BRD4通过靶向GSTP1调控食管癌TE-1细胞顺铂敏感性

BRD4 regulates cisplatin sensitivity of esophageal cancer TE-1 cells by targeting GSTP1

目的揭示食管癌组织中溴结构域蛋白4(BRD4)表达水平,并分析其与谷胱甘肽硫基转移酶P1(GSTP1)分子的联系,探究BRD4在人食管癌细胞(TE-1细胞)顺铂敏感性调控中的作用。方法基于癌症基因组图谱(TCGA)数据利用R语言包解析BRD4在食管癌及癌旁正常组织中表达情况;基因表法普交互分析(GEPIA2)在线评估食管癌肿瘤组织中BRD4与化疗药物解毒酶GSTP1表达的联系;通过小干扰RNA(siRNA)和靶向抑制剂(+)-JQ1(25、50和100 nM)分别处理食管癌TE-1细胞,通过蛋白免疫印迹法(Western blot)验证食管癌TE-1细胞中BRD4及GSTP1蛋白表达变化;TE-1细胞(+)-JQ1预处理(50 nM,24 h)前后及结合顺铂(DDP,5μg/ml,24 h),运用Western blot实验检测凋亡相关蛋白Bcl-2关联X蛋白(Bax)、淋巴细胞瘤-2(Bcl-2)、半胱氨酸天冬氨酸蛋白酶3(Caspase-3)及半胱氨酸天冬氨酸蛋白酶3切割体(cleaved-caspase-3)表达变化;采用CCK-8法检测TE-1细胞经(+)-JQ1(50 nM,24 h)抑制BRD4作用后DDP敏感性的改变。结果TCGA数据解析后结果显示,食管癌肿瘤组织中BRD4的mRNA总体表达水平相较于癌旁正常组织有明显升高[(5.61±0.57)VS(4.80±0.59)],差异有统计意义(P<0.001)。进一步通过对比配对肿瘤和癌旁正常组织样本发现,食管癌患者食管癌组织中BRD4表达水平(5.57±0.31)普遍显著高于其癌旁正常组织的(4.77±0.44),差异有统计学意义(P<0.01)。GEPIA2数据库发现,在食管癌化疗药物耐药形成过程中关键解毒结合酶GSTP1与肿瘤组织中BRD4表达呈正相关(r=0.26,P=0.00029<0.001)。结合靶向siRNA处理Western blot结果显示,siRNA-BRD4处理后食管癌TE-1细胞中BRD4和GSTP1的表达水平较NC组均有明显升高,而siRNA-GSTP1后TE-1细胞中GSTP1表达下调未对BRD4蛋白产生明显影响。进一步采取BRD4靶向抑制剂的梯度浓度处理证实,伴随(+)-JQ1浓度升高TE-1细胞中GSTP1蛋白的表达水平较阴性对照组出现明显下降趋势,而由于(+)-JQ1主要是通过结合于BRD4的溴结构域发挥作用,仅在最高浓度处理(100 nM)组食管癌细胞中BRD4出现表达抑制作用。低浓度的(+)-JQ1(50 nM)处理可引起食管癌TE-1细胞抗凋亡蛋白Bcl-2的表达下调以及凋亡相关蛋白Bax表达上升,但对caspase-3并无明显影响,而在与DDP联合应用时可见caspase-3的活化形式cleaved-caspase-3的相比DDP单独使用组细胞有显著表达上调。Western blot结果表明,(+)-JQ1预处理抑制BRD4的功能可以提高DDP对食管癌细胞的杀伤作用。CCK-8结果显示,食管癌TE-1细胞经抑制BRD4蛋白功能[(+)-JQ1,50 nM,24 h]预处理后可显著提高对化疗药物DDP的敏感性,DMSO对照组DDP的半数抑制浓度(IC50)值为(19.43±0.59)μg/ml,而(+)-JQ1预处理后降至(10.46±0.24)μg/ml,差异有统计学意义(P<0.05)。结论食管癌中高表达的BRD4可能通过GSTP1分子参与DDP敏感性调控。

