CN117187391A - Application of plicamycin in pituitary corticotropin adenoma - Google Patents
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- CN117187391A CN117187391A CN202311194454.2A CN202311194454A CN117187391A CN 117187391 A CN117187391 A CN 117187391A CN 202311194454 A CN202311194454 A CN 202311194454A CN 117187391 A CN117187391 A CN 117187391A
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Abstract
The invention relates to the technical field of biology and medicine, and discloses application of plicamycin in pituitary adrenocorticotropic hormone adenoma. According to the invention, the molecular mechanism of pituitary adrenocorticotropic hormone adenoma generation is studied by taking chromatin openness as an entry point for the first time, an ATAC-seq technology is utilized to draw an open chromatin map of the pituitary adrenocorticotropic hormone adenoma, the obvious difference of the pituitary adrenocorticotropic hormone adenoma and tissue beside the tumor in chromatin openness is revealed, the influence of open chromatin on RNA expression is explored, and further, the transcriptional factor Sp1 regulatory gene and the chromatin openness are found to be obviously increased in the pituitary adrenocorticotropic hormone adenoma. Further, it was demonstrated by cytostatic experiments: the Sp1 inhibitor plicamycin is a potential drug for treating pituitary adrenocorticotropic hormone adenoma. The invention expands the new field of research of the pituitary adrenocorticotropic hormone adenoma in epigenetic science, and provides a new thought and theoretical basis for searching pituitary adrenocorticotropic hormone adenoma treatment targets.
Description
Technical Field
The invention relates to the technical field of biology and medicine, in particular to an application of plicamycin in pituitary adrenocorticotropic hormone adenoma.
Background
Pituitary corticotropin adenomas are a rare endocrine tumor that results in hypersecretion of corticotropin, leading to hyperplasia of the adrenal cortex, hypercortisolism, and in turn, a series of metabolic disorders and pathological changes of the substance. The clinical manifestations of the medicine mainly include cushing's syndrome, including metabolic abnormality, water electrolyte disorder, sexual dysfunction and the like, which seriously reduce the life quality of patients and increase the mortality of the patients. At present, clinical treatment is mainly surgery, but a considerable proportion of patients relapse, and corticotropin hypersecretion always persists after surgery. Although many new drugs are currently developed for treatment, they only can alleviate clinical manifestations in a short time, and can not realize cure of diseases, requiring additional treatment strategies. There is therefore an urgent need to more accurately and comprehensively understand the molecular biology characteristics associated with the development of pituitary corticotropin adenomatogenesis and to develop new therapeutic targets.
Disclosure of Invention
In view of the above, the present invention provides the use of plicamycin in pituitary adrenocorticotropic hormone adenomas. The invention discovers that the transcriptional factor Sp1 regulatory gene and the regional opening degree are obviously increased in pituitary adrenocorticotropic hormone adenoma, and the activity of tumor cells is obviously inhibited after the administration treatment of the mouse pituitary adrenocorticotropic hormone adenoma cell line AtT20 by using the Sp1 inhibitor plicamycin.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the invention provides the use of an agent for detecting the degree of openness of an Sp1 regulatory gene and a region in the preparation of a product for diagnosing pituitary adrenocorticotropic hormone adenoma.
Further, the product is a reagent or a kit.
In a second aspect, the invention provides an agent for diagnosing pituitary adrenocorticotropic hormone adenoma, the agent comprising an agent for detecting Sp1 regulatory genes and the degree of regional openness.
In a third aspect, the invention provides a kit for diagnosing pituitary adrenocorticotropic hormone adenoma, the kit comprising reagents for detecting Sp1 regulatory genes and regional openness.
In a fourth aspect, the invention provides the use of an Sp1 inhibitor in the manufacture of a product for the treatment of pituitary adrenocorticotropic hormone adenoma.
Further, the Sp1 inhibitor is plicamycin.
Further, the concentration of the plicamycin is 50-1000nmol/L.
Further, the product is a medicament or a kit.
In a fifth aspect, the invention provides a medicament for the treatment of pituitary adrenocorticotropic hormone adenoma, the medicament comprising the Sp1 inhibitor plicamycin.
In a sixth aspect, the invention provides a kit for the treatment of pituitary adrenocorticotropic hormone adenoma, the kit comprising the Sp1 inhibitor plicamycin.
