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BCL11B expression in hepatocellular carcinoma relates to chemosensitivity and clinical prognosis
肝细胞癌中 BCL11B 的表达与化疗敏感性和临床预后有关

Hiroyuki Abe | Kenya Kamimura | Shujiro Okuda | Yu Watanabe |
阿部浩之 | 上村 健也 {{1}| 奥田修次郎 | 渡边雄 { |
Jun Inoue | Yutaka Aoyagi | Toshifumi Wakai | Ryo Kominami | Shuji Terai
井上纯 {{0}| 青柳 丰 { | 若井 俊文 {{2}| 小凑亮 { | 寺内修二

Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata, Japan
日本新潟县新潟市新潟大学医学和牙科学研究生院胃肠病学和肝病学部
Department of General Medicine, Niigata University School of Medicine, Niigata, Niigata, Japan
日本新泻县新泻市新泻大学医学院全科医学系
Division of Bioinformatics, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata, Japan
日本新潟县新潟市新潟大学医学牙科学研究生院生物信息学研究科
Department of Agricultural Chemistry, Faculty of Applied Biosciences, Tokyo University of Agriculture, Tokyo, Japan
日本东京农业大学应用生物科学学院农业化学系,日本东京
Department of Gastroenterology and Hepatology, Niigata Medical Center, Niigata, Niigata, Japan
日本新泻县新泻市医疗中心肠胃病学和肝病学部
Division of Digestive and General Surgery, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata, Japan
日本新潟县新潟市新潟大学医学和牙科学研究生院消化和普通外科学部
Department of Molecular Genetics, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata, Japan
日本新潟县新潟市新潟大学医学牙科研究生院分子遗传学系

Correspondence 通信

Kenya Kamimura, Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Sciences, Niigata University, 1-757 Asahimachi-dori, Chuo-ku, Niigata, Niigata, 951-8510, Japan.
Kenya Kamimura,日本新泻县新泻市中央区朝日町通 1-757 号,新泻大学医学和牙科学研究生院胃肠病学和肝病学部,邮编 951-8510。
Email: kenya-k@med.niigata-u.ac.jp
电子邮件: kenya-k@med.niigata-u.ac.jp

Funding information 资金信息

Ichiro Kanehara Foundation for the Promotion of Medical Sciences and Medical Care
金原一郎医学科学和医疗促进基金会

Abstract 摘要

Introduction: B-cell lymphoma/leukemia 11B (BCL11B) is a subunit of SWI/ SNF chromatin remodeling complexes and functions in cell cycle regulation and apoptosis upon DNA replication stress and damages via transcription. Many malignancies were reported to exhibit changes in BCL11B gene expression; however, no study has focused on the relationship between BCL11B and hepatocellular carcinoma, which potentially exhibits DNA replication stress and damages upon its oncogenesis. Thus, in this study, we examined the molecular characterization of BCL11B expression in hepatocellular carcinoma.
导言:B细胞淋巴瘤/白血病11B(BCL11B)是SWI/ SNF染色质重塑复合物的一个亚基,通过转录在DNA复制压力和损伤时调节细胞周期和细胞凋亡。据报道,许多恶性肿瘤都表现出 BCL11B 基因表达的变化;但是,还没有研究关注 BCL11B 与肝细胞癌之间的关系,因为肝细胞癌在其肿瘤发生过程中可能表现出 DNA 复制应激和损伤。因此,在本研究中,我们考察了肝细胞癌中 BCL11B 表达的分子特征。

Methods and Results: The cumulative progression-free survival and overall survival were significantly longer in the clinical cases of BCL11B-negative hepatocellular carcinoma than BCL11B-positve cases. Microarray and real-time PCR analyses in hepatocellular carcinoma cell lines indicated a correlation between BCL11B and GATA6, a gene reported to be correlated with oncogenic activities and resistance to anthracycline, which is often used for hepatocellular carcinoma chemotherapy. Consequently, BCL11B-overexpressing cell lines exhibited resistance to anthracycline in cell growth assays and the resistance has been evidenced by the increased expression of BCL-xL in cell lines. The results were supported by the analyses of human HCC samples showing the correlation between BCL11B and GATA6 expressions.
方法和结果:BCL11B阴性肝细胞癌临床病例的累积无进展生存期和总生存期明显长于BCL11B阳性病例。肝癌细胞系的微阵列和实时 PCR 分析表明,BCL11B 与 GATA6 之间存在相关性,据报道,GATA6 与致癌活性和抗蒽环类药物相关,而蒽环类药物常用于肝癌化疗。因此,在细胞生长实验中,BCL11B 高表达的细胞系表现出对蒽环类药物的耐药性,细胞系中 BCL-xL 的表达增加证明了这种耐药性。对人类 HCC 样本的分析表明,BCL11B 和 GATA6 的表达之间存在相关性,这也支持了上述结果。

Discussions and Conclusion: Our results indicated that overexpression of BCL11B amplifies GATA6 expression in hepatocellular carcinoma in vitro and in vivo that leads to anti-apoptotic signal activation, and induces resistance to chemotherapy, which influenced the postoperative prognosis.
讨论与结论:我们的研究结果表明,BCL11B在体外和体内的过表达会扩大肝细胞癌中GATA6的表达,从而导致抗凋亡信号激活,诱导化疗耐药,影响术后预后。

KEYWORDS 关键词

BCL11B, drug sensitivity, GATA6, hepatocellular carcinoma, prognosis
BCL11B、药物敏感性、GATA6、肝细胞癌、预后

