JQ1 And PI3K Inhibition Synergistically Reduce Salivary Adenoid Cystic Carcinoma Malignancy By Targeting The C-myc And EGFR Signaling Pathways

Salivary adenoid cystic carcinoma(SACC)is one of the common malignant tumors of salivary glands,which is found in the small salivary glands and parotid glands,accounting for about 1%of the malignant tumors in the head and neck[1].At present,the treatment of this tumor is mainly based on surgery.However,because of its high malignancy,strong invasiveness and high metastasis rate,the prognosis of the patients is poor.In the past,many target genes including Ki-67,p53 and Bcl-2 have been studied extensively,but the results are very disappointed.The therapeutic effect of this disease is also not satisfactory.In recent years,with the rapid development of molecular biology technology,the research of new molecular markers and molecular targets is in the ascendant.Therefore,in order to improve the curative effect of adenoid cystic carcinoma,it is still important to continue to explore new molecular markers and gene targets of the disease.Chapter 1 high expression of BRD4 in salivary adenoid cystic carcinomaObjective:To invest e expression of BRD4 in SACC and corresponding para-cancerous tissues,analyze the role of it in SACC.Methods:The expression of BRD4 in 30 cases of SACC and corresponding para-cancerous tissues was detected by immunohistochemical technique(SP method).Results:Immunohistochemical results showed that the expression rate of BRD4 in SACC tissues was up to 80%,but no expression was found in para-cancerous tissues.Conclusion:The high expression of BRD4 in SACC may play an important role in the occurrence and development of tumor.The second chapter adenoid cystic carcinoma of salivary gland is insensitive to JQ1 treatmentObjective:What is the effect of JQ1 in the treatment of SACC?The sensitivity of SACC to the treatment of JQ1 will be detected,and a suitable concentration of JQ1 will be found to prepare for the follow-up experiment.Methods:The Acc-83 cells were incubated for 24 hours after were given 0.2?M,1?M,5?M dose of JQ1 for 4 days,the proliferation of tumor cells was detected by CCK8.The apoptosis level of tumor cells was detected by flow cytometry.Western Blot was used to evaluate the protein expression level of the apoptotic signaling pathway cleaved caspase-3.Results:JQ1 can inhibit Acc-83 cell activity,but when the JQ1 concentration reached 5?M,the activity of Acc-83 cells lower to the maximum,only 30%of the control group.Conclusion:SACC is insensitive to JQ1 treatmentThe third chapter JQ1 inhibits the expression of BRD4 and c-Myc and activates the EGFR signaling pathwayObjective:Is it possible to explore the role of the c-Myc and EGFR signaling pathways in the treatment of SACC by JQ1,What is the relationship.Methods:The use of Western blot were detected BRD4,c-Myc,pEGFR and EGFR related protein expression level of JQ1 in treatment 0.2?M,1?M,5?M dose of JQ1 for 4 days,the mRNA expression level of c-Myc and EGFR was detected by qRT-PCR.Results:Western blot results showed that with the increasing of JQ1 concentration,the expression of BRD4 and c-Myc gradually decreased,while the expression of EGFR and pEGFR protein increased;qRT-PCR results showed:with increasing concentrations of JQ1,c-Myc expression gradually decreased,while the expression level of EGFR increased,which is consistent with the results of Western blotConclusion:In SACC,JQ1 can play a role in anti-cancer through the c-Myc signaling pathway and activates the EGFR signaling pathwayThe fourth chapter Combine the treatment of JQ1 and BKM120 to improve the efficacy of adenoid cystic carcinoma of salivary glandObjective:What is the effect of combined use of JQ1 and PI3K inhibitor BKM120 for the treatment of SACC,And explore its potential mechanisms.Methods:The experiment was divided into control group,JQ1(5?M)group,BKM120(1?M)group and combined treatment group,using CCK8 and flow cytometry to detect the proliferation and apoptosis of tumor cells;Western blot was detected the expression protein level of BRD4,c-Myc,EGFR and pEGFR,EGFR and c-Myc measurement the mRNA with the qRT-PCR test.Results:The results of CCK8 test showed that compared with JQ1 and BKM120 alone,the combined treatment group significantly reduced the activity of ACC-83 cells(P<0.05).Western blot results showed that the protein expression of BRD4 and c-Myc in the combined treatment group decreased significantly(P<0.05),the expression of EGFR in the combined treatment group was significantly lower than that in the JQ1 treatment group(P<0.05),but slightly higher than that in the BKM120 treatment group.Conclusion:JQ1 and BKM120 can improve the therapeutic effect of SACC by simultaneously inhibiting the EGFR and c-Myc signaling pathways.The fifth chapter:the expression of EGFR affects the sensitivity of adenoid cystic carcinoma of salivary glands to JQ1Objective:To explore the effect of EGFR expression on the therapeutic effect of JQ1 in SACC.Methods:We used siRNA to silence EGFR first,and the experiment was divided into siEGFR group,JQ1 5 pM group,siEGFR+JQ1 5?M group and control group.After 2 days of successful transfection of ACC-83 cells,CCK8 detected the proliferation of tumor cells in 1-4 days,and the expression of cleaved caspase-3 was used to examine the apoptosis level of tumor cells in each group.QRT-PCR and Western-blot were used to detect the expression level of BRD4 and c-Myc.Results:The results of CCK8 detection showed that the cell proliferation in JQl+siEGFR group was significantly lower than that of other groups(P<0.05),and the expression level of cleaved caspased-3 increased significantly.The Western blot results showed that the expression of BRD4 and c-Myc in the JQl+siEGFR group was significantly reduced,and the qRT-PCR results confirmed the western blot results.Conclusion:The expression of EGFR can significantly affect the sensitivity of SACC to JQ1.