CN117187391A - Application of plicamycin in pituitary adrenocorticotroph adenoma - Google Patents
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Abstract
Description
技术领域Technical field
本发明涉及生物与医药技术领域，具体为普卡霉素在垂体促肾上腺皮质激素腺瘤中的应用。The present invention relates to the field of biological and medical technology, specifically the application of plicamycin in pituitary adrenocorticotroph adenoma.
背景技术Background technique
垂体促肾上腺皮质激素腺瘤是一种罕见的内分泌肿瘤，其导致促肾上腺皮质激素分泌过多，引起肾上腺皮质增生，产生皮质醇增多症，进而导致一系列物质代谢紊乱和病理变化。其临床表现主要为库欣综合征，包括代谢异常、水电解质紊乱及性功能异常等，严重降低患者的生活质量，增加患者死亡率。目前临床治疗主要以手术为主，但相当一部分比例的患者出现复发，且促肾上腺皮质激素分泌过多总是在术后持续存在。虽然目前开发了较多新的药物进行治疗，但它们只能在短时间内缓解临床表现，不能实现疾病的治愈，需要额外的治疗策略。因此迫切需要更准确和全面地理解垂体促肾上腺皮质激素腺瘤发生相关的分子生物学特征，开发新的治疗靶点。Pituitary adrenocorticotroph adenoma is a rare endocrine tumor that leads to excessive secretion of adrenocorticotropic hormone, adrenal cortex hyperplasia, and hypercortisolism, which in turn leads to a series of substance metabolism disorders and pathological changes. The main clinical manifestations are Cushing's syndrome, including metabolic abnormalities, water and electrolyte disorders, and sexual dysfunction, which seriously reduce the patient's quality of life and increase patient mortality. Current clinical treatment is mainly based on surgery, but a considerable proportion of patients experience recurrence, and excessive secretion of adrenocorticotropic hormone always persists after surgery. Although many new drugs have been developed for treatment, they can only alleviate clinical manifestations in a short period of time and cannot cure the disease, requiring additional treatment strategies. Therefore, there is an urgent need to more accurately and comprehensively understand the molecular biology characteristics related to the occurrence of pituitary adrenocorticotroph adenomas and to develop new therapeutic targets.
发明内容Contents of the invention
针对上述问题，本发明提供了普卡霉素在垂体促肾上腺皮质激素腺瘤中的应用。本发明发现转录因子Sp1调控基因及区域开放程度在垂体促肾上腺皮质激素腺瘤中显著上升，在使用Sp1抑制剂普卡霉素对小鼠垂体促肾上腺皮质激素腺瘤细胞系AtT20进行给药处理后，发现肿瘤细胞活性受到明显的抑制。In view of the above problems, the present invention provides the application of plicamycin in pituitary adrenocorticotroph adenoma. The present invention found that the transcription factor Sp1 regulatory gene and the degree of regional opening increased significantly in pituitary adrenocorticotroph adenomas, and the Sp1 inhibitor plicamycin was used to administer the mouse pituitary adrenocorticotroph adenoma cell line AtT20. Afterwards, it was found that tumor cell activity was significantly inhibited.
为了达到上述目的，本发明采用了下列技术方案：In order to achieve the above objects, the present invention adopts the following technical solutions:
第一方面，本发明提供了检测Sp1调控基因及区域开放程度的试剂在制备诊断垂体促肾上腺皮质激素腺瘤产品中的应用。In a first aspect, the present invention provides the use of reagents for detecting the openness of Sp1 regulatory genes and regions in the preparation of products for diagnosing pituitary adrenocorticotroph adenomas.
进一步，所述产品为试剂或试剂盒。Further, the product is a reagent or a kit.
第二方面，本发明提供了一种诊断垂体促肾上腺皮质激素腺瘤的试剂，所述试剂包含检测Sp1调控基因及区域开放程度的试剂。In a second aspect, the present invention provides a reagent for diagnosing pituitary adrenocorticotroph adenoma, which reagent includes a reagent for detecting the Sp1 regulatory gene and the degree of opening of the region.
