Inhibition Of YAP On Apoptosis And Migration In Different Subtypes Of Breast Cancer Cells

ObjectiveBreast cancer is one of the most common malignancies among women worldwide,which is divided into four subtypes of Luminal A,Luminal B,HER-2 overexpression and Basal-like according to the expression levels of histiocytic receptor,progesterone receptor,human epidermal growth factor receptor 2 and Ki-67.The molecular mechanism of breast cancer has always been the focus of international research because its development process is regulated by multiple signaling systems.Hippo signaling plays an indispensable role in breast cancer,but the underlying mechanisms are not deep yet.The YAP gene is a terminal effector of the upstream kinase of Hippo signaling pathway with potential carcinogenicity.Activated YAP induces overexpression of growth factors,apoptosis inhibitors and low expression of tumor suppressor,leading to malignant transformation of cells.In this study,three subtypes of Luminal A,Luminal B and Basal-like cells were selected in vitro,and YAP was used to explore these three molecular subclasses at the molecular level by utilizing gene knockout technology or YAP gene inhibitors.The effect of breast cancer cell line on apoptosis and migration is to reveal the mechanism of YAP gene in the development of breast cancer,and provide a scientific theoretical basis for the treatment of breast cancer targeting YAP.Methods1.Human Luminal A breast cancer cell line MCF-7,Luminal B breast cancer cell line BT-474 and TNBC cell line BT-549 were cultured in vitro and total cellular protein were extracted out.Western blot was used to observe the expression of YAP in different molecular subtypes of breast cancer cell lines.2.Transfected MCF-7,BT-474,BT-549 cells with vector and filtered out to construct YAP silenced breast cancer cell lines.The knockdown efficiency of YAP gene was detected by Western blot and Real-time PCR.3.The following experiments were performed using the three different molecular subtypes of YAP gene silencing cell lines: CCK8 method was used to detect the effects in different concentration of YAP silencing on the proliferation of three different molecular subtypes of breast cancer cells;Western blot and Real-time PCR were used to detect the protein and m RNA expression of AXL,CYR61,CTGF;Western blot was used to observe the expression of p-YAP,TEAD and apoptosis-related proteins such as Survivin,BCL-2,BAX,Caspase-9,PARP,Cleaved Caspase-9 and Cleaved PARP protein expression;Transwell migration and scratch test to detect the effect of silencing YAP on the influence of MCF-7,BT474,BT-549 cells migration ability.4.Human Luminal A breast cancer cell line MCF-7,Luminal B breast cancer cell line BT-474 and TNBC cell line BT-549 were cultured in vitro.The following experiments were performed together with cells treated with various VP concentration: CCK8 method was used to detect the effects in different concentration of VP on the proliferation of three different molecular subtypes of breast cancer cells;TUNEL staining was used to detect the effects of VP on the apoptosis in three different molecular subtypes of breast cancer cells;Western blot and Real-time PCR was used to detect different concentration of YAP inhibitor VP on YAP downstream target genes AXL,CYR61,CTGF protein and m RNA expression levels;Detection of phosphorylated YAP,TEAD and apoptosis-related proteins such as Survivin,BCL-2,BAX,Caspase-9,PARP,Cleaved Caspase-9 and Cleaved PARP expression after treated different concentration of inhibitors in different subtype BC cell lines by Western blot;Transwell migration and scratch test were used to detect the effect of VP on the influence of MCF-7,BT474,BT-549 cells migration ability.Results1.Western blot results showed that the expression of YAP gene in Luminal B cell line BT-474 and BT-549 was significantly higher than Luminal A breast cancer MCF-7 cell line(P<0.001),and the expression of YAP on BT-474 was the highest among the three subtype cell lines(P<0.01).2.Compared with the control group,knockdown of YAP in three different molecular subtypes of breast cancer cell lines resulted in inhibition of proliferation(P<0.05).3.Silencing YAP gene can reduce the expression of p-YAP,TEAD and downstream target gene AXL,CYR61 in these three different breast cancer cells(P<0.05),and also decreased the expression of Survivin and BCL-2/BAX(P<0.05),and meanwhile,increased the expression of Cleaved Caspase-9 and Cleaved PARP protein(P<0.01).Real-time PCR results showed that the expression of AXL,CYR61 m RNA were decreased compared with the control group(P<0.05),the expression of CTGF protein and m RNA in BT-474、BT-549 cells were also decreased(P<0.05).The result also showed that Silencing the YAP gene inhibited the migration of MCF-7,BT-474 and BT-549 breast cancer cells(P<0.05).4.CCK-8 assay showed that the proliferation of MCF-7,BT-474 and BT-549 breast cancer cell were decreased by Verteporfin in a dose-dependent manner while compared with the control group.TUNEL experiments showed that VP can induce the apoptosis of MCF-7,BT-474 and BT-549 cell.5.Western blot analysis showed that VP can reduce the expression of YAP,p-YAP,TEAD and downstream target gene AXL,CYR61 in MCF-7,BT-474,BT-549 cells Hippo pathway(P<0.05).Western blot results also showed that VP can decrease the expression of Survivin and BCL-2/BAX(P<0.05),and meanwhile increase the expression of Cleaved Caspase-9 and Cleaved PARP protein compared with the control group(P<0.05).Real-time PCR results showed that the expression of AXL and CYR61 m RNA were decreased compared with the control group in three different subtypes of breast cancer cell lines after the treat by VP(P<0.01).The expression of CTGF m RNA in BT-474,BT-549 cells also decreased after the treat by VP(P<0.01).Transwell migration and scratch test showed that VP will influence migration ability of MCF-7,BT474,BT-549 cells(P<0.01).The result showed VP could inhibit the proliferation of breast cancer induced by YAP gene,and induce the apoptosis of MCF-7,BT-474,BT-549 cells by influencing the expression of apoptosis related protein.Conclusions1.YAP was expressed in Luminal A,Luminal B and TNBC cells.YAP was highest expressed in Luminal B BT-474 cells,suggesting that YAP as an oncogene promoted the occurrence and development of breast cancer.2.Silencing YAP gene could significantly down-regulate the expression of YAP downstream target AXL,CYR61 protein and m RNA in MCF-7,BT-474,BT-549 breast cancer cells by inhibiting the expression of YAP and TEAD,and also inhibited the proliferation and migration ability of different molecular subtypes breast cancer cells in vitro.3.After YAP gene silencing,the expression of apoptosis-related proteins Survivin,BCL-2/BAX were decreased,BAX,caspase-9 and PARP were increased,which could induce the apoptosis of MCF-7,BT-474 and BT-549 cells.4.Vitepofen,a YAP inhibitor,could down-regulate the expression of YAP downstream target AXL,CYR61 protein and m RNA in MCF-7,BT-474,BT-549 breast cancer cells by inhibiting the expression of YAP and TEAD,and also inhibited the proliferation and migration ability of different molecular subtypes breast cancer cells.Vitepofen could also induce cell apoptosis by regulating the expression of Survivin,BCL-2/BAX,Cleaved Caspase-9 and Cleaved PARP.