Effect Of BRD4 Protein Inhibitor JQ1 On The Development And Metastasis Of Tongue Squamous Cell Carcinoma

Background and Objectives:Oral and pharyngeal malignant tumors accounted for the sixth highest incidence of malignant tumors in the whole body.Oral malignant tumors account for about 5%of all malignant tumors in the whole body.Tongue squamous cell carcinoma(TSCC)is the most common malignant tumour of the ora]cavity.lt often netastases in the early stage of tumor.With postoperative recurrence feature,the treatment and prognosis of patients is seriously affected.Bromodomain-containing Protein 4(BRD4)is one menber of the bromodomain and extraterminal domain(BET)gene family.It can recognize acetyllysine residues in histone H3 and H4 through two tandem bromine domain modules,involved in regulating multiple cell process such as DNA replication,repair,transcription,chromatin remodeling and cell cycle.JQ1,a a small lolecule inhibitor of BRD4,which can competently bind to the bromine domain in BRD4 to separating the acetylated lysine in BRD4 protein and histone,thereby interfering with the biological function of BRD.At present,JQ1 has been found to inhibit the activity of BRD4 effectively in a variety of cancer cells,thus producing a more definite anti-cancer effect.c-Myc,the member of myc gene fanily,is one of the earliest proto-oncogenes.which play the role in transcriptional regulation function by binding to promoter regulatory regions of some coding and non-coding RNA.The expression of c-Myc is high in many malignant tumors,which is considered to be closely related to the occurrence,development and prognosis of tumors.Epithelial-mesenchymal transformation(EMT)refers to the biological process of epithelial cells transforming into mesenchymal phenotypic cells under specific conditions,which play significant role for the ability of tumor cells to migrate and invade.Numerous studies have shown that Twist gene is a major factor in inducing epithelial-mesenchymal transformation(EMT)in cancer cells.Twist protein structure is highly conservative and is a member of the basic helix-loop-helix(bHLH)transcription factor gene family.With the development of the relationship between Twist-related signaling pathways and malignant tumors,its role in the diagnosis,treatment and prognosis evaluation of tumors has been as highly regarded.In this research.the expressions of BRD4?c-Myc and Twist in tongue squamous cell carcinoma were analysed,and the relationship and relevance among BRD4,c-Myc and Twist the occurrence,and development and prognosis of tongue squamous cell carcinoma were conducted.Different concentrations of JQ1 were used to treat tongue squamous cell carcinoma cell lines,and the effects of JQ1 treatment on the proliferation,apoptosis and invasion of tongue squamous cell carcinoma were·detected.To detect the effect of JQ1 on the expression of BRD4,c-Myc and Twist,and to explore the molecular biological mechanism of JQ1 in the treatment of tongue squamous cell carcinoma,to deeply understand the biological mechanism of JQ1 on tongue squamous cell carcinoma.To verify the feasibility of BRD4 inhibitor as a treatment for tongue squamous cell carcinoma,and to provide new ideas for the development of treatment methods for tongue squamous cell carcinoma.Materials and Methods1.Expression of BRD4 in tongue squamous cell carcinomaThe expression of BRD4 in tongue squamous cell carcinoma tissues was detected by immunohistochemistry.The relationship between BRD4 expression and sex,age,clinical stage,metastasis(lymph node metastasis and distant metastasis)and pathological differentiation was analyzed.The expression of BRD4 in tongue squamous cell carcinoma cell line Cal27 was detected by immunocytochemistry and fluorescent immunocytochemistry.2.Effect of BRD4 inhibitor JQ1 on invasion and metastasis of tongue squamous cell carcinomaCCK8 kit was used to detect the effect of JQI with different concentration on the proliferation of Ca27 and HN6 cell line;Transwell experiment was used to detect the change of invasive ability of Ca27 cell line with different concentration of JQI;flow cytometry was used to detect cell cycle and apoptosis,and Western blotting was used to quantitative analysis of apoptosis-related protein caspase-3.3.Molecular Mechanisms of BRD4 inhibitor JQI affecting the development and