第三方面，本发明提供了一种诊断垂体促肾上腺皮质激素腺瘤的试剂盒，所述试剂盒包括检测Sp1调控基因及区域开放程度的试剂。In a third aspect, the present invention provides a kit for diagnosing pituitary adrenocorticotroph adenoma, which kit includes reagents for detecting the Sp1 regulatory gene and the degree of openness of the region.
第四方面，本发明提供了Sp1抑制剂在制备治疗垂体促肾上腺皮质激素腺瘤产品中的应用。In a fourth aspect, the present invention provides the use of Sp1 inhibitors in the preparation of products for treating pituitary adrenocorticotroph adenomas.
进一步，所述Sp1抑制剂为普卡霉素。Further, the Sp1 inhibitor is plicamycin.
进一步，所述普卡霉素的浓度为50-1000nmol/L。Further, the concentration of plicamycin is 50-1000 nmol/L.
进一步，所述产品为药物或试剂盒。Further, the product is a medicine or a kit.
第五方面，本发明提供了一种用于治疗垂体促肾上腺皮质激素腺瘤的药物，所述药物包含Sp1抑制剂普卡霉素。In a fifth aspect, the present invention provides a medicament for treating pituitary adrenocorticotroph adenoma, the medicament comprising the Sp1 inhibitor plicamycin.
第六方面，本发明提供了一种用于治疗垂体促肾上腺皮质激素腺瘤的试剂盒，所述试剂盒包括Sp1抑制剂普卡霉素。In a sixth aspect, the present invention provides a kit for treating pituitary adrenocorticotroph adenoma, which kit includes the Sp1 inhibitor plicamycin.
与现有技术相比本发明具有以下优点：Compared with the prior art, the present invention has the following advantages:
本发明首次以染色质开放性为切入点研究垂体促肾上腺皮质激素腺瘤发生的分子机制，利用ATAC-seq技术绘制了垂体促肾上腺皮质激素腺瘤的开放染色质图谱，揭示了垂体促肾上腺皮质激素腺瘤与瘤旁组织在染色质开放性的显著差异，探究了开放染色质对于RNA表达的影响，进而发现转录因子Sp1调控基因及区域开放程度在垂体促肾上腺皮质激素腺瘤中显著升高。进一步，通过对小鼠促肾上腺皮质激素腺瘤细胞系AtT20进行抑制实验证明：Sp1抑制剂普卡霉素是垂体促肾上腺皮质激素腺瘤治疗的潜在药物。本发明拓展了垂体促肾上腺皮质激素腺瘤在表观遗传学研究的新领域，为寻找垂体促肾上腺皮质激素腺瘤治疗靶点提供了新的思路和理论依据。For the first time, the present invention uses chromatin openness as an entry point to study the molecular mechanism of pituitary adrenocorticotroph adenoma. It uses ATAC-seq technology to draw an open chromatin map of pituitary adrenocorticotroph adenoma, revealing the pituitary adrenocorticotropic hormone There is a significant difference in chromatin openness between hormonal adenomas and paratumoral tissues. The impact of open chromatin on RNA expression was explored. It was then found that the transcription factor Sp1 regulated genes and the degree of regional openness was significantly increased in pituitary adrenocorticotroph adenomas. . Furthermore, inhibition experiments on the mouse ACTH adenoma cell line AtT20 demonstrated that the Sp1 inhibitor plicamycin is a potential drug for the treatment of pituitary ACTH adenomas. The present invention expands the new field of epigenetic research on pituitary adrenocorticotroph adenoma, and provides new ideas and theoretical basis for finding treatment targets for pituitary adrenocorticotroph adenoma.