1 | INTRODUCTION 1 简介

The B-cell lymphoma/leukemia 11B (BCL11B) gene encodes a lineage-specific -type zinc finger transcription factor protein. BCL11B expressed in various types of cells and contributes to the development of neuron, cells, and others. Furthermore, BCL11B is known as an adipogenesis regulator and a haploinsufficient tumor suppressor. The BCL11B allele loss results in the occurrence of human T-cell acute lymphoblastic leukemia and mouse thymic lymphoma. On the other hand, several studies have reported the effect BCL11B on cell proliferation and chemoresistance in cancer as an oncogene. For instance, BCL11B upregulation has been reported in Ewing sarcoma to maintain its oncogenic character ; the expression of BCL11B has been found to be correlated with the poorly differentiated tumor status of head and neck squamous cell carcinoma ; enriched expression of BCL11B in glioma cells promotes cell growth ; and BCL11B suppression using RNA interference technique inhibited leukemic cells proliferation by apoptosis. This oncogenic mechanism might involve the apoptosis resistance accompanied by the delay of cell cycle by accumulating cells at G1 associated with the upregulation of p27, p57, and p18. The mechanism is also supported by the report that BCL11B contributes to the maintenance of genomic integrity at cell cycle as a result of the lack of the gene that causes the failure of checkpoint kinase 1 activity in thymocytes. These data support the fact that BCL11B-overexpressing malignant cells may obtain resistance characteristics to chemotherapy and radiotherapy to induce apoptosis. As a fact, various cancer cells activate DNA repair pathways to gain chemo- and radio-resistance characteristics. Based on this evidence, BCL11B could be a new therapeutic target in the refractory of malignancy to the conventional therapeutics.
B细胞淋巴瘤/白血病11B(BCL11B)基因编码一种系特异性 型锌指转录因子蛋白。-型锌指转录因子蛋白。BCL11B 在多种细胞中表达,有助于神经元、 细胞等的发育。 此外,BCL11B 还是一种已知的脂肪生成调节因子 和单倍体肿瘤抑制因子。 BCL11B 等位基因缺失会导致人类 T 细胞急性淋巴细胞白血病和小鼠胸腺淋巴瘤的发生。 另一方面,一些研究报道了BCL11B作为癌基因对癌症细胞增殖和化疗抗性的影响。例如,有报道称BCL11B在尤文肉瘤中上调可保持其致癌特性 ;发现BCL11B的表达与头颈部鳞状细胞癌的分化不良肿瘤状态相关 ;BCL11B在胶质瘤细胞中的富集表达可促进细胞生长 ;利用RNA干扰技术抑制BCL11B可通过凋亡抑制白血病 细胞的增殖。 这种致癌机制可能涉及细胞凋亡抵抗,以及细胞周期的延迟,即细胞在 G1 阶段的积累与 p27、p57 和 p18 的上调有关。 有报告称,由于胸腺细胞中缺乏导致检查点激酶1活性失效的基因,BCL11B有助于维持细胞周期中基因组的完整性,这也为该机制提供了支持。 这些数据证明,BCL11B过表达的恶性细胞可能对化疗和放疗诱导凋亡产生抗药性。事实上,各种癌细胞都会激活DNA修复途径,从而获得对化疗和放疗的抵抗特性。 基于这些证据,BCL11B可能成为传统疗法难治性恶性肿瘤的新治疗靶点。
Hepatocellular carcinoma (HCC) is a malignancy related to DNA damage and mutagenesis caused by various hepatitis caused by virus, fatty infiltration, and alcoholic liver disease, which leads to carcinogenesis. Some studies have recently reported that DNA repair signaling pathway in HCC cells contributed to worse prognosis ; however, no detailed mechanisms have been reported in this regard. Based on the role of BCL11B in biology, it is reasonable to hypothesize that BCL11B is involved in the clinical course of HCC as it is treated with various DNA-damaging chemotherapy and radiotherapy agents. Regarding the relationship between BCL11B and HCC, only a few studies have reported BCL11B gene modification by mutation, deletion, amplification, truncation, gain, and copy number aberrations, immune evasion mechanisms, and retention of cancer stem cell traits in HBV-related HCC. Based on the aforementioned backgrounds, we have investigated the effect of BCL11B expression in HCC cells on resistance to various therapies and on HCC prognosis.
肝细胞癌(HCC)是一种恶性肿瘤,与病毒引起的各种肝炎、脂肪浸润、酒精性肝病等导致的DNA损伤和诱变有关,从而引发癌变。 最近有研究报告称,HCC细胞中的DNA修复信号通路会导致预后恶化 ;然而,这方面的详细机制尚未见报道。根据 BCL11B 在生物学中的作用,我们可以合理地推测,在使用各种 DNA 损伤性化疗和放疗药物治疗 HCC 时,BCL11B 与 HCC 的临床病程有关。关于BCL11B与HCC之间的关系,仅有少数研究报道了BCL11B基因的突变、缺失、扩增、截断、增益和拷贝数畸变等修饰, 免疫逃避机制,{{3}以及在HBV相关HCC中保留癌症干细胞特质。 基于上述背景,我们研究了BCL11B在HCC细胞中的表达对各种疗法的耐药性和HCC预后的影响。

2 MATERIALS AND METHODS
2 材料和方法

2.1 Clinical course and BCL11B expression in vivo
2.1 临床病程和 BCL11B 在体内的表达

With a written informed consent, tissue samples were collected from the HCC cases who were diagnosed with imaging studies including magnetic resonance imaging and computed tomography, and underwent surgical resection in Niigata University Hospital. The tissues were stained with hematoxylin and eosin or immunohistochemical stains: Rat anti-Ctip2 (Bcl11b) antibody (ab18465; Abcam), Rabbit anti-GATA6 antibody (ab175349; Abcam), Vectastain Elite ABC Rat immunoglobulin G kit (PK6104; Vector Laboratories), Vectastain Elite ABC Rabbit immunoglobulin G kit (PK-6101; Vector Laboratories, Burlingame, CA), and 3,3'-diaminobenzidine chromogen tablets (Muto Pure Chemicals). The expression of BCL11B and GATA6 was confirmed via RT-PCR in recently resected 70 cases using the aforementioned procedure. The tumor tissues of these cases were immunohistochemically stained to determine the relationship between BCL11B and GATA6. Images were captured randomly, and analyzed quantitatively, using the ImageJ software (version 1.8.0_172; National Institutes of Health, Bethesda, MD).
在获得书面知情同意的情况下,收集了经磁共振成像和计算机断层扫描等影像学检查确诊并在新潟大学医院接受手术切除的 HCC 病例的组织样本。组织经苏木精和伊红染色或免疫组化染色:大鼠抗Ctip2(Bcl11b)抗体(ab18465;Abcam)、兔抗GATA6抗体(ab175349;Abcam)、Vectastain Elite ABC大鼠免疫球蛋白G试剂盒(PK6104;Vector Laboratories)、Vectastain Elite ABC兔免疫球蛋白G试剂盒(PK-6101;Vector Laboratories,Burlingame,CA)和3,3'-二氨基联苯胺显色剂片(Muto Pure Chemicals)。在最近切除的 70 个病例中,采用上述方法通过 RT-PCR 确认了 BCL11B 和 GATA6 的表达。对这些病例的肿瘤组织进行免疫组化染色,以确定 BCL11B 和 GATA6 之间的关系。采用ImageJ软件(版本1.8.0_172;马里兰州贝塞斯达,美国国立卫生研究院)随机采集图像并进行定量分析。

2.2 Microarray and bioinformatic analyses
2.2 微阵列和生物信息学分析

Mock- and BCL11B-transfected HLE cell lines were compared for the gene expression using SurePrint G3 Human Gene Expression (v2) Microarray Kit and GeneSpring GX version 14.5.1 (Agilent Technologies, Inc.). Among the 50,599 genes analyzed, 1052, which exhibited -fold differences in expression, were assessed with related gene expressions using the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology database. The gene orthology terms were selected based on Fisher's exact test, followed by the Benjamini-Yekutieli correction method.
使用 SurePrint G3 Human Gene Expression (v2) Microarray Kit 和 GeneSpring GX 14.5.1 版(安捷伦科技公司)比较了模拟和 BCL11B 转染的 HLE 细胞系的基因表达情况。在分析的 50,599 个基因中,有 1052 个基因表现出 -倍的表达差异。-倍差异的 1052 个基因,利用京都基因组百科全书(KEGG)的基因同源数据库评估了相关基因的表达情况。根据费舍尔精确检验和本杰明-耶库泰利校正法选择了基因同源词。