metastasis of Tongue Squamous Cell CarcinomaTestl BRD4 inhibitor JQI affects the development and metastasis of tongue squamous cell carcinoma by affecting oncogene c-MycThe expression of c-Myc in 122 specimens of tongue squamous cell carcinoma was detected by immunohistochemistry.The relationship among c-Myc expression and sex,age,clinical stage,metastasis and pathological differentiation of tongue squamous cell carcinoma was analyzed.The correlation among c-Myc expression and BRD4 was also analyzed.The expression of c-Myc in tongue squamous cell carcinoma cell line Cal27 was detected by immunocytochemistry and immunofluorescence cytochemistry.Cal27 cells were transfected with BRD4 over-expression vector and shRNA to construct over-expression BRD4 or shRNA interference expression cell lines.The effects of different concentrations of BRD4 inhibitor JQ1 on c-Myc,Twist gene expression in tongue squalous cell carcinoma cell line Cal27 were detected by qRT-PCR,and the changes of c-Myc and Twist gene expression in shRNA Cal27 cell line or over-expression were detected.The effects of different concentrations of BRD4 inhibitor JQI on tongue squamous cell carcinoma cell line Cal27 were detected by Western blotting.The effect of c-Myc protein expression was detected,and the changes of c-Myc protein expression in shRNA Cal27 cell line were detected.Test2 BRD4 inhibitor JQI affects the development and metastasis of tongue squamous cell carcinoma by influencing Twist to promote epithelial mesenchymal transformationThe expression of Twist in 122 specimens of tongue squamous cell carcinoma was detected by immunohistochemistry.The relationship among Twist expression and sex,age,clinical stage,metastasis and pathological differentiation of tongue squamous cell carcinoma was analyzed.The correlation among Twist expression and BRD4 was also analyzed.The expression of Twist in tongue squamous cell carcinoma cell line Cal27 was detected by immunocytochemistry and immunofluorescence cytochemistry.Cal27 cells were transfected with BRD4 over-expression vector and shRNA to construct over-expression BRD4 or shRNA interference expression cell lines.The effects of different concentrations of BRD4 inhibitor JQI on Twist gene expression in tongue squamous cell carcinoma cell line Cal27 were detected by qRT-PCR,and the changes of Twist gene expression in shRNA Cal27 cell line or over-expression were detected.The effects of different concentrations of BRD4 inhibitor JQ1 on tongue squamous cell carcinoma cell line Cal27 were detected by Western blotting.The effect of Twist protein expression was detected,and the changes of Twist protein expression in shRNA Cal27 cell line were detected.Results[Expression of BRD4 in tongue squamous cell carcinomaImmunohistochemical results showed that BRD4 was positive in 66 of 122 tongue squamous cell carcinoma cell lines.The expression of BRD4 was riot related to sex and age,but related to clinical stage,metastasis and pathological differentiation of tongue squamous cell carcinoma.Immunocytochemistry showed that BRD4 was positive in tongue squamous cell carcinoma cell line Cal27.2.Effect of BRD4 inhibitor JQ1 on invasion and metastasis of tongue squamous cell carcinoma(1)CCK8 was used to detected cell proliferation.Compared with the control group,after adding different concentrations of JQ1 to Cal27 and HN6 cell line for l day,2 days,3 days,4 days and 5 days.JQ1 of different concentrations had obvious inhibitory effect on tumor cells,and the cell proliferation effect of experimental group increased with the increase of drug concentration and the prolongation of action time.It was significantly increased,showing a significant time-dose dependence.(2)After 48 hours of incubation with JQ1 of different concentration in Cal27 and HN6 cell line,Transwell experiment was carried out.The results showed that JQ1 of different concentration had obvious inhibitory effect on the invasiveness of tongue squamous cell carcinoma compared with the control group,and the effect increased with the increase of drug concentration.(3)The cell cycle of tongue squamous cell carcinoma cell line Cal27 and HN6 was detected by flow cytometry after adding JQ1 of different concentration for 24 hours.The results showed that the percentage of cells in Gl phase increased significantly,while that in S phase decreased significantly.