附图说明Description of the drawings
图1人类垂体促肾上腺皮质激素腺瘤的开放染色质图谱。(A)ATG起始密码子的染色质开放性密度图。(B)肿瘤组织和瘤旁组织的开放染色质峰值的主成分分析。(C)峰值中心信号的热图显示出肿瘤组织和瘤旁组织中所有峰值的信号平均值。(D)在肿瘤组织和瘤旁组织中，整个mRNA转录本范围内的开放染色质和封闭染色质的分布统计。(E)柱状图显示肿瘤组织和瘤旁组织中开放和封闭染色质峰值的数量。Figure 1 Open chromatin map of human pituitary corticotroph adenomas. (A) Chromatin openness density map of the ATG start codon. (B) Principal component analysis of open chromatin peaks in tumor tissue and paratumor tissue. (C) Heat map of the peak center signal showing the signal average of all peaks in tumor tissue and paratumoral tissue. (D) Distribution statistics of open chromatin and closed chromatin within the entire mRNA transcript range in tumor tissue and paratumor tissue. (E) Histogram showing the number of open and closed chromatin peaks in tumor tissue and paratumor tissue.
图2垂体促肾上腺皮质激素腺瘤中染色质开放和封闭基因的功能分析。(A)肿瘤组织中染色质开放性增加的区域主要涉及细胞周期、DNA损伤刺激、高尔基囊泡泛素介导的蛋白降解和小GTP酶介导的信号传导等信号通路。(B)肿瘤组织中染色质开放性降低的区域主要涉及B细胞增殖调节、对糖皮质激素的反应、cAMP降解过程和钙离子转运。Figure 2 Functional analysis of chromatin open and closed genes in pituitary adrenocorticotroph adenomas. (A) Areas with increased chromatin openness in tumor tissues are mainly involved in signaling pathways such as cell cycle, DNA damage stimulation, Golgi vesicle ubiquitin-mediated protein degradation, and small GTPase-mediated signaling. (B) The areas of reduced chromatin openness in tumor tissues are mainly involved in the regulation of B cell proliferation, response to glucocorticoids, cAMP degradation process, and calcium ion transport.
图3基因开放程度与其表达之间的相关性分析。(A)散点图显示肿瘤组织与瘤旁组织相比，开放染色质峰值富集(富集倍数log2)与RNA丰度(FPKM log2)之间的相关性。(B)表达上调基因的功能分析。(C)表达下调基因的功能分析。Figure 3 Correlation analysis between gene openness and expression. (A) Scatter plot showing the correlation between open chromatin peak enrichment (enrichment fold log2) and RNA abundance (FPKM log2) in tumor tissue compared with paratumoral tissue. (B) Functional analysis of genes with up-regulated expression. (C) Functional analysis of down-regulated genes.
图4垂体促肾上腺皮质激素腺瘤开放染色质区域的基序富集。(A)开放染色质区域的前50个最富集的开放染色质峰的热图展示。(B)开放染色质区域的基序富集情况。(C)封闭染色质区域的基序富集情况。(D)列举垂体促肾上腺皮质激素腺瘤致病基因USP8,USP48和POMC基因染色质开放区域及其结合的转录因子。Figure 4 Motif enrichment of open chromatin regions in pituitary adrenocorticotroph adenomas. (A) Heatmap representation of the top 50 most enriched open chromatin peaks for open chromatin regions. (B) Motif enrichment in open chromatin regions. (C) Motif enrichment of closed chromatin regions. (D) List of open chromatin regions of pituitary adrenocorticotroph adenoma-causing genes USP8, USP48 and POMC genes and their binding transcription factors.
图5Sp1抑制剂显著抑制小鼠垂体促肾上腺皮质激素腺瘤细胞系的增殖情况。Figure 5 Sp1 inhibitor significantly inhibits the proliferation of mouse pituitary adrenocorticotroph adenoma cell line.