2.3 Cells 2.3 细胞

HLE and HepG2 cell lines were obtained from the Japanese Collection of Research Bioresources Cell Bank (National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, Japan). The cells were cultured in Minimum Essential Medium with fetal bovine serum
HLE 和 HepG2 细胞系来自日本研究生物资源细胞库(日本大阪茨城国立生物医学创新、健康和营养研究所)。细胞在含有胎牛血清的最低限度基本培养基中培养。
(10%) and of penicillin and streptomycin (100U/mL). The BCL11B complementary DNA was cloned in pCMV6Entry Tagged Cloning Vector (OriGene Technologies, Inc.). Either mock or BCL11B-cloned vectors were transfected into the cells using FuGENE HD Transfection Reagent (Promega) and selected using G418 sulfate. Three independent clones were isolated from each of the two cell lines for the analyses.
(10%)以及青霉素和链霉素(100U/mL)。BCL11B 的互补 DNA 被克隆到 pCMV6Entry 标记克隆载体(OriGene Technologies, Inc.)使用 FuGENE HD 转染试剂(Promega)将模拟载体或 BCL11B 克隆载体转染到细胞中,并使用硫酸 G418 进行筛选。从两种细胞系中各分离出三个独立的克隆进行分析。

2.4 Gene and protein expression in the cells
2.4 细胞中基因和蛋白质的表达

and GAPDH genes expression was confirmed via reverse transcription-polymerase chain reaction (RTPCR). RNA was prepared from the cells (RNA Easy Mini Kit) and the complementary DNA was synthesized from 1 to of RNA (SuperScript II Reverse Transcriptase, Invitrogen). Complementary DNA products (1-2 aliquots) were used for PCR. The primers used are (F, forward; R, reverse):
通过反转录聚合酶链反应(RTPCR)确认 和 GAPDH 基因的表达。从细胞中制备 RNA(RNA Easy Mini Kit),用 1 到 的 RNA 合成互补 DNA(SuperScript II 逆转录酶,Invitrogen 公司)。互补 DNA 产物(1-2 份)用于 PCR。所用引物为(F,正向;R,反向):
  1. BCL11B (F): CACTCATCCGTGATCACTTC
    BCL11B (F):cactcatccgtgatcacttc
  2. BCL11B (R): CGATGAGATTGCTCTGGAAC
    BCL11B(R):CGATGAGATGCTCTAGAAC
  3. GATA6 (F): GCGGGAGAGAGCACCAATC
    GATA6 (F):GCGGAGAGAGCACCAATC
  4. GATA6 (R): GAGCCCATCTTGACCCGAAT
    GATA6(R): GAGCCCATCTTGACCCGAAT
  5. GAPDH (F): GGTCGGAGTCAACGGATTTGGTCG
    GAPDH (F):GGTCGAGTCAACGATTGTCCG
  6. GAPDH (R): CCTCCGACGCCTGCTTCACCAC
    GAPDH(R):CCTCCGACGCCTGCTTCCAC
PCR protocol: for , followed by 30 cycles of for for , and for ) followed by for of extension.
PCR 协议: 。然后是 的30个循环。 ),然后进行 的循环。然后进行 延伸。
The protein expression of BCL11B, BCL-xL, BCL2 , BAX, GATA6, and -actin was examined via western blotting. Cells were suspended in phosphate-buffered saline and mixed with an equal volume of lysis buffer, tris sucrose, sodium dodecyl sulfate, 2-mercaptoethanol, and bromophenol blue. The extract was subjected to gel (4%-15% Mini-PROTEAN TGX Stain-Free Protein Gels, Bio-Rad Laboratories) and blotted onto Hybond membranes (GE Healthcare Life Sciences) and the bands were visualized (ECL plus Western Blotting Detection System, GE Healthcare Life Sciences).
通过蛋白印迹检测了 BCL11B、BCL-xL、BCL2、BAX、GATA6 和 -actin 的蛋白表达。-肌动蛋白的表达。将细胞悬浮在磷酸盐缓冲液中,然后与等体积的裂解缓冲液( 蔗糖、{{2}十二烷基硫酸钠、 2-巯基乙醇)混合。2-巯基乙醇和 {{4} 溴酚蓝。提取物经凝胶(4%-15% Mini-PROTEAN TGX 无染色蛋白凝胶,Bio-Rad 实验室)处理后,印迹到 Hybond 膜(通用电气医疗生命科学公司)上,然后用 ECL plus Western 印迹检测系统(通用电气医疗生命科学公司)显现条带。
Antibodies used were: 使用的抗体有
  1. Rabbit anti-Ctip2 (Bcl11b) antibody (ab28448; Abcam, Cambridge, UK)
    兔抗 Ctip2(Bcl11b)抗体(ab28448;Abcam,英国剑桥)
  2. Rabbit anti-Bcl-xL antibody (ab98143; Abcam)
    兔抗 Bcl-xL 抗体(ab98143;Abcam)
  3. Rabbit anti-Bcl-2 antibody (ab196495; Abcam)
    兔抗 Bcl-2 抗体(ab196495;Abcam)
  4. Rabbit anti-Bax antibody (ab104156; Abcam)
    兔抗 Bax 抗体(ab104156;Abcam)
  5. Rabbit anti-GATA6 antibody (ab175349; Abcam)
    兔抗 GATA6 抗体(ab175349;Abcam)
  6. Rabbit anti- -actin antibody (ab8227; Abcam)
    兔抗 -肌动蛋白抗体(ab8227; Abcam-肌动蛋白抗体(ab8227;Abcam)
  7. Anti-rabbit immunoglobulin horseradish peroxidase (NA934-1ML; GE Healthcare Life Sciences, PA, USA)
    抗兔免疫球蛋白 辣根过氧化物酶(NA934-1ML;美国宾夕法尼亚州通用电气医疗生命科学公司)
Expression of BCL11B protein in the cells was confirmed immunohistochemically, using Rat anti-Ctip2 (Bcl11b) antibody (ab18465; Abcam), Vectastain Elite ABC Rat immunoglobulin G kit (PK-6104; Vector Laboratories), and 3,3'-diaminobenzidine chromogen tablets (Muto Pure Chemicals).
使用大鼠抗 Ctip2(Bcl11b)抗体(ab18465;Abcam)、Vectastain Elite ABC 大鼠免疫球蛋白 G 试剂盒(PK-6104;Vector Laboratories)和 3,3'-二氨基联苯胺显色剂片剂(Muto Pure Chemicals)对细胞中 BCL11B 蛋白的表达进行免疫组化确认。

2.5 Cell growth assay
2.5 细胞生长试验

The cells were plated in 96-well tissue culture dishes at a concentration of cells per well in medium. Then, they were treated with CDDP or epirubicin using doses determined based on previously reported studies. The cell growth was assessed using the Premix WST-1 Cell Proliferation Assay System (Takara Inc.).
细胞以每孔 个的浓度在 培养基中培养到96孔组织培养皿中。然后,用 CDDP 或表柔比星处理细胞,剂量根据之前的研究确定。 细胞生长情况使用 Premix WST-1 细胞增殖检测系统(Takara Inc.)