Compared with the prior art, the invention has the following advantages:
according to the invention, the molecular mechanism of pituitary adrenocorticotropic hormone adenoma generation is studied by taking chromatin openness as an entry point for the first time, an open chromatin map of the pituitary adrenocorticotropic hormone adenoma is drawn by utilizing an ATAC-seq technology, the obvious difference of the pituitary adrenocorticotropic hormone adenoma and tissues beside the tumor in chromatin openness is revealed, the influence of open chromatin on RNA expression is explored, and further, the regulation gene of a transcription factor Sp1 and the regional openness are found to be obviously increased in the pituitary adrenocorticotropic hormone adenoma. Further, the inhibition experiment on the mouse corticotropin adenoma cell line AtT20 proves that: the Sp1 inhibitor plicamycin is a potential drug for treating pituitary adrenocorticotropic hormone adenoma. The invention expands the new field of research of the pituitary adrenocorticotropic hormone adenoma in epigenetic science, and provides a new thought and theoretical basis for searching pituitary adrenocorticotropic hormone adenoma treatment targets.
Drawings
FIG. 1 is an open chromatin map of human pituitary corticotropin adenomas. (A) A chromatin opening density map of ATG initiation codon. (B) Principal component analysis of open chromatin peaks in tumor tissue and paraneoplastic tissue. (C) The heat map of the peak center signal shows the signal average of all peaks in tumor tissue and paraneoplastic tissue. (D) Distribution statistics of open chromatin and closed chromatin throughout the mRNA transcript range in tumor tissue and paraneoplastic tissue. (E) The histogram shows the number of open and closed chromatin peaks in tumor tissue and paraneoplastic tissue.
FIG. 2 functional analysis of chromatin opening and closing genes in pituitary corticotropin adenomas. (A) The areas of increased chromatin patency in tumor tissue are primarily involved in signal 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 patency in tumor tissue are primarily involved in B cell proliferation regulation, response to glucocorticoids, cAMP degradation processes and calcium ion transport.
FIG. 3 correlation analysis between the degree of gene opening and its expression. (A) The scatter plot shows the correlation between open chromatin peak enrichment (fold enrichment log 2) and RNA abundance (FPKM log 2) for tumor tissue compared to paraneoplastic tissue. (B) functional analysis of expression upregulated genes. (C) functional analysis of expression downregulating genes.
FIG. 4 motif enrichment of the open chromatin region of pituitary corticotropin adenoma. (A) The heat map of the first 50 most enriched open chromatin peaks of the open chromatin region is displayed. (B) motif enrichment of open chromatin regions. (C) motif enrichment of the blocked chromatin region. (D) The pituitary corticotropin adenomatous disease genes USP8, USP48 and POMC gene chromatin opening regions and transcription factors bound thereto are listed.
Figure 5Sp1 inhibitors significantly inhibited proliferation of the mouse pituitary adrenocorticotropic hormone adenoma cell line.
Detailed Description
The technical scheme of the invention is specifically and specifically described below with reference to the embodiment of the invention and the attached drawings. It should be noted that the following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Example 1 preparation and sequencing of ATAC-seq library
We obtained samples from tumor tissues of 9 cushing patients, digested into single cells, followed the previous ATAC-seq library construction method using 1X 10 5 The individual cell particles were subjected to library construction, specifically: the cell particles are resuspended in lysis buffer and after centrifugation, the nuclei are pelleted. The supernatant was discarded and the nuclei resuspended in reaction buffer containing Tn5 transposase and TD buffer. After incubation at 37 ℃ for 30 minutes, DNA was isolated using the MinElute PCR purification kit. The amplified library was then purified using 2×spri purification. Finally, library size was confirmed by LabChip GXII Touch HT and sequencing was performed on the Illumina platform.
Example 2 pituitary corticotropin adenoma chromatin patency mapping
The paired-end ATAC-seq fragment was aligned with human reference genome hg38 using BWA-MEM. PCR duplicates were removed using Picard, reads mapped to mitochondria were discarded, leaving only uniquely mapped paired-end reads with fragment lengths less than 2000bp and mapping quality greater than 30. The ATAC-seq peak region for each sample is called using MACS2, excluding the blacklist region from the peaks called. The open chromatin region from TCGA was converted to hg38 genome by liftdover. To generate a consistent set of unique peaks, a 200bp sliding window and 100bp step size were used to scan across the genome. Adjacent open windows that appear in at least one sample are merged into a set of common open area lists. For each sample, single-ended reads were used for bioinformatics analysis. Sequencing reads from RNA-seq and ATAC-seq were filtered using Trimmomatic, and sequencing quality was assessed using FastQC tools before and after the read filtering step. Next, clean reads were aligned to human reference genomes using the STAR alignment program, using default parameters. Only reads with mapping quality greater than or equal to 20 are retained for subsequent analysis.
The bam file generated from the uniquely mapped reads is used as an input file, callpeak is performed using MACS2 software, and the q-value threshold is set to <0.05. Peaks were annotated using the tanotatepeak function of the ChIPseeker.
Results: the THSS region was found by ATAC-seq to be concentrated mainly within 1KB upstream of the translation initiation codon (ATG) (fig. 1A); differences in chromatin patency were found between tumor tissue and paraneoplastic tissue by unsupervised principal component analysis (fig. 1B); peak levels of tumor tissue were significantly increased compared to paraneoplastic tissue (fig. 1A and 1C); tumor tissues exhibited more up-regulated differential open chromatin peaks (fig. 1D and 1E).