具体实施方式Detailed ways
下面结合本发明实施例和附图，对本发明的技术方案进行具体、详细的说明。应当指出，以下实施例仅用于说明本发明，不用来限制本发明的保护范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。The technical solution of the present invention will be described in detail below with reference to the embodiments of the present invention and the accompanying drawings. It should be noted that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
实施例1ATAC-seq文库的制备和测序Example 1 Preparation and sequencing of ATAC-seq library
我们从9位库欣病患者的肿瘤组织中获得样本，消化成单个细胞后，遵循先前的ATAC-seq文库构建方法，使用1×105个细胞颗粒进行文库构建，具体为：细胞颗粒在裂解缓冲液中重新悬浮，经离心后，细胞核沉淀。废弃上清液，将细胞核重新悬浮于包含Tn5转座酶和TD缓冲液的反应缓冲液中。在37℃孵育30分钟后，利用MinElute PCR纯化试剂盒分离DNA。接着使用2×SPRI纯化扩增和纯化文库。最后，借助LabChip GXII Touch HT确认文库大小，并在Illumina平台上进行测序。We obtained samples from tumor tissues of 9 patients with Cushing's disease. After digesting them into single cells, we followed the previous ATAC-seq library construction method and used 1×10 5 cell pellets for library construction. The specifics are: the cell pellets were lysed Resuspend in buffer and centrifuge to pellet the nuclei. Discard the supernatant and resuspend the nuclei in reaction buffer containing Tn5 transposase and TD buffer. After incubation at 37°C for 30 minutes, DNA was isolated using the MinElute PCR purification kit. The amplified and purified library was then purified using 2×SPRI. Finally, the library size was confirmed with LabChip GXII Touch HT and sequenced on the Illumina platform.
实施例2垂体促肾上腺皮质激素腺瘤染色质开放性图谱绘制Example 2 Drawing of open chromatin map of pituitary adrenocorticotroph adenoma
使用BWA-MEM将配对末端ATAC-seq片段与人类参考基因组hg38进行比对。使用Picard去除PCR重复，映射到线粒体的读段被丢弃，只保留片段长度小于2000bp且映射质量大于30的唯一映射配对末端读段。使用MACS2调用每个样本的ATAC-seq峰值区域，从调用的峰值中排除黑名单区域。将来自TCGA的开放染色质区域通过liftOver转换为hg38基因组。为了生成一组一致的唯一峰值，使用200bp滑动窗口和100bp步长在基因组上进行扫描。在至少一个样本中出现的相邻开放窗口被合并为一组共识开放区域列表。对于每个样本，单端读段被用于生物信息学分析。使用Trimmomatic对来自RNA-seq和ATAC-seq的测序读段进行过滤，在读段过滤步骤之前和之后，使用FastQC工具评估测序质量。接下来，使用STAR比对程序将干净的读段与人类参考基因组进行比对，使用默认参数。只保留映射质量大于等于20的读段，以供后续分析使用。Paired-end ATAC-seq fragments were aligned to the human reference genome hg38 using BWA-MEM. PCR duplicates were removed using Picard, and reads mapping to mitochondria were discarded, leaving only uniquely mapped paired-end reads with fragment lengths less than 2000 bp and mapping quality greater than 30. Use MACS2 to call the ATAC-seq peak region of each sample, excluding blacklisted regions from the called peaks. Open chromatin regions from TCGA were converted to the hg38 genome by liftOver. To generate a consistent set of unique peaks, scans were performed across the genome using a 200bp sliding window and a 100bp step size. Adjacent open windows that occurred in at least one sample were combined into a set of consensus open area lists. For each sample, single-end reads were used for bioinformatic analysis. Sequencing reads from RNA-seq and ATAC-seq were filtered using Trimmomatic, and sequencing quality was assessed using the FastQC tool before and after the read filtering step. Next, clean reads were aligned to the human reference genome using the STAR alignment program, using default parameters. Only reads with mapping quality greater than or equal to 20 are retained for subsequent analysis.
由唯一映射的读段生成的bam文件作为输入文件，使用MACS2软件进行callpeak，设置q值阈值<0.05。使用ChIPseeker的annotatePeak功能对峰值进行注释。The bam file generated from the uniquely mapped reads was used as the input file, and MACS2 software was used for callpeak, setting the q value threshold to <0.05. Peaks were annotated using ChIPseeker's annotatePeak function.
