Use of jun in diagnosis and treatment of functional pituitary adrenocorticotrophic hormone adenoma and hormone hypersecretion

Abstract

The application discloses application of JUN in diagnosis of functional pituitary adrenocorticotropic hormone adenoma and treatment of hormone hypersecretion of the adenoma. It is found that JUN is significantly reduced in expression in the functional pituitary adrenocorticotropic hormone adenoma, has binding and regulation on POMC and ACTH, and a plasmid with overexpression of JUN has a significant inhibitory effect on hormone secretion.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to the application of JUN in the diagnosis of functional pituitary adrenocorticotropic hormone adenoma and the treatment of excessive hormone secretion in such adenoma. Background Technology

[0002] Functional pituitary adrenocorticotropic hormone (ACTH) adenoma is a rare endocrine tumor that causes excessive secretion of ACTH, leading to adrenal hyperplasia and Cushing's syndrome. Cushing's disease, caused by this adenoma, is a severe endocrine disorder resulting from long-term, autonomous, and excessive cortisol secretion. Patients with this condition have a standardized mortality rate 2-4 times higher than normal, and their quality of life is severely reduced. The core cause of the increased mortality is the multi-system damage caused by chronic hypercortisolism, including: metabolic syndrome characterized by systemic hypertension, visceral obesity, glucose metabolism disorders, and dyslipidemia; electrolyte imbalances; myopathy, osteoporosis, and musculoskeletal disorders such as fractures; cognitive impairment; neuropsychiatric disorders such as depression or mania; reproductive and sexual dysfunction; and skin manifestations such as acne, hirsutism, and alopecia. Late-stage patients often die from complications such as severe infections and cardiovascular diseases.

[0003] Treatment options for functional pituitary ACTH adenomas include surgery, radiation therapy, and medication. However, even with standardized treatment, approximately 30% of patients with primary ACTH-PA still experience persistent high ACTH secretion post-surgery, and the remission rate for recurrent patients is only 50%. Excessive ACTH secretion leads to persistent hypercortisolism, inducing severe metabolic disorders, cardiovascular events, and infections, resulting in high mortality and disability rates. Currently, medications for pituitary ACTH adenomas (Cushing's disease) are mainly divided into pituitary-targeted drugs (such as parrevitide) and adrenal-targeted drugs (such as oxozostel and ketoconazole). While pituitary-targeted drugs attempt to inhibit ACTH secretion from the tumor at its source, parrevitide has an efficacy rate of only about 40%-50%, and a high percentage (70%-80%) of patients experience severe side effects such as hyperglycemia. Although adrenal-targeted drugs can control cortisol levels relatively quickly, oxozostel faces the risk of discontinuation due to the manufacturer's withdrawal from the Chinese market, and ketoconazole and other drugs are used off-label, severely limiting their clinical application. Overall, existing drugs generally suffer from insufficient efficacy, significant side effects, or difficulty in obtaining them for commercial reasons, and they are difficult to achieve long-term stable hormone level control, which falls far short of meeting clinical needs.

[0004] Therefore, there is an urgent need to develop new therapies for functional pituitary ACTH adenomas that can effectively inhibit the secretion of pituitary ACTH hormones. Summary of the Invention

[0005] Based on this, one embodiment of this application provides the use of JUN in the preparation of a medicament for treating excessive secretion of functional pituitary adrenocorticotropic hormone adenoma.

[0006] The specific technical solution of this application includes:

[0007] Application of JUN in the preparation of drugs for the treatment of excessive secretion of functional pituitary adrenocorticotropic hormone adenoma.

[0008] Application of JUN in the preparation of drugs for the prevention or treatment of diseases related to excessive secretion of functional pituitary adrenocorticotropic hormone (ACTH) adenoma hormone.

[0009] In some embodiments, the associated disease includes hypercortisolism. In some embodiments, the associated disease includes Cushing's disease.

[0010] In some implementations, JUN is overexpressed to treat excessive secretion of functional pituitary adrenocorticotropic hormone (ACTH) adenoma hormone.