2.6 Statistical analyses
2.6 统计分析

Data were analyzed by paired -test or one- or two-way ANOVA followed by Bonferroni's multiple comparison test. The cumulative PFS and OS curves were generated by the Kaplan-Meier method, and the occurrence rates were compared using a log-rank test. GraphPad Prism 9 software (version 9.3.1; GraphPad) was used for the analyses. was considered statistically significant.
数据分析采用配对 检验或单因子或双因子方差分析,然后进行 Bonferroni 多重比较检验。-检验或单向或双向方差分析,然后进行Bonferroni多重比较检验。累积 PFS 和 OS 曲线采用 Kaplan-Meier 法生成,发生率的比较采用 log-rank 检验。分析使用 GraphPad Prism 9 软件(9.3.1 版;GraphPad)。 被认为具有统计学意义。

3 | RESULTS 3 结果

3.1 Effect of BCL11B expression in HCC tumor on prognosis
3.1 BCL11B 在 HCC 肿瘤中的表达对预后的影响

To examine the effect of BCL11B expression in HCC tumor cells on the prognosis of the reported cases, the PFS and OS were assessed in BCL11B-positive (>5% positive in IHC analyses, ) and BCL11B-negative groups. The characteristics of the patients and their clinical information are summarized in Table 1. No significant differences were observed in age, gender, body mass index, etiology of the liver disease, histological classification of HCC, complication of liver cirrhosis, hepatic reserve function, tumor markers, nutrition, and number of postoperative treatments. The PFS ( 2608 vs. 480 days, ) and OS (3570 vs. 1564 days, were significantly longer in the BCL11B-negative tumor cell group (Figure 1). These results indicated that the BCL11B expression in HCC helps gain resistance to various postoperative therapeutic options and leads to poor prognosis.
为了研究BCL11B在HCC肿瘤细胞中的表达对所报道病例预后的影响,我们对BCL11B阳性组(IHC分析中阳性率>5%, )和BCL11B阴性 组的PFS和OS进行了评估。表1总结了患者的特征及其临床信息。在年龄、性别、体重指数、肝病病因、HCC 组织学分类、肝硬化并发症、肝储备功能、肿瘤标志物、营养状况和术后治疗次数等方面均无明显差异。BCL11B阴性肿瘤细胞组的PFS(2608天 vs. 480天, )和OS(3570天 vs. 1564天, )明显长于BCL11B阴性肿瘤细胞组(图1)。这些结果表明,BCL11B在HCC中的表达有助于获得对各种术后治疗方案的耐受性,并导致不良预后。
TABLE 1 Patient characteristics.
表 1 患者特征
BCL11B
-
+
MWW
Test
-value
Characteristics
Age (years) 0.39
Median 65.5 69.0
Range
Gender 0.09
Female 23 24
Male 75 43
Body mass index 0.87
Median 22.7 23.1
Range
Etiology 0.11
HBV infection 29 12
HCV infection 27 19
Alcoholic 14 9
Nonalcoholic
steatohepatitis
23 25
AIH, PBC 5 2
Histology 0.93
Well-differentiated
tumor
29 21
Moderately
differentiated
tumor
57 36
Poorly
differentiated
tumor
12 10
Cirrhosis 0.14
Yes/No
Child-Pugh Score 0.80
Stage 0.37
I/II/III/IV 16/15/22/14
0.19
Median 8.3 12.3
Range
0.21
Median 27.8 37.0
Range
0.20
Median 171.0 187.0
Range
TG (mg/dL) 0.53
Median 100.0 107.0
Range
HbA1c (%) 0.87
TABLE 1 (Continued) 表 1(续)
MWW
BCL11B -value
Median 5.5 5.5
Range
Postoperative 0.06
Treatments
(Number of
Options)
0/1/2/3/4/5/6
Note: The values are expressed as median and range. MWW test, MannWhitney-Wilcoxon test.
注:数值以中位数和范围表示。MWW 检验,MannWhitney-Wilcoxon 检验。
Abbreviations: AFP, alpha fetoprotein; AIH, autoimmune hepatitis; DCP, des- -carboxy prothrombin; HBV, hepatitis B virus; , hepatitis virus; , primary biliary cholangitis; , total cholesterol; TG, triglyceride.
缩写:缩写: AFP,甲胎蛋白;AIH,自身免疫性肝炎;DCP,去 -羧基凝血酶原;HBV,乙型肝炎病毒; ,乙型肝炎。-羧基凝血酶原;HBV,乙型肝炎病毒;{{1} ,肝炎{{2}病毒; 。乙型肝炎病毒;{{1} ,肝炎{{2}病毒;{{3} ,原发性胆汁性胆管炎。}原发性胆汁性胆管炎;{{4}.TG,甘油三酯。

3.2 | Development of BCL11Boverexpressing cells
3.2 表达 BCL11Bov 的 细胞的发育

To examine the molecular function of BCL11B on the viability of , we produced -overexpressing cell lines by transfecting plasmid DNA-expressing human BCL11B into HCC cell lines, that are, HLE and HepG2. Figure presents the gene and BCL11B protein expression in the cells comparing with that of Molt4 cell line, which is known to express BCL11B. Quantitative analyses of multiple clones revealed a significantly higher expression of BCL11B in HLE and HepG2 cells by RTPCR, WB, and IHC (Figure 2B,C). Under normal cell culture condition with fetal bovine serum, BCL11Boverexpressing cells exhibited no significant difference in growth rate compared with the mock-transfected cells (Figure 2D).
为了研究 BCL11B 对 活力的分子功能,我们制备了 。为了研究BCL11B对 活力的分子功能,我们通过将表达人BCL11B的质粒DNA转染到HCC细胞系中,制备了{{1-我们将表达人BCL11B的质粒DNA转染到HCC细胞系(即HLE和HepG2)中,制备了 -外表达细胞系。图 显示了细胞中 基因和BCL11B蛋白的表达情况,并与已知能表达BCL11B的Molt4细胞系进行了比较。通过RTPCR、WB和IHC对多个克隆的定量分析发现,HLE和HepG2细胞中BCL11B的表达量明显更高(图2B,C)。在 胎牛血清的正常细胞培养条件下,与模拟转染细胞相比,BCL11Berexpressing细胞的生长速度没有明显差异(图2D)。

3.3 Effect of BCL11B expression on gene expression modification
3.3 BCL11B 的表达对基因表达修饰的影响

To determine the molecular mechanism of BCL11B, the gene expressions in mock-transfected HLE and BCL11Boverexpressing HLE were compared via DNA microarray analyses (Figure 3A,B). Analysis of KEGG orthology terms, after the hierarchical clustering of genes, revealed higher gene expression in terms of various oncogenic pathways, including the Jak-STAT, MAPK, Wnt, and Hippo signaling pathways, apoptosis, cell cycle, p53 signaling, and DNA replication in BCL11B-overexpresing cells than in mock-transfected cells. The genes upregulated and downregulated in BCL11B-overexpressing cells are presented in Tables 2 and 3. Based on the analyses, in order to examine the mechanisms of BCL11B on
为了确定BCL11B的分子机制,我们通过DNA芯片分析比较了模拟转染HLE和BCL11Boverexpressing HLE的基因表达(图3A,B)。对基因进行分层聚类后的 KEGG 同源词分析表明,与模拟转染细胞相比,BCL11B 高表达细胞中各种致癌通路的基因表达量更高,包括 Jak-STAT、MAPK、Wnt 和 Hippo 信号通路、细胞凋亡、细胞周期、p53 信号转导和 DNA 复制。表2和表3列出了BCL11B高表达细胞中上调和下调的基因。根据上述分析,为了研究 BCL11B 对细胞增殖的影响机制,研究人员对细胞进行了以下研究
(A)
(B)
FIGURE 1 Effect of BCL11B expression on the prognosis of HCC.
图 1 BCL11B 表达对 HCC 预后的影响。
Kaplan-Meier estimates of the progression-free survival (A) and overall survival (B) following the surgical treatment for HCC. MST, median survival time. .
HCC 手术治疗后无进展生存期(A)和总生存期(B)的 Kaplan-Meier 估计值。MST,中位生存时间。 .