结果：通过ATAC-seq发现THSS区域主要集中在翻译起始密码子(ATG)上游1KB内(图1A)；通过无监督主成分分析发现肿瘤组织和瘤旁组织的染色质开放性存在差异(图1B)；与瘤旁组织相比，肿瘤组织的峰值水平显著增加(图1A和1C)；肿瘤组织表现出更多的上调差异性开放染色质峰值(图1D和1E)。Results: Through ATAC-seq, it was found that the THSS region is mainly concentrated within 1KB upstream of the translation start codon (ATG) (Figure 1A); through unsupervised principal component analysis, it was found that there are differences in the chromatin openness of tumor tissue and paratumor tissue (Figure 1A) 1B); compared with paratumor tissue, the peak levels in tumor tissue were significantly increased (Figures 1A and 1C); tumor tissues showed more up-regulated differential open chromatin peaks (Figures 1D and 1E).
实施例3差异峰值和基因功能分析Example 3 Difference peaks and gene function analysis
使用bedtools软件合并每个样本的峰值文件。接着使用bedtools multicov为每个样本确定峰值上的读取计数。最后，使用DESeq2评估差异峰值，通过GO数据库进行差异基因功能分析。Use bedtools software to merge peak files for each sample. The read count at the peak was then determined for each sample using bedtools multicov. Finally, DESeq2 was used to evaluate differential peaks and differential gene function analysis was performed through the GO database.
结果：GO分析显示，肿瘤组织中染色质开放性增加的区域主要涉及细胞周期、DNA损伤刺激、高尔基囊泡泛素介导的蛋白降解和小GTP酶介导的信号传导等信号通路(图2A)，降低的区域主要涉及B细胞增殖调节、对糖皮质激素的反应、cAMP降解过程和钙离子转运(图2B)。Results: GO analysis showed that areas with increased chromatin openness in tumor tissues were mainly involved in signaling pathways such as cell cycle, DNA damage stimulation, Golgi vesicle ubiquitin-mediated protein degradation, and small GTPase-mediated signaling (Figure 2A ), the reduced areas mainly involve B cell proliferation regulation, response to glucocorticoids, cAMP degradation process and calcium ion transport (Figure 2B).
实施例4差异峰值、表达量和基因功能分析Example 4 Analysis of differential peak values, expression levels and gene functions
使用R软件包clusterProfiler进行了差异基因分析，背景基因设定为每个条件下的表达基因。P值<0.05被视为统计学显著。通过GO数据库进行差异基因功能分析。Differential gene analysis was performed using the R package clusterProfiler, and the background genes were set as the expressed genes under each condition. A P value <0.05 was considered statistically significant. Differential gene function analysis was performed through the GO database.
结果：通过结合ATAC-seq和RNA-seq的数据显示，186个开放染色质峰值显示出基因表达上调，128个显示出表达下调(图3A)；上调的基因主要涉及跨膜转运、细胞分化、谷氨酸受体信号通路和蛋白激酶c激活的G蛋白偶联受体信号通路(图3B)；下调的基因涉及上皮的形态生成、血管生成、细胞迁移、神经元死亡和凋亡过程，以及细胞增殖通路(图3C)。在具有上调基因表达和增加峰值的基因中，包括MERTK、INSR、MYCL和STK17b，这些基因与肿瘤的发生和发展有着密切联系，表明由开放染色质调节的基因表达在垂体促肾上腺皮质激素腺瘤中发挥了重要作用。Results: Combining ATAC-seq and RNA-seq data showed that 186 open chromatin peaks showed up-regulation of gene expression, and 128 showed down-regulation of expression (Figure 3A); the up-regulated genes were mainly involved in transmembrane transport, cell differentiation, Glutamate receptor signaling pathway and protein kinase c-activated G protein-coupled receptor signaling pathway (Figure 3B); down-regulated genes are involved in epithelial morphogenesis, angiogenesis, cell migration, neuronal death and apoptosis processes, and Cell proliferation pathway (Figure 3C). Among the genes with upregulated gene expression and increased peaks, including MERTK, INSR, MYCL, and STK17b, these genes are closely related to tumor initiation and progression, indicating that gene expression regulated by open chromatin is important in pituitary adrenocorticotroph adenomas. played an important role.
