肝母细胞瘤表达BRD4的促肿瘤机制及靶向抑制研究

Tumor-promoting mechanism and targeted inhibition of BRD4 in hepatoblastoma

目的探讨溴结构域蛋白4 (bromodomain-containing protein 4, BRD4)促进肝母细胞瘤(hepatoblastoma, HB)机制及靶向抑制效应。方法收集未经化疗的45例HB穿刺活检或手术切除组织石蜡标本。将未经化疗的45例具有特征性肿瘤细胞的组织石蜡标本作为HB组;在45例石蜡标本中有30例组织边缘可见瘤旁肝组织肝小叶结构正常、可见小叶中央静脉和汇管区的组织, 该30例作为瘤旁对照组。采用免疫组织化学半定量检测BRD4的表达水平, 并对BRD4的表达水平与患儿临床病理特点的相关性进行统计学分析。运用实时定量聚合酶链反应(quantificational real-time polymerase chain reaction, qRT-PCR)、蛋白质印迹法和免疫荧光技术检测人HB细胞株HepG2对照组(未经处理的HepG2细胞)、空载组(转染Si-NC的HepG2细胞)以及Si-BRD4敲低组(转染Si-BRD4的HepG2细胞)的BRD4表达水平, 检测Yes相关蛋白1 (Yes-associated protein 1, YAP1)和c-Myc的mRNA和蛋白表达水平。运用CCK-8法和流式细胞术分别检测HepG2对照组和Si-BRD4敲低组的细胞活力和凋亡率。使用JQ1、长春新碱(vincristine, VCR)、JQ1联合VCR分别处理HepG2细胞48 h后, 通过CCK-8法、EDU-555细胞增殖试剂盒和TUNEL染色检测各组细胞的活力、增殖能力及凋亡率。将使用30 nmol/L JQ1干预的HepG2细胞作为JQ1组, 将使用70 μg/ml VCR干预的HepG2细胞作为VCR组, 将使用30 nmol/L JQ1和70 μg/ml VCR干预的HepG2细胞作为JQ1联合VCR组。结果①免疫组织化学检测结果显示BRD4在HB细胞核阳性率为97.8%(44/45), 瘤旁肝细胞核阳性率为0, 差异具有统计学意义(P<0.001 )。②根据美国儿童肿瘤协作组(Children’’s Oncology Group, COG)分期, Ⅰ~Ⅱ期低表达8例, 高表达4例, Ⅲ~Ⅳ期低表达3例, 高表达30例, BRD4表达水平与肿瘤的COG分期有关(P<0.001)。低表达患儿无转移发生, 高表达患儿转移11例, BRD4表达水平与肿瘤转移相关(P=0.042)。③qRT-PCR检测结果显示YAP1和c-Myc的mRNA表达水平在Si-BRD4敲低组较HepG2对照组和空载组明显下降;蛋白质印迹法及免疫荧光检测结果也显示YAP1和c-Myc的蛋白表达水平在Si-BRD4敲低组较HepG2对照组和空载组显著下降。④CCK-8法检测结果显示HepG2细胞48 h的光密度(optical density, OD)值在Si-BRD4敲低组明显低于HepG2对照组, 0.423±0.015比0.532±0.026, 细胞活力明显下降, 两组之间的差异具有统计学意义(t=5.03, P= 0.007)。流式细胞术检测结果显示SiRNA敲低BRD4处理48 h后, Si-BRD4敲低组HepG2细胞的凋亡率为(24.58±3.95)%, 显著高于HepG2对照组的(6.46±2.13)%, 差异具有统计学意义(t=7.13, P=0.002)。⑤CCK-8检测结果显示JQ1组、VCR组和JQ1联合VCR组的HepG2细胞活力较HepG2对照组显著降低, 分别为0.364±0.020、0.383±0.014、0.269±0.019和0.943±0.014, 和HepG2对照组之间的差异均具有统计学意义(P<0.001), JQ1联合VCR组细胞的活力低于JQ1组和VCR组, 分别为0.269±0.019、0.364±0.020、0.383±0.014, 差异具有统计学意义, (t=5.88, P=0.004)和(t= 8.26, P=0.002)。EDU检测结果显示运用JQ1、VCR及JQ1联合VCR分别作用于HepG2细胞后, 明显抑制了HepG2细胞增殖, 提升了细胞凋亡率, JQ1联合VCR对细胞增殖的抑制和细胞凋亡的促进作用更明显。TUNEL染色结果显示JQ1联合VCR用药的细胞凋亡率高于单独使用JQ1和VCR。结论 BRD4在HB中高表达且与肿瘤的COG分期及转移相关, 可通过YAP1、c-Myc发挥促肿瘤作用, JQ1联合VCR用药作用效果强于单独使用JQ1和VCR。