Objective To investigate the expression level of bromodomain-containing protein 4(BRD4)in esophageal cancer tissues,analyze its association with glutathione-S-transferase P1(GSTP1),and explore the role of BRD4 in the regulation of cisplatin sensitivity in human esophageal cancer cells.Methods Based on The Cancer Genome Atlas(TCGA)data,R language package was used to analyze the expression of BRD4 in esophageal carcinoma and adjacent normal tissues.Gene Expression Profiling Interactive Analysis(GEPIA2)online evaluated the relationship between BRD4 and the expression of chemotherapeutic detoxifying enzyme GSTP1 in esophageal cancer tissue.Small interfering RNA(siRNA)and targeted inhibitor(+)-JQ1(25,50 and 100 nM)were used to treat esophageal cancer TE-1 cells.Western blot was used to verify the expression changes of BRD4 and GSTP1 in esophageal cancer TE-1 cells.After(+)-JQ1 pretreatment(50 nM,24 h)and binding cisplatin(DDP,5μg/ml,24 h),the expression of apoptosis-related proteins of Bel-2 associated X(BAX),B-cell lymphoma-2(Bcl-2),cysteinyl aspartate specific proteinase 3(Caspase-3),cleaved-cysteinyl aspartate specific proteinase 3(cleaved-caspase-3)were detected by Western blot assay.CCK-8 assay was used to detect the change of DDP sensitivity of TE-1 cells after(+)-JQ1(50 nM,24 h)inhibition of BRD4.Results The analysis of TCGA data showed that the overall expression level of BRD4 mRNA in esophageal cancer was significantly higher than that in adjacent normal tissues[(5.61±0.57)VS(4.80±0.59)],and the difference was statistically significant(P<0.001).Further,by comparing the samples of paired tumors and adjacent normal tissues,it was found that the expression level of BRD4 in esophageal cancer tissues of patients(5.57±0.31),which was generally significantly higher than that of(4.77±0.44)in adjacent normal tissues,and the difference was statistically significant(P<0.01).The GEPIA2 database found that the key detoxification binding enzyme GSTP1 was positively correlated with the expression of BRD4 in tumor tissues during the formation of chemotherapy drug resistance in esophageal cancer(r=0.26,P=0.00029<0.001).Combined with the targeting siRNA treatment Western blot results showed that the expression levels of BRD4 and GSTP1 in esophageal cancer TE-1 cells after sIRNA-BRD4 treatment were significantly higher than those in NC group.However,the down-regulation of GSTP1 expression in TE-1 cells after siRNA-GSTP1 had no significant effect on BRD4 protein.Further treatment with graded concentrations of BRD4-targeting inhibitors confirmed that the expression level of GSTP1 protein in TE-1 cells showed a significant decrease in comparison with that in the negative control group along with the increase in(+)-JQ1 concentration,since(+)-JQ1 acted mainly by binding to the bromodomain of BRD4,the expression inhibition of BRD4 was only observed in the esophageal cancer cells of the highest concentration treatment(100 nM)group.Low concentration of(+)-JQ1(50 nM)treatment can cause the down-regulation of anti-apoptotic protein Bcl-2 and the increase of apoptosis-related protein Bax expression in TE-1 cells,but has no significant effect on caspase-3,whereas the activated form of caspase-3,cleaved-caspase-3,was significantly expressed in cells of the TE-1 group compared to those of the DDP-alone group.Western blot results indicated that inhibition of BRD4 function by(+)-JQ1 pretreatment could enhance the killing effect of DDP on esophageal cancer cells.The results of CCK-8 showed that the sensitivity of TE-1 cells to chemotherapy drug DDP could be significantly improved after pretreatment with inhibition of BRD4 protein function[(+)-JQ1,50 nM,24 h].The median inhibitory concentration(IC50)of DDP in DMSO control group was(19.43±0.59)μg/ml.After pretreatment with(+)-JQ1,it decreased to(10.46±0.24)μg/ml,and the difference was statistically significant(P<0.05).Conclusion The high expression of BRD4 in esophageal cancer may be involved in the regulation of DDP sensitivity through GSTP1 molecule.