(B)
(D)
FIG URE 2 Development of BCL11B-overexpressing hepatocellular carcinoma (HCC) cell lines. (A) Representative images of the reverse transcription-polymerase chain reaction (RT-PCR) of BCL11B and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as well as Western blotting of BCL11B and -Actin in BCL11B-overexpressing HLE. (B) Summary of relative expression ratios of the BCL11B gene and protein. The values are expressed as means standard deviations on Student's -test. (C) Representative immunohistochemical staining of BCL11B-overexpressing HLE. The T lymphoblast cell line of Molt4 was shown as a positive control, and the Mock-transfected HLE was used as a negative control. (D) Growth of BCL11B-overexpressing cell lines under a normal culture condition with 10% fetal bovine serum. Mock HLE and Mock HepG2 indicate Mock-transfected HLE and HepG2 cell lines. BCL11B-HLE and BCL11BHepG2 indicate BCL11B overexpression.
图URE 2 BCL11B过表达肝细胞癌(HCC)细胞系的发展。(A) BCL11B 和甘油醛-3-磷酸脱氢酶(GAPDH)的反转录聚合酶链反应(RT-PCR)以及 BCL11B 和 -Actin 的 Western 印迹的代表性图像。-Actin。(B) BCL11B基因和蛋白的相对表达比汇总。数值以学生 检验的均值 标准差 表示。-检验。(C) BCL11B过表达HLE的代表性免疫组化染色。Molt4的T淋巴母细胞系为阳性对照,Mock转染的HLE为阴性对照。(D)在 10%胎牛血清的正常培养条件下,BCL11B 高表达细胞系的生长情况。模拟 HLE 和模拟 HepG2 表示模拟转染的 HLE 和 HepG2 细胞系。BCL11B-HLE 和 BCL11BHepG2 表示 BCL11B 过表达。
(A)
KEGG Orthology KEGG 正选
High in BCL11B-overexpressed HLE
在 BCL11B 过表达的 HLE 中含量高
Chemokine signaling pathway 5
趋化因子信号通路 5
cell receptor signaling pathway Toll-like receptor signaling pathway
细胞受体信号转导途径 Toll 样受体信号转导途径

NOD-like receptor signaling pathway Leukocyte transendothelial migration Insulin signaling pathway
NOD 样受体信号通路 白细胞跨内皮细胞迁移 胰岛素信号通路

Oxytocin signaling pathway -
催产素信号途径
Thyroid hormone synthesis -
甲状腺激素合成
PPAR signaling pathway PPAR 信号通路
Thyroid hormone signaling pathway
甲状腺激素信号途径
GnRH signaling pathway GnRH 信号通路
Prolactin signalin secretion
催乳素信号素分泌
Prolactin signaling pathway
催乳素信号通路
Vascular smooth muscle contraction
血管平滑肌收缩
Drug metabolism - other enzymes
药物代谢--其他酶
yrimidine metabolism - 嘧啶代谢
Steroid hormone biosynthesis
类固醇激素的生物合成
Primary bile acid biosynthesis
初级胆汁酸的生物合成
Glycosylphosphatidylinositol(GPI)-anchor biosynthesis -
糖基磷脂酰肌醇(GPI)锚生物合成
Other glycan degradation
其他聚糖降解
Oxidative phosphorylation -
氧化磷酸化
Non-alcoholic fatty liver disease (NAFLD)
非酒精性脂肪肝(NAFLD)
Viral carcinogenesis 病毒致癌
Pathways in cancer -
癌症的发病途径
Choline metabolism in cancer -
癌症中的胆碱代谢
Mroteoglycans in cancer 癌症中的蛋白聚糖
RNA transport RNA 运输
Aminoacyl-tRNA biosynthesis
氨基酰-tRNA 的生物合成

Nucleotide excision repair
核苷酸切除修复
RNA polymerase - RNA 聚合酶
Homologous recombination -
同源重组
Protein processing in endoplasmic reticulum Mismatch repair
内质网中的蛋白质加工 错配修复
Ubiquitin mediated proteolysis -
泛素介导的蛋白水解作用
Base excision repair 碱基切除修复
Cytokine-cytokine receptor interaction -
细胞因子与细胞因子受体的相互作用--细胞因子与细胞因子受体的相互作用
Hippo signaling pathway -
河马信号通路
Sphingolipid signaling pathway
鞘脂信号传导途径
FoxO signaling pathway FoxO 信号通路
MAPK signaling pathway MAPK 信号通路
MKA-Akt signaling pathway
MKA-Akt 信号通路
Ras signaling pathway -
Ras 信号通路
Rap1 signaling pathway -
Rap1 信号通路
mTOR signaling pathway -
mTOR 信号通路
Jak-STAT signaling pathway -
Jak-STAT 信号通路
AMPK signaling pathway -
AMPK 信号通路
CAM signaling pathway CAM 信号通路
cGMP - PKG signaling pathway
cGMP - PKG 信号通路
NF-kappa Bignaling pathway
NF-kappa 信号通路
Phosphatidylinositol signaling system
磷脂酰肌醇信号系统
Signaling pathways regulating pluripotency of stem cells -
调节干细胞多能性的信号通路 - 干细胞的多能性
p53 signaling pathway -
p53 信号通路
Cell cycle 细胞周期
Lysosome- 溶酶体
Apoptosis 细胞凋亡
Adherens junction 粘连交界处
(B)
Flycosaminoglycan biosynthesis - heparan sulfate / heparin-
蝇类氨基多糖的生物合成--硫酸肝素/肝素--蝇类氨基多糖的生物合成--硫酸肝素/肝素--蝇类氨基多糖的生物合成。
unsaturated fatty acids-
不饱和脂肪酸
Histidine metabolism- 组氨酸代谢
Oxidative phosphorylation-
氧化磷酸化
Inositol phosphate metabolism=
磷酸肌醇代谢=
Arachidonic acid metabolism-
花生四烯酸代谢--
Glycosphingolipid biosynthesis - globo series-
糖磷脂的生物合成--球状系列--糖磷脂的生物合成
Fatty acid biosynthesis-
脂肪酸的生物合成
Lysine degradation - 赖氨酸降解
Fatty acid metabolism- 脂肪酸代谢
Phenylalanine, tyrosine and tryptophan biosynthesis-
苯丙氨酸、酪氨酸和色氨酸的生物合成--苯丙氨酸、酪氨酸和色氨酸的生物合成

Tyrosine metabolism- 酪氨酸代谢
Phenylalanine metabolism-
苯丙氨酸代谢--
Phenylalanine, tyrosine and tryptophan biosynthesis-
苯丙氨酸、酪氨酸和色氨酸的生物合成--苯丙氨酸、酪氨酸和色氨酸的生物合成