实施例5差异开放染色质区域上的转录因子结合基序分析Example 5 Analysis of transcription factor binding motifs on differentially open chromatin regions
使用HOMER的findMotifsGenome.pl工具进行基序分析。输入文件为峰值文件和基因组fasta文件。根据峰值文件提取DNA序列，并与基序数据库进行比较，以获得基序信息。这些基序分析有助于确定峰值区域中可能的DNA结合蛋白结合位点。Motif analysis was performed using HOMER's findMotifsGenome.pl tool. The input files are peak files and genome fasta files. DNA sequences are extracted based on peak files and compared with the motif database to obtain motif information. These motif analyzes help identify possible DNA-binding protein binding sites in the peak region.
结果：鉴定出15个在染色质开放性增加区域中富集程度最高的转录因子(图4B)，其中NFY、Sp1、Sp5、KLF3和Sp2的基序在垂体促肾上腺皮质激素腺瘤中富集最为显著，证明其在垂体促肾上腺皮质激素腺瘤发生中发挥关键作用。此外，在垂体促肾上腺皮质激素腺瘤发生相关基因(如USP8、USP48等)的TSS周围的开放染色质区域中鉴定出了Sp1和KLF5基序(图4D)。Results: 15 transcription factors were identified that were most enriched in regions of increased chromatin openness (Figure 4B), among which the motifs of NFY, Sp1, Sp5, KLF3, and Sp2 were enriched in pituitary adrenocorticotroph adenomas. Most notably, it has been shown to play a key role in the development of pituitary adrenocorticotroph adenomas. In addition, Sp1 and KLF5 motifs were identified in open chromatin regions around the TSS of genes involved in pituitary adrenocorticotroph adenomatogenesis (such as USP8, USP48, etc.) (Fig. 4D).
实施例6Sp1抑制剂功能分析Example 6 Sp1 inhibitor functional analysis
基于Sp1及其调节基因在垂体促肾上腺皮质激素腺瘤发生中的重要性，对其进行细胞功能分析。将小鼠垂体促肾上腺皮质激素腺瘤细胞系AtT20进行细胞计数后，分配到96孔板培养皿的不同孔中，每孔5000个细胞，至少6个复孔。细胞过夜贴壁后，添加适当浓度(20-1000nmol/L)的普卡霉素。随后，在给药处理的48小时后对细胞进行CCK-8活性分析，来测量细胞的活率。Based on the importance of Sp1 and its regulatory genes in the occurrence of pituitary adrenocorticotroph adenomas, cell function analysis was performed. After counting the cells of the mouse pituitary adrenocorticotroph adenoma cell line AtT20, they were distributed into different wells of a 96-well plate culture dish, with 5,000 cells per well and at least 6 duplicate wells. After the cells adhere overnight, add plicamycin at an appropriate concentration (20-1000 nmol/L). Subsequently, the cells were analyzed for CCK-8 activity 48 hours after the drug treatment to measure the cell viability.
结果：在浓度为50-1000nmol/L内，普卡霉素处理后，肿瘤细胞的增殖活性明显受到抑制(图5)。表明Sp1抑制剂普卡霉素是垂体促肾上腺皮质激素腺瘤治疗的潜在药物。Results: Within the concentration range of 50-1000 nmol/L, the proliferation activity of tumor cells was significantly inhibited after treatment with plicamycin (Figure 5). It is suggested that the Sp1 inhibitor plicamycin is a potential drug for the treatment of pituitary adrenocorticotroph adenomas.
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。In addition, it should be understood that although this specification is described in terms of implementations, not each implementation only contains an independent technical solution. This description of the specification is only for the sake of clarity, and those skilled in the art should take the specification as a whole. , the technical solutions in each embodiment can also be appropriately combined to form other implementations that can be understood by those skilled in the art.
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