The Mechanism Of YAP On The Proliferation And Apoptosis Of Oral Squamous Cell Carcinoma Cells Under Metformin Treatment

Background and ObjectiveMetformin is a biguanide small molecule compound which can be used to treat type 2 diabetes patients,Recently,researchers have found that metformin could reduce the risk of oral squamous cell carcinoma(OSCC)in patients with mellitus.It was confirmed that metformin has an inhibitory effect on the OSCC in vitro and in vivo.Besides,the combined use of metformin and anti-oral cancer chemotherapy drugs could increase the toxic effect of chemotherapy drugs on OSCC cells.Thus,metformin may become one of promising anticancer drugs in OSCC treatment.Yes-associated protein(YAP),with gene position of 11 q22,is one of the major transcriptional co-activators of the Hippo signaling pathway which could regulate the proliferation,cycle,apoptosis,invasion and migration of OSCC cells.It has been confirmed that YAP activation could promote the OSCC cells proliferation by accelerating the cell cycle and inhibiting cell apoptosis.YAP activation could promote the OSCC cells migration by increasing the epithelial-mesenchymal transition of OSCC cells.YAP activation also promotes OSCC cells ability of chemoresistance.Besides of those,YAP overexpression is positively correlated with the malignant change of OSCC.Our previous study also has confirmed that YAP translation into nucleus could inhibit cell apoptosis and promote cell proliferation by accelerating c-myc and bcl-2 gene transcription.Meanwhile,the OSCC cells proliferation and invasion could be inhibited by decreasing YAP protein expression by verteporfin.Therefore,YAP may be used as a potential carcinogen target for OSCC.The purpose of this study was to investigate whether metformin could inhibit the proliferation of OSCC cells or promote the apoptosis of OSCC cells by inhibiting the protein expression of YAP.This study also explored the specific molecular mechanism of YAP in the anti-OSCC action of metformin.Thus,this study would provide a theoretical basis for the molecular mechanism of metformin as an anti-OSCC drug and give a choice for the screening of OSCC anticancer drugs.Methods1 CAL27 and SCC25 cells were treated with range of four different concentrations with Ommol/L?5mmol/L?15mmol/L and 30mmol/L of metformin.The CAL27 and SCC25 cells viability were detected after 24h,48h,and 72h drug treatment by the CCK-8 cell viability detection kit.24h later,CAL27 and SCC25 cell cycle and apoptosis changes were detected by flow cytometry.2 CAL27 and SCC25 cells were treated with range of four different concentrations with Ommol/L?5mmol/L,15mmol/L and 30mmol/L of metformin.24h later,CAL27 and SCC25 cells total protein were extracted.The protein expression of MST1,p-MSTl,LATS1,p-LATS1,MOB1,SAV1,YAP,p-YAP,mTOR,p-mTOR,CDK4,CDK6,p21,BCL-2 and BAX in CAL27 and SCC25 cells were detected by Western blotting.3 Overexpression YAP CAL27 and SCC25 cells line were constructed by overexpressing YAP gene lentiviral vector.The CAL27 and SCC25 cells total protein and RNA were extracted after transfection for 72h.The transfection efficiency was assessed by Western blotting and Real-time fluorescent quantitative PCR.4 Cells treated without metformin or overexpression YAP gene lentiviral vector were served as the control group;Cells treated with metformin were served as the experimental group;Cells treated with overexpression YAP gene lentiviral vector were served as experimental group;Cells treated with metformin and overexpression YAP gene lentiviral vector were served as experimental group.The cells viability of each groups was detected after 24h using the CCK-8 cell viability detection kit.The cell cycle and cell apoptosis changes in each group were detected by flow cytometry.The mTOR,p-mTOR,CDK4,CDK6,BCL-2 and BAX protein expression of each groups were detected by Western blotting.5 Cells treated without metformin or verteporfin were served as the control group;Cells treated with metformin were served as the experimental group;Cells treated with verteporfin were served as the experimental group;Cells treated with metformin and verteporfin were served as the experimental group.24 later,the cell cycle and cell apoptosis changes in each group were detected by flow cytometry.The mTOR,p-mTOR,CDK4,CDK6,BCL-2 and BAX protein expression of each groups were detected by Western blotting.Results1 CAL27 and SCC25 cells proliferative activity decreased as the drugs concentration progressively increased.But the CAL27 and SCC25 cells proportion of G1 phase and apoptosis were down-regulated with the increasing of drugs concentration.Data differences were statistically significant.Meanwhile,the protein expression of CDK4,CDK6 and BCL-2 reduced while the protein expression of p21 and BAX increased in CAL27 and SCC25 cells.2 The protein expression of p-MST,p-LATS,MOB1 and SAV1 which all of them belong to Hippo signal pathway were increased with the progressive increase of metformin concentration.The phosphorylated YAP protein expression was up-regulated and unphosphorylated YAP protein was down-regulated at the same time.The protein expression of mTOR,p-mTOR and C-MYC reduced with the progressive increased concentration of metformin.3 The cell proliferative activity in group of CAL27 and SCC25 cells treated with metformin and overexpression YAP gene lentiviral vector were higher than that in the group of cells only treated with metformin.However,the rate of G1 cell cycle phase and apoptosis in the group of CAL27 and SCC25 cells treated with metformin and overexpression YAP gene lentiviral vector were lower than that in the group of cells only treated with metformin.Data differences were statistically significant.The protein expression of CDK4,CDK6 and BCL-2 in the group of CAL27 and SCC25 cells treated with metformin and overexpression YAP gene lentiviral vector were higher than that in the group of cells only treated with metformin.4 The rate of G1 cell cycle phase,early and lately apoptosis in the group of CAL27 and SCC25 cells treated with metformin and verteporfin were lower than that of cells only treated with metformin.Data differences were statistically significant.The protein expression of CDK4,CDK6 and BCL-2 in the group of CAL27 and SCC25 cells treated with metformin and verteporfin were less than that in the group of cells only treated with metformin.5 The metformin inhibitory effect on the protein expression of mTOR,p-mTOR and C-MYC could be weakened by YAP overexpression.By contrary the metformin inhibitory effect on the protein expression of mTOR,p-mTOR and C-MYC could be enhanced by YAP inhibition.ConclusionsIt could be confirmed from this study that metformin could block cell cycle at G1 phase,promote cell apoptosis,and then reduce cell proliferative activity by inhibiting YAP protein in OSCC.On the other hand,YAP could regulate metformin's inhibitory effect on mTOR,p-mTOR and C-MYC in OSCC.