Tyrosine metabolism- 酪氨酸代谢
Phenylalanine metabolismUbiquinone and other terpenoid-quinone biosynthesis-
苯丙氨酸代谢联醌和其他萜类化合物--醌的生物合成--苯丙氨酸代谢
Amyotrophic lateral sclerosis (ALS)-
肌萎缩性脊髓侧索硬化症(ALS)--
Staphylococcus aureus infection-
金黄色葡萄球菌感染--
Chagas disease (American trypanosomiasis)
查加斯病(美洲锥虫病)
Salmonella infection 沙门氏菌感染
HTLV-I infection- 感染 HTLV-I-
Amoebiasis 阿米巴病
Primary immunodeficiency
原发性免疫缺陷
Non-alcoholic fatty liver disease (NAFLD)
非酒精性脂肪肝(NAFLD)
MicroRNAs in cancer- 癌症中的微RNA
Central carbon metabolism in cancer-
癌症中的中枢碳代谢
Transcriptional misregulation in cancers-
癌症中的转录失调--
Non-small cell lung cancer-
非小细胞肺癌
Basal cell carcinoma- 基底细胞癌
Endometrial cancer- 子宫内膜癌
Choline metabolism in cancer-
癌症中的胆碱代谢
Pathways in cancer- 癌症治疗途径
Acute myeloid leukemia- 急性髓性白血病
Ubiquitin mediated proteolysis-
泛素介导的蛋白水解--
Fanconi anemia pathway 范可尼贫血症途径
Sulfur relay system- 硫中继系统
Neuroactive ligand-receptor interaction-
神经活性配体-受体相互作用--
Hedgehog signaling pathway-
刺猬信号通路
NF-kappa B signaling pathway-
NF-kappa B 信号通路--
Rap1 signaling pathway Rap1 信号通路
Phosphatidylinositol signaling system-
磷脂酰肌醇信号系统--
Hippo signaling pathway-
河马信号通路
ErbB signaling pathway- ErbB 信号通路
Apoptosis - 细胞凋亡
Endocytosis 内吞
Focal adhesion  病灶粘附 {{0}
FIGURE 3 Microarray and bioinformatic analyses. KEGG orthology classification for the genes with more than 2-fold differences in the expression. Higher (A) and lower (B) expressions in BCL11B-overexpressing HLE than mock-transfected HLE.
图 3 微阵列和生物信息学分析。表达差异超过 2 倍的基因的 KEGG 亲缘关系分类。与模拟转染的 HLE 相比,BCL11B 高表达(A)和低表达(B)。
prognosis of HCC cases, we have focused on the correlation of GATA binding protein 6 (GATA6), which showed 78905.9-fold increase upon BCL11B-overexpression, and apoptosis-related gene expression. Moreover, GATA6 has been reported to be related to the differentiation and progression of the malignant disease, such as pancreatic cancer, colorectal carcinoma, and The BCL11B-overexpressing HLE and HepG2 cell lines demonstrated a significantly higher GATA6 expression than the mock-transfected cells (Figure 4A,B). Furthermore, the BCL11B expression contributed to BCL-xL increase in both cell lines and BCL-2 increase in HLE but not to BAX expression. Altogether, these results indicated that BCL11B overexpression in HCC cells contributes to the induction of GATA6 expression and anti-apoptotic phenotype (Figure 4C).
为了预测HCC病例的预后,我们重点研究了GATA结合蛋白6(GATA6)与凋亡相关基因表达的相关性,GATA6在BCL11B过表达时增加了78905.9倍。此外,有报道称 GATA6 与胰腺癌、结直肠癌、 等恶性疾病的分化和进展有关。BCL11B过表达的HLE和HepG2细胞系的GATA6表达明显高于模拟转染细胞(图4A,B)。此外,BCL11B的表达导致了两种细胞系中BCL-xL的增加和HLE中BCL-2的增加,但与BAX的表达无关。这些结果表明,BCL11B 在 HCC 细胞中的过表达有助于诱导 GATA6 的表达和抗凋亡表型(图 4C)。

3.4 Effect of BCL11B expression on cell growth under a cytotoxic condition
3.4 细胞毒性条件下 BCL11B 的表达对细胞生长的影响

To examine the effect of BCL11B on cell viability under a cytotoxic condition, cell growth assay was conducted using BCL11B-overexpressing cells exposed to either epirubicin hydrochloride or cisplatin, which are often used in chemotherapy for HCC (Figure 5). While mocktransfected HLE and HepG2 cell lines exhibited dosedependent cell growth inhibition when treated with epirubicin hydrochloride (Figure 5A,C) or cisplatin (Figure 5B,D), BCL11B-overexpressing HLE and HepG2 showed a significantly milder inhibition with epirubicin hydrochloride (Figure 5A,C). These results indicated that BCL11B overexpression induced resistance to epirubicin hydrochloride in HCC cells.
为了研究 BCL11B 在细胞毒性条件下对细胞活力的影响,我们使用 BCL11B 表达过高的细胞暴露于盐酸表柔比星或顺铂中进行细胞生长试验,这两种药物通常用于 HCC 化疗(图 5)。模拟转染的HLE和HepG2细胞系在接受盐酸表柔比星(图5A,C)或顺铂(图5B,D)治疗时表现出剂量依赖性细胞生长抑制,而BCL11B高表达的HLE和HepG2在接受盐酸表柔比星(图5A,C)治疗时表现出明显较弱的抑制作用。这些结果表明,BCL11B过表达会诱导HCC细胞对盐酸表柔比星产生耐药性。
TABLE 2 Upregulated genes in BCL11B-overexpressing HCC cell (>10-fold differences).
表 2 BCL11B 过表达 HCC 细胞中的上调基因(差异大于 10 倍)。
TABLE 2 (Continued) 表 2(续)
No Gene symbol Gene name
Fold change BCL11B-HLE
versus Mock-HLE
41 SPAG6 Sperm associated antigen 6 10.731
42 TDRD1 Tudor domain containing 1 10.634
43 VSIG10L V-set and immunoglobulin domain containing 10 like 10.551
44 IAH1
Isoamyl acetate-hydrolyzing esterase 1 homolog (S.
cerevisiae)
10.509
45 UCK2 Uridine-cytidine kinase 2 10.399
46 HKR1 HKR1, GLI-Kruppel zinc finger family member 10.398
47 SOHLH2 Spermatogenesis and oogenesis specific basic helix-loop- 10.364
48 NFIA huclix 2 factor I/A 10.312
TABLE 3 Downregulated genes in BCL11B-overexpressing HCC cell (>10-fold differences).
表 3 BCL11B 过表达 HCC 细胞中的下调基因(差异大于 10 倍)。
No Gene symbol Gene name
Fold change BCL11B-HLE
versus Mock-HLE
1 TCP11L2 T-complex 11 (mouse)-like 2 41.370
2 TRAPPC9 Trafficking protein particle complex 9 22.601
3 NLRC3 NLR family, CARD domain containing 3 22.437
4 ADAM5P ADAM metallopeptidase domain 5, pseudogene 21.112
5 SPATA19 Spermatogenesis associated 19 19.573
6 PML Promyelocytic leukemia 17.612
7 WDR31 WD repeat domain 31 16.985
8 ZNF319 Zinc finger protein 319 16.857
9 KLK14 Kallikrein-related peptidase 14 15.325
10 HSBP1L1 Heat shock factor binding protein 1-like 1 15.132
11 AREG Amphiregulin 14.431
12 FTLP10 Ferritin, light polypeptide pseudogene 10 14.351
13 GOLGA6L6 Golgin A6 family-like 6 14.257
14 CEACAM3 Carcinoembryonic antigen-related cell adhesion molecule 3 13.712
15 HTR2C 5-hydroxytryptamine (serotonin) receptor 2C 13.123
16 FAM207A Family with sequence similarity 207, member A 12.942
17 ABLIM1 Actin binding LIM protein 1 12.457
18 DHRS4L2 Dehydrogenase/reductase (SDR family) member 4 like 2 12.452
19 MGC39372 Serpin peptidase inhibitor, clade B (ovalbumin), member 9 pseudogene 12.266
20 THNSL2 Threonine synthase-like 2 (S. cerevisiae) 12.248
21 CPLX4 Complexin 4 11.574
22 MBD1 Methyl-CpG binding domain protein 1 11.509
23 ALK Anaplastic lymphoma receptor tyrosine kinase 11.051
24 FPR1 Formyl peptide receptor 1 10.717
25 RPUSD2 RNA pseudouridylate synthase domain containing 2 10.500
26 AHNAK AHNAK nucleoprotein 10.249
27 ZNHIT1 Zinc finger, HIT-type containing 1 10.037