(4)JQ1(0.5 ?M)was added to Cal27 and HN6 cell line of tongue squamous cell carcinoma for 48 hours.Flow cytometry was used to detect apoptosis.The results showed that the percentage of early apoptosis in experimental group was significantly higher than that in control group.(5)The expression of Caspase-3 in tongue squamous cell carcinoma cell line Cal27 and HN6 were significantly increased by adding JQ1 of different concentration for 24 hours,and the expression of Caspase-3 was gradually increased with the increase of drug concentration.3.The Molecular mechanisms of BRD4 affecting the development and metastasis of tongue squamous cell carcinomaTestl BRD4 affects the development and metastasis of tongue squamous cell carcinoma by oncogene c-Myc(1)Immunohistochemical results showed that 87 of 122 tongue squamous cell carcinomas were positive tor c-Myc,which was not related to sex and age,but related to clinical stage,distant lymph node metastasis and pathological differentiation.Statistical analysis showed that c-Myc expression was positively correlated with BRD4 expression.(2)Compared with the control group(DMSO).co-culture with JQ1 at different concentrations for 24 and 48 hours,the mRNA and protein expression of c-Myc decreased significantly,and the effect increased significantly with the increase of the concentration of JQ1.(3)The gene and protein expression of c-Myc increased significantly in BRD4 Over-expression Cal27 cells.(4)The gene and protein expression of c-Myc decreased significantly in BRD4 shRNA Cal27 cells.Test2 BRD4 affects the development and metastasis of tongue squamous cell carcinoma by influencing Twist to promote epithelial mesenchymal transformation(1)Immunohistochenical results showed that 78 of 122 tongue squamous cell carcinomas were positive for Twist,which was not related to sex and age,but related to clinical stage,distant lymph node metastasis and pathological differentiation.Statistical analysis showed that Twist expression was positively correlated with BRD4 expression.(2)Compared with the control group(DMSO),co-culture with JQ1 at different concentrations for 24 and 48 hours,the mRNA and protein expression of Twist decreased significantly,and the effect increased significantly with the increase of the concentration of JQ1.(3)The gene and protein expression of Twist increased significantly in BRD4 Over-expression Cal27 cells.(4)The gene and protein expression of Twist decreased significantly in BRD4 shRNA Cal27 cells.(5)After JQ1 treatment,the expression of interstitial marker gene Fibronectin and MMP9 decreased,while the expression of epithelial marker gene E-cadherin increased in Cal27 cells.(6)The expression of interstitial marker gene Fibronectin and MMP9 increased,while the expression of epithelial marker gene E-cadherin decreased in BRD4 Over-expression Cal27 cells.(7)The expression of interstitial marker gene Fibronectin and MMP9 decreased,while the expression of epithelial marker gene E-cadherin increased in BRD4 shRNA Ca127 cells.Conclusions 1.BRD4 is highly expressed in tongue squamous cell carcinoma,which may be a target for treatment of tongue squamous cell carcinoma.2.JQ1,an inhibitor of BRD4,can effectively inhibit the expression of BRD4 in tongue squamous cell carcinoma cells,inhibit the proliferation and invasion of tongue squamous cell carcinoma cells,promote their apoptosis,and affect their cell cycle.3.JQ1,an inhibitor of BRD4,can effectively inhibit the expression of c-Myc in tongue squamous cell carcinoma cells,suggesting that the effect of JQI on the proliferation,invasion,apoptosis and cell cycle of tongue squamous cell carcinoma cells may be mediated by the effect of BRD4 on c-Myc.4.JQ1,an inhibitor of BRD4,can effectively inhibit the expression of Twist in tongue squamous cell carcinoma cells.Overexpression of BRD4 increased the expression of interstitial marker genes Fibronectin and MMP9,and decreased the expression of epithelial marker gene E-cadherin in Cal27 cells.JQ1 treatment and BRD4 inhibition decreased the expression of interstitial marker genes Fibronectin and MMP9,and increased the expression of epithelial marker gene E-cadherin in Cal27 cells.These results suggested that BRD4 may play an important role in the invasion of tongue squamous cell carcinoma cells by influencing the regulation of EMT by Twist.