied to determine the protein expression of Med19,Atg3,Atg5,P62,HMGB1 and the transformation from LC3-I to LC3-II.The accumulation of RFP-LC3 puncta was detected with immunofluorescent technique.Results Compared with parental MCF-7 cells,the IC50 values for ADM,taxinol(TAX),cisplatin(DDP)in MCF-7/ADM cells were significantly increased(P<0.05),and the m RNA and protein expression of MDR1 were increased simultaneously,which suggest the multi-drug resistant characteristic of MCF-7/ADM cell lines.MCF-7/ADM cells exhibited significantly higher mRNA and protein expression of Med19,LC3 B,Atg3,Atg5,LC3?/LC3?ratio,RFP-LC3 puncta accumulation,and lower P62 protein expression level than MCF-7 cells(P<0.05),which suggest a higher Med19 expression and a higher autophagy level in MCF-7/ADM cells than the parental cells.Neoadjuvant chemotherapy(NACT)group exhibited increased Med19 expression and autophagy levels relative to control group.MCF-7 and MCF-7/ADM cells were treated with ADM or 3-Methyladenine(3-MA),an autophagy inhibitor,and we found an increased level of autophagy with the treatment of ADM.However,3-MA treated group showed the opposite tendency,and besides,the cell viabilities of MCF-7/ADM and MCF-7 cells were significantly decreased after treating with 3-MA(P<0.05).These results suggest that ADM induces autophagy,and inhibiting autophagy could increase the sensitivity to ADM in breast cancer cells.Rapamycin(Rap),a general autophagy inducer,was used for exploring the relationship between Med19,autophagy and chemotherapeutic drugs.We found a lower level of autophagy in the groups of KD,KD+ADM and KD+Rap than the corresponding groups of NC or CON,NC+ADM,NC+Rap,respectively.These findings indicate that knocking down Med19 could inhibit autophagy.In the KD group,the viabilities of MCF-7/ADM and MCF-7 cells to ADM,TAX and DDP were significantly decreased(P<0.05).Besides,functional recovery experiments showed the level of autophagy and cell viabilities were obviously increased in the group of KD+Rap when compared with KD group(P<0.05),and tend to NC group.It is indicated that inhibition the expression of Med19 can inhibit autophagy and increase chemosensitivity of breast cancer cells.Furthermore,we investigated the mechanisms of Med19 in regulating autophagy and found the mRNA and protein expression levels of HMGB1 in KD group were decreased significantly in comparing with NC and CON groups;the group of H-KD exhibited a lower level of autophagy and cell viabilities(P<0.05).However,knocking down the expression of HMGB1 would not affect the expression of Med19(P<0.05).Besides,functional recovery experiments showed the autophagy level and cell viabilities of KD+H-LV group was significantly increased when compared with the KD+H-LV-NC group,and tend to NC+H-LV-NC group.These suggest that up-regulating of HMGB1 could eliminate the chemotherapy increase-sensitivity induced by knocking down Med19,and we suspect that Med19 may play a role in mediating autophagy and chemoresistance through HMGB1.Conclusions1.MCF-7/ADM with multidrug resistance and NACT breast cancer tissue exhibited a high expression level of Med19 and a high level of autophagy.2.Med19 inhibition could significantly suppresses the levels of basal autophagy and ADM-induced autophagy in breast cancer cells.3.Med19 inhibition could significantly enhances the sensitivity to chemotherapy by blocking autophagy in MCF-7 and MCF-7/ADM cells.4.Med19 might play a role in regulating autophagy and chemoresistance through HMGB1 signaling in breast cancer.