3.5 | Expression of BCL11B and GATA6 in tissues
3.5 | BCL11B 和 GATA6 在 组织中的表达量

To determine whether BCL11B-induced GATA6 expression contributes to cytotoxic agent tolerance and affects prognosis, the gene and protein expressions in the HCC and surrounding liver tissues were tested by RT-PCR, and immunohistochemical testing was conducted on the HCC tissues. RT-PCR revealed a significant correlation between the BCL11B and GATA6 expressions
为了确定BCL11B诱导的GATA6表达是否有助于细胞毒药物耐受性并影响预后,我们通过RT-PCR检测了HCC和周围肝组织中的基因和蛋白表达,并对HCC组织进行了免疫组化检测。RT-PCR发现BCL11B和GATA6的表达之间存在明显的相关性
(C)
(B)

FIGURE 4 Effect of BCL11B expression on the changes in GATA6 and apoptosis-related proteins in HCC cells. (A) Representative Western blotting images of GATA6, BCL-xL, BCL-2, BAX, and -Actin proteins in the cells. (B) Relative expression ratio of the GATA6 gene and protein expression. Molt4 cell was used as a positive control. (C) Relative expression ratios of the BCL-xL, BCL-2, and BAX proteins. The values are expressed as means standard deviations , and N.S., not significant on Student's -test.
图 4 BCL11B 表达对 HCC 细胞中 GATA6 和凋亡相关蛋白变化的影响。(A) 细胞中 GATA6、BCL-xL、BCL-2、BAX 和 -Actin 蛋白的代表性 Western 印迹图像。-Actin蛋白。(B)GATA6 基因和蛋白表达的相对表达比。Molt4 细胞作为阳性对照。(C) BCL-xL、BCL-2 和 BAX 蛋白的相对表达比。数值以均值 标准差 表示。N.S.表示经学生 检验无显著性意义。-检验。
in tumor and non-tumor tissues (Figure 6A,B), and the IHC analysis supported the results in tumor tissue (Figure 6C,D). These findings indicated that GATA6 upregulation in the BCL11B-positive HCC tissue might be related to chemosensitivity to epirubicin hydrochloride, an anthracycline.
图 6A,B),IHC 分析证实了肿瘤组织中的结果(图 6C,D)。这些发现表明,BCL11B阳性HCC组织中GATA6的上调可能与蒽环类药物盐酸表柔比星的化疗敏感性有关。

4 DISCUSSION 4 讨论

Our study demonstrated the influence of the amplified expression of BCL11B upon prognosis. The BCL11B expression in HCC induces resistance to epirubicin hydrochloride, an anthracycline, and results in poor postoperative prognosis. The amplification of GATA6 in BCL11B overexpressed cells and BCL11B-positive HCC tissues might be related to the anthracycline resistance. The mechanisms involved activation of anti-apoptotic proteins, including BCL-xL. It is important to analyze factors involved in anthracycline chemosensitivity, since for intermediate stage HCC, chemoembolization is recommended and anthracycline has been reported to be more effective than other chemicals for chemoembolization. The results obtained in the current study are supported by previous study demonstrating that BCL-xL expression is inhibited in BCL11B knockdown cells and thymocytes of BCL11B knockout mice due to transcriptional repression. A different expression changes between HLE and HepG2 cell lines might be due to the characteristics of the cells, that is, invasive and poorly differentiated HLE cells and noninvasive and hepatoblastoma-oriented HepG2 cells.
我们的研究证明了BCL11B的扩增表达对预后的影响。BCL11B在HCC中的表达会诱导患者对蒽环类药物盐酸表柔比星产生耐药性,并导致术后预后不良。BCL11B过表达细胞和BCL11B阳性HCC组织中GATA6的扩增可能与蒽环类耐药有关。其机制涉及抗凋亡蛋白的激活,包括 BCL-xL。分析蒽环类药物化疗敏感性的相关因素非常重要,因为对于中晚期HCC,建议采用化疗栓塞疗法 ,而据报道,蒽环类药物比其他化学药物的化疗栓塞效果更好。 先前的研究表明,由于转录抑制,BCL11B敲除细胞和BCL11B敲除小鼠胸腺细胞中BCL-xL的表达受到抑制 。HLE细胞系和HepG2细胞系的表达变化不同可能与细胞的特性有关,即HLE细胞具有侵袭性且分化较差,而HepG2细胞具有非侵袭性且以肝母细胞瘤为主。
Furthermore, our study demonstrated the increased expression of GATA6 in BCL11B-overexpressing cells and highly BCL11B-expressing human tissues of HCC and surrounding liver tissues. The GATA family consists of six
此外,我们的研究表明,在 BCL11B 高表达细胞和高表达 BCL11B 的 HCC 人体组织及周围肝组织中,GATA6 的表达量增加。GATA 家族由六个
FIGURE 5 Effect of BCL11B expression on HCC cell growth. Cell growth rate of permanent clones of mocktransfected and BCL11B-overexpressing HCC cell lines determined via WST-1 assay. HLE (A, B) and HepG2 (C, D) cells after culture in the medium with epirubicin hydrochloride (A, C) and cisplatin (B, D). The values are expressed as means standard deviations , triplet with three clones), , **** on Student's -test.
图 5 BCL11B 表达对 HCC 细胞生长的影响。通过 WST-1 试验测定模拟转染和 BCL11B 表达的 HCC 细胞系永久克隆的细胞生长率。HLE(A, B)和 HepG2(C, D)细胞 在含有盐酸表柔比星(A, C)和顺铂(B, D)的培养基中培养后。数值以平均值 标准差 表示。有三个克隆的三倍体)、 、{{4}、{{5}和{{6}。**** 学生{{5}-检验。}-检验。