Example 3 differential peak and Gene function analysis
The peak files for each sample were combined using bedtools software. A read count on the peak is then determined for each sample using bedtools multicov. Finally, the differential peak was assessed using DESeq2 and differential gene function analysis was performed by GO database.
Results: GO analysis showed that areas of increased chromatin patency in tumor tissue are mainly involved in cell cycle, DNA damage stimulation, golgi vesicle ubiquitin-mediated protein degradation and small gtpase-mediated signaling etc. signaling pathways (fig. 2A), and the reduced areas are mainly involved in B cell proliferation regulation, response to glucocorticoids, cAMP degradation process and calcium ion transport (fig. 2B).
Example 4 differential peak, expression level and Gene function analysis
Differential gene analysis was performed using the R software package clusterifier, with background genes set as expressed genes for each condition. P values <0.05 were considered statistically significant. And performing differential gene function analysis through a GO database.
Results: data from both ATAC-seq and RNA-seq combined show that 186 open chromatin peaks show up-regulation of gene expression and 128 show down-regulation of expression (fig. 3A); upregulated genes are primarily involved in transmembrane transport, cell differentiation, glutamate receptor signaling pathway and protein kinase c activated G protein coupled receptor signaling pathway (fig. 3B); downregulated genes are involved in the morphogenesis of the epithelium, angiogenesis, cell migration, neuronal death and apoptotic processes, and in the cell proliferation pathway (fig. 3C). Among genes having up-regulated gene expression and increased peak values, MERTK, INSR, MYCL and STK17b are included, and these genes are closely related to the occurrence and development of tumors, suggesting that gene expression regulated by open chromatin plays an important role in pituitary corticotropin adenomas.
Example 5 transcription factor binding motif analysis on differential open chromatin regions
Motif analysis was performed using the findMotifsGenome.pl tool of HOMER. The input files are peak files and genomic fasta files. DNA sequences are extracted from the peak files and compared to a base sequence database to obtain motif information. These motif analyses help to determine the likely DNA binding protein binding sites in the peak region.
Results: 15 transcription factors with the highest enrichment in the region of increased chromatin patency were identified (FIG. 4B), with the motifs NFY, sp1, sp5, KLF3 and Sp2 being most significantly enriched in pituitary corticotropin adenomas, demonstrating a key role in pituitary corticotropin adenogenesis. In addition, sp1 and KLF5 motifs were identified in the open chromatin region around the TSS of pituitary corticotropin adenogenesis associated genes (e.g., USP8, USP48, etc.) (FIG. 4D).
EXAMPLE 6Sp1 inhibitor functional assay
Based on the importance of Sp1 and its regulatory genes in pituitary corticotropin adenomatogenesis, it was analyzed for cellular function. After cell counting the mouse pituitary adrenocorticotropic adenoma cell line AtT20, it was distributed into different wells of 96-well plate culture dishes with 5000 cells per well, at least 6 multiple wells. After overnight attachment of the cells, appropriate concentrations (20-1000 nmol/L) of plicamycin were added. Subsequently, the cells were subjected to CCK-8 activity analysis 48 hours after the administration treatment to measure the cell viability.
Results: at concentrations of 50-1000nmol/L, proliferation activity of tumor cells was significantly inhibited after the plicamycin treatment (FIG. 5). The Sp1 inhibitor plicamycin is shown to be a potential drug for treating pituitary adrenocorticotropic hormone adenoma.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (10)
1. The application of the reagent for detecting the Sp1 regulatory gene and the regional openness in the preparation of the product for diagnosing pituitary adrenocorticotropic hormone adenoma.
2. The use according to claim 1, characterized in that: the product is a reagent or a kit.
3. An agent for diagnosing pituitary adrenocorticotropic hormone adenoma, characterized in that: the reagent comprises a reagent for detecting Sp1 regulatory genes and regional openness.
4. A kit for diagnosing pituitary adrenocorticotropic hormone adenoma, characterized in that: the kit comprises a reagent for detecting Sp1 regulatory genes and regional openness.
Use of an inhibitor of sp1 for the preparation of a product for the treatment of pituitary adrenocorticotropic hormone adenoma.
6. The use according to claim 5, characterized in that: the Sp1 inhibitor is plicamycin.
7. The use according to claim 6, characterized in that: the concentration of the plicamycin is 50-1000nmol/L.
8. The use according to claim 5, characterized in that: the product is a drug or a kit.
9. A medicament for the treatment of pituitary corticotropin adenoma, characterized in that: the medicament comprises the Sp1 inhibitor plicamycin.
10. A kit for treating pituitary corticotropin adenoma, comprising: the kit comprises the Sp1 inhibitor plicamycin.
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