Objective To explore the tumor-promoting mechanism of BRD4 and to examine the effect of BRD4 inhibitor on hepatoblastoma cells.Methods Paraffin samples of 45 HB needle biopsies or surgically resected tissues without chemotherapy were harvested.Forty-five paraffin tissue samples with characteristic tumor cells without chemotherapy were selected as HB group;30 specimens with normal hepatic lobular structure,central lobular vein and portal area of paratumoral liver tissue were observed at the edge of 45 paraffin samples and these 30 cases as paratumoral control group.The expression level of BRD4 was detected semiquantitatively by immunohistochemistry and the correlation between expression level of BRD4 and clinicopathological characteristics statistically examined.The BRD4 expression levels of human HB cell line HepG2 control group(untreated HepG2 cells),unloaded group(HepG2 cells transfected with Si-NC)and Si-BRD4 knockdown group(HepG2 cells transfected with Si-BRD4)were detected by real-time quantitative polymerase chain reaction(qRT-PCR),Western blot and immunofluorescent techniques.mRNA and protein expression levels of Yes-associated protein 1(YAP1)and c-Myc were detected.Cell viability and apoptotic rate were measured by CCK-8 assay and flow cytometry in HepG2 control and Si-BRD4 knockdown groups.After HepG2 cells were treated for 48 h with JQ1,vincristine(VCR)and JQ1 plus VCR,viability,proliferation ability and cellular apoptotic in each group were detected by CCK-8 assay,EDU-555 cell proliferation kit and TdT-mediated dUTP nick end labeling(TUNEL)staining.HepG2 cells treated with 30 nmol/L JQ1 served as JQ1 group,HepG2 cells treated with 70μg/ml VCR as VCR group and HepG2 cells treated with 30 nmol/L JQ1 and 70μg/ml VCR as JQ1 plus VCR group.Results Immunohistochemistry showed that the positive rate of BRD4 was 97.8%(44/45)in HB nuclei and the positive rate of paratumoral hepatocyte nuclei zero.The difference was statistically significant(P<0.001).According to the Children's Oncology Group(COG)stage,there were low expression(n=8)and high expression(n=4)at stageⅠ-Ⅱand low expression(n=3)and high expression(n=30)at stageⅢ-Ⅳ.BRD4 expression level was correlated with COG tumor stage(P<0.001).No metastasis occurred in low-expression children and high expression(n=11)and BRD4 expression levels were correlated with tumor metastasis(P=0.042).qRT-PCR indicated that the mRNA expression levels of YAP1 and c-Myc declined markedly in Si-BRD4 knockdown HepG2 control and unloaded groups.Also Western blot and immunofluorescence implied that the protein expression levels of YAP1 and c-Myc declined markedly in Si-BRD4 knockdown HepG2 control and unloaded groups.And CCK-8 assay revealed that optical density(OD)at 48 h was significantly lower in Si-BRD4 knockdown group than in HepG2 control group[(0.423±0.015)vs.(0.532±0.026)].And cellular viability dropped obviously and inter-group difference was statistically significant(t=5.03,P=0.007).The results of flow cytometry showed that after a 48 h treatment of SiRNA knockdown BRD4,cellular apoptosis was significantly higher in Si-BRD4 knockdown group than that in HepG2 control group[(24.58±3.95)%vs(6.46±2.13)%]and the difference was statistically significant(t=7.13,P=0.002).The results of CCK-8 assay showed that cellular viability was significantly lower in JQ1/VCR group than that in control group[(0.364±0.020),(0.383±0.014),(0.269±0.019)and(0.943±0.014)]and the differences were statistically significant between JQ1 plus VCR and control groups(P<0.001).Cellular viability was lower in JQ1 plus VCR group than that in JQ1 plus VCR group[(0.269±0.019),(0.364±0.020)and(0.383±0.014)]and the differences were statistically significant[(t=5.88,P=0.004),(t=8.26,P=0.002)].And 5-ethynyl-2'-deoxyuridine(EDU)showed that JQ1,VCR and JQ1 plus VCR significantly suppressed cellular proliferation and accelerated the apoptotic rate after treatment and JQ1 plus VCR arrested cellular proliferation and promoted apoptosis more significantly.TUNEL staining showed that apoptotic rate of JQ1 plus VCR was higher than that of JQ1/VCR alone.Conclusions Highly expressed in HB,BRD4 is correlated with COG stage and tumor metastasis and plays a tumor-promoting role through YAP1/c-Myc.And the effect of JQ1 plus VCR is more potent than that of JQ1/VCR alone.