Molecular Mechanism Of DACT2 Suppresses Growth Of Glioma Throuth Activation Of YAP

Objective: To explore the expression of DACT2(Dishevelled-binding antagonist of beta-catenin 2)in glioma tissues and non-tumor brain tissues and the impact of DACT2 expression on the prognosis in glioma patients.The effects of DACT2 on the proliferation and apoptosis of glioma and the underlying molecular mechanisms were investigated.Methods: 1.The data of the expression of DACT2 in different grade glioma from TCGA database was used to analyzed the the relationship between DACT2 expression and various clinic-pathological features of glioma and the impact of DACT2 expression on the prognosis in glioma patients.IHC was examined the expression of DACT2 in 80 glioma tissues and 10 non-tumor brain tissues that were collected from department of pathology,Chongqing Medical University from 2008 to 2012.2.Real-time PCR and WB were used to test the expression levels of DACT2 in eight glioma tissues and the paired adjacent tissues which were collected from department of neurosurgery,the second affiliated hospital of Chongqing Medical University from 2015 to 2016.3.The expression of DACT2 in glioma cell lines was assessed by Real-time PCR and WB.Over-DACT2-U251 and U87 cell lines,Sh-DACT2-SHG44 and A172 cell lines were established by lentivirus transfection.Transfection efficiency was evaluated by Real-time PCR and WB.4.CCK8 and BrdU were used to investigate the proliferation of glioma cells.FCM was used to analyze cell cycle of glioma cells.FCM and TUNEL were used to measure glioma cell apoptosis.5.The activation of YAP and the expression of downstream target genes(PCNA,CyclinD1 and Bax)were evaluated by WB.IF was used to investigate the effect of DACT2 on the nuclear transfer of YAP.6.U251 and U87 glioma cells were co-transfected with Over-DACT2 and Over-YAP,WB was used to evaluate transfection efficiency and assess the expression of PCNA,CyclinD1 and Bax.CCK8 and BrdU were used to investigate the proliferation of glioma cells.FCM was used to analyze cell cycle of glioma cells.FCM and TUNEL were used to measure glioma cell apoptosis.7.The subcutaneous tumor formation in nude mice was established,and the volume and weight of the tumor were analyzed.The expression of YAP,PCNA,CyclinD1 and Bax were tested by IHC.Result: 1.The expression of DACT2 was significantly downexpressed in glioma tissues than in non-tumor brain tissues,and it negatively correlated with WHO grade.Univariate and multivariate analyses showed that were independent prognostic parameters.2.The expression of DACT2 was significantly downexpressed in glioma tissues compared with that in the paired adjacent tissues.3.The expression of DACT2 m RNA and protein in Over-DACT2-U251 and U87 were increased than that in Control cells,and they were decreased in Sh-DACT2-SHG44 and A172 than that in Sh-Control cells.4.Overexpression of DACT2 significantly suppressed the proliferation,induced cell cycle arrest and promoted apoptosis.Whereas knockdown of DACT2 increased the proliferation,promoted cell cycle and reduced the rate of apoptosis in glioma cells.5.DACT2 reduced the expression of YAP,PCNA and CyclinD1,increased Bax and p-YAP expression,and suppressed the nuclear transfer of YAP.6.YAP overexpression could partially reverse the DACT2-induced biological effect,including cell growth inhibition and apoptosis induction.7.DACT2 has a negative effect on the growth of human glioma cells in vivo and IHC revealed that DACT2 upregulated the expression of Bax and downexpressed the expression of YAP,PCNA and CyclinD1 and in glioma tissues.Conclusion: 1.DACT2 was downexpressed in glioma tissues,and its expression was related with WHO grade and prognosis of glioma patients.2.DACT2 suppressed proliferation,induced cell cycle arrest and promoted apoptosis of glioma cells.3.DACT2 increased the expression of p-YAP,suppressed the nuclear transfer of YAP,promoted degradation of YAP,and then inhibited the growth and apoptosis of glioma.In conclusion,our findings suggest that DACT2 is a novel tumor suppressor gene and may be a potential therapeutic target in gliomas.