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Epirubicin Hydrochloride ( )
盐酸表柔比星 ( )
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members (GATA1 to GATA6), which are transcriptional factors with a zinc finger structure. GATA1 to GATA3 are important for differentiation of hematopoietic stem cells and GATA4 to GATA6 are expressed in the various organs including heart, gut, pancreas, lung, and ovary. Among them, the GATA6 gene is transcribed in a pattern overlapping GATA4, and its expression is additionally found in liver tissues. Moreover, it works as an oncogenic factor in various types of tumors, including stomach, pancreatic, and colorectal cancers and
成员(GATA1 至 GATA6),它们是具有锌指结构的转录因子。 GATA1 至 GATA3 对造血干细胞的分化非常重要,而 GATA4 至 GATA6 则在心脏、肠道、胰腺、肺和卵巢等多个器官中表达。 其中,GATA6 基因的转录模式与 GATA4 重叠,而且在肝脏组织中也有表达。 此外,它还是多种肿瘤的致癌因子,包括胃癌、 胰腺癌、 和结肠直肠癌{{5}和{{6}。
Recently, the oncogenic role of GATA6 on HCC has been reported in an in vitro study to counter the tumorsuppressive effect of miR-143. Interestingly, GATA4 and GATA6 have been reported to contribute to cardiac hypertrophy when overexpressed in cardiomyocytes, interacting with numerous cofactors under complex mechanisms. Such mechanisms involve anti-apoptotic signal activation, supported by the findings that overexpression of GATA4 or GATA6 attenuates cardiac muscle cell apoptosis induced by anthracyclines. In addition, GATA4 has been reported to positively regulate anti-apoptotic protein BCL-2. These findings support the results of the present study, which indicated that GATA6 expression in hepatocytes may positively regulate BCL-xL and BCL-2 proteins to attenuate the apoptotic signal with epirubicin hydrochloride; and support HCC growth. The fact that BCL11B is also involved in the mechanisms of the cardiac hypertrophy suggests the potential relationship between BCL11B and GATA6.
最近,一项体外研究报道了 GATA6 对 HCC 的致癌作用,以对抗 miR-143 的抑瘤作用。 有趣的是,有报道称 GATA4 和 GATA6 在心肌细胞中过表达时会导致心肌肥大,并在复杂的机制下与多种辅助因子相互作用。 过表达 GATA4 或 GATA6 可减轻蒽环类药物诱导的心肌细胞凋亡。 此外,据报道 GATA4 对抗凋亡蛋白 BCL-2 有正向调节作用。 这些发现支持了本研究的结果,即 GATA6 在肝细胞中的表达可能正向调节 BCL-xL 和 BCL-2 蛋白,从而减弱盐酸表柔比星的凋亡信号;并支持 HCC 的生长。BCL11B也参与了心肌肥大 的机制,这表明BCL11B和GATA6之间存在潜在的关系。
Regarding the relationship between BCL11B and the GATA family, it has been reported that BCL11B and GATA3 collaborate to play a crucial role in T-cell development by repressing cyclin-dependent kinase inhibitor Moreover, it is known that the BCL11B/ GATA3 complex is involved in this sequence and that BCL11B controls the GATA3-mediated gene activation. These reports support our present results indicating the BCL11B-associated GATA6 expression in HCC which activates anti-apoptotic characteristics and led to the poorer postoperative prognosis than non-BCL11B expressed HCC cases. These findings suggest that BCL11B expression in HCC might be the potential therapeutic target; however, further studies focusing on the direct relationship between BCL11B and GATA6 using Bcl11b conditional knockout mice and its contribution in HCC pathology are essential. In addition, as there are several genes up- and down-regulated by the BCL11B overexpression and involved in the key roles of various oncogenic pathways (Figures 3 and 4; Tables 2 and 3), further analyses are important to explore the therapeutic target in BCL11B-expressing HCC, which is also an important factor in various organs and cells.
关于 BCL11B 与 GATA 家族的关系,有报道称 BCL11B 和 GATA3 相互合作,通过抑制细胞周期蛋白依赖性激酶抑制剂 在 T 细胞发育过程中发挥关键作用。此外,已知 BCL11B/ GATA3 复合物参与了这一序列,并且 BCL11B 控制了 GATA3 介导的基因激活。 这些报道支持了我们目前的研究结果,即BCL11B相关的GATA6在HCC中的表达激活了抗凋亡特性,导致术后预后较无BCL11B表达的HCC病例差。这些研究结果表明,BCL11B 在 HCC 中的表达可能是潜在的治疗靶点;然而,利用 Bcl11b 条件性基因敲除小鼠进一步研究 BCL11B 和 GATA6 之间的直接关系及其在 HCC 病理中的作用至关重要。此外,由于BCL11B过表达导致多个基因上调或下调,并参与了各种致癌通路的关键作用(图3和4;表2和3),因此进一步的分析对于探索BCL11B表达的HCC的治疗靶点非常重要,BCL11B在各种器官和细胞中也是一个重要因素。
The current study had several limitations: The number of cases involved is small, more than of our cases are associated with viral hepatitis. Therefore, based on the results obtained in this study, further analyses for larger number of HCC cases with various clinical stages and on various liver diseases including non-alcoholic fatty liver and autoimmune liver diseases, in a multicenter study is encouraged. With the development of systemic treatment including immunotherapy and molecular target therapy for the advanced stage combination
目前的研究存在一些局限性:本研究涉及的病例数量较少, 以上的 病例与病毒性肝炎有关。因此,基于本研究的结果,我们鼓励在多中心研究中对更多不同临床分期的 HCC 病例和各种肝病(包括非酒精性脂肪肝和自身免疫性肝病) 进行进一步分析。随着晚期 合并症的免疫疗法和分子靶向疗法等全身治疗方法的发展,{{4}.
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BCL11B
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FIG U RE 6 BCL11B and GATA6 expressions in HCC tissues. Relationship between the BCL11B and GATA6 gene expressions in the tissues. (A) non-tumor tissue surrounding HCC and (B) HCC tissue. (C) Representative images of the immunohistochemical staining of BCL11B and GATA6 in HCC tumors. (D) Relationship between the BCL11B- and GATA6-stained areas , , Pearson's correlation test. , correlation coefficient. Continuous lines are the best hit lines, accompanied by dotted lines showing confidence intervals.
图 U RE 6 BCL11B 和 GATA6 在 HCC 组织中的表达。各组织中 BCL11B 和 GATA6 基因表达的关系。(A) HCC 周围的非肿瘤组织,(B) HCC 组织。(C) HCC 肿瘤中 BCL11B 和 GATA6 免疫组化染色的代表性图像。(D) BCL11B 和 GATA6 染色区域 和{{2}之间的关系。, ,皮尔逊相关性检验。 ,相关系数。连续线为最佳命中线,虚线表示 置信区间。
of various agents and chemoembolization is considered to improve the prognosis of Furthermore, the molecular mechanisms for these therapeutic impacts have been reported. For example, recent studies have shown the therapeutic effectiveness of capecitabine when used as a second-line treatment after sorafenib failure by inducing T-cell apoptosis.