YY1 Activates EMI2 And Promotes The Progression Of Cholangiocarcinoma Through The PI3K/AKT Signaling Axis

Objective To investigate the expression and biological behavior of early mitotic inhibitor 2（EMI2）in cholangiocarcinoma（CCA）.To explore the mechanism of YY1 regulating EMI2 and promoting the progression of CCA through PI3K/AKT axis.Methods The expression of EMI2 in cancer and adjacent tissues of patients with CCA was verified by public database.The tissues of patients with CCA were collected,and the expression of EMI2 in cancer and adjacent tissues was verified by immunohistochemistry and western blotting.Kaplan-meier curve was used to detect the effect of high expression of EMI2 on patients with CCA.Subsequently,EMI2 expression was verified in bile duct cancer cell lines and normal bile duct cells.QBC939 and RBE with the highest expression were selected as follow-up studies to construct EMI2-silencing lentiviral vectors,and q RT-PCR and western blot were used to detect the silencing efficiency.Then the effects of proliferation,metastasis,cell cycle and apoptosis in both groups were detected.EMI2 transcription factors were predicted using JASPAR and PROMO databases.Among the predicted transcription factors,Yin-Yang transcription factor 1（YY1）was rarely reported in CCA,which was subsequently verified by luciferase reporter gene assay,followed by TCGA to verify the expression level of YY1.After silencing and overexpressing YY1,the expression level was detected by western blot,and proliferation,metastasis,cell cycle and apoptosis were detected in each group.The effect of EMI2 silencing on subcutaneous tumors in nude mice was verified in vivo,and ki67 expression was detected by immunohistochemistry.Finally,in order to verify the specific mechanism of EMI2,the expression of star molecules in 19 pathways after EMI2 silencing was detected by PCR and verified by western blotting.Results EMI2 is highly expressed in CCA.EMI2 silencing can inhibit the proliferation,invasion and migration of bile duct cancer cells.After EMI2 silencing,bile duct cancer cell cycle arrest increased in G1 phase,while after EMI2 silencing,bile duct cancer cell early apoptosis increased.The response results of overexpression of EMI2 confirm the previous experimental conclusion.Subsequently,luciferase reporter gene detection results showed that YY1 was bound to the EMI2 promoter region,and after YY1 was silenced,the expression of EMI2 was decreased,and the proliferation and invasion ability of bile duct cancer cells were inhibited after YY1 was silenced.YY1 and EMI2 groups had the best inhibition efficiency.Overexpression of YY1 and EMI2 promoted the progression of bile duct cancer cells.In vivo experiment results showed that in vivo imaging fluorescence intensity was decreased in EMI2 silencing group,tumor size was smaller than that in the control group,and ki67 expression was lower in the silencing group.In addition,EMI2 silencing reduced the contents of p-AKT,p-PI3 K and MDM2,key proteins of PI3K/AKT signaling pathway.Conclusion EMI2 gene is highly expressed in the tumor species of patients with CCA,and high expression of EMI2 is associated with poor prognosis of CCA.Subsequent cell experiments verified that EMI2 is overexpressed in CCA cell lines,and promotes proliferation,metastasis and apoptosis escape of CCA cells.Meanwhile,YY1,as a transcription factor regulating EMI2,can promote the expression of EMI2 and promote the progression of CCA through PI3K/AKT signaling pathway.