[0011] A drug for treating excessive secretion of functional pituitary adrenocorticotropic hormone (JUN) adenoma, said drug comprising a substance that overexpresses JUN.

[0012] In some embodiments, the drug also includes pharmaceutically acceptable excipients.

[0013] Application of reagents for detecting JUN expression levels in the preparation of products for the identification or diagnosis of functional pituitary adrenocorticotropic hormone adenomas.

[0014] In some implementations, JUN is expressed at a lower level in patients with functional pituitary adrenocorticotropic hormone adenomas compared to non-functional pituitary adenomas.

[0015] In some embodiments, the detection reagent is selected from one or more of the following methods: sequencing, PCR, liquid chromatography, mass spectrometry, and immunoassay.

[0016] In some embodiments, the immunoassay includes one or more of Western blot analysis, radioimmunoassay, immunofluorescence assay, immunoprecipitation, immunodiffusion, electrochemiluminescence immunoassay, ELISA assay, and immunopolymerase chain reaction.

[0017] In some embodiments, the detection reagent comprises a specific antibody against JUN or a functional fragment thereof.

[0018] In some embodiments, the product includes one or more of the following: a chip, a reagent kit, a reagent, a test strip, and a device.

[0019] A diagnostic or screening system or device for functional pituitary adrenocorticotropic hormone-secreting adenomas, characterized in that it comprises:

[0020] The result determination unit is used to analyze the level of markers in the detected samples and output the results, wherein the markers include JUN.

[0021] In some implementations, the sample is selected from tissue or blood.

[0022] In some implementations, a detection unit is also included for detecting the level of the marker in the sample.

[0023] In some implementations, the result determination unit includes an input section for inputting the level of the marker.

[0024] In some embodiments, the result determination unit further includes an analysis section for analyzing, based on the level of the biomarker, whether the subject has a functional pituitary adrenocorticotropic hormone adenoma or a non-functional pituitary adenoma (such as a silent pituitary adenoma).

[0025] In some embodiments, the result determination unit further includes an output section for outputting the analysis results of the analysis section.

[0026] A method for the diagnosis or auxiliary diagnosis of functional pituitary adrenocorticotropic hormone adenoma, the method comprising:

[0027] (1) Determine the expression level of JUN in the subjects' samples;

[0028] (2) The difference in JUN expression levels between the subjects and the control group with non-functional pituitary adenoma was used to determine whether the subjects belonged to functional pituitary adrenocorticotropic hormone adenoma or non-functional pituitary adenoma.

[0029] In some implementations, a significantly reduced level of JUN protein expression compared to a non-functional pituitary adenoma control indicates that the subject has a functional pituitary adrenocorticotropic hormone adenoma.

[0030] Compared with the prior art, the beneficial effects of this application are as follows:

[0031] This application experimentally revealed that JUN expression is significantly reduced in functional pituitary adrenocorticotropic hormone (ACTH) adenomas. JUN binds to and regulates POMC and ACTH, and its overexpression plasmid significantly inhibits hormone secretion. JUN and its corresponding target genes can help researchers gain a deeper understanding of the pathological role of pituitary adenomas, contributing to the theoretical study of ACTH adenomas. Based on this, a new therapeutic strategy has been discovered, providing a new direction for controlling excessive hormone secretion in functional pituitary ACTH adenomas. Attached Figure Description

[0032] Figure 1 1. The pseudo-time series analysis defines the different states of secretory and non-secreting epithelial cells; (AC) Transition trajectory of active secretory cells or quiescent cells in functional pituitary ACTH adenomas (A&B) and quiescent pituitary ACTH adenomas (C); (D) Gene clusters and main functions of pseudo-time series changes in different states; (E) List of marker genes highly expressed in pseudo-time series changes in different states; (F) Representative marker genes of pseudo-time series changes in different states; (G) Taking the intersection of pseudo-time series and SCENIC analysis to obtain the key transcription factors for the transition from quiescent (prebrunch) to active secretory (State 1) cell states; (H) ROC curve of JUN expression in epithelial cells of NF adenoma and ACTH adenoma samples.

[0033] Figure 2 Comparison of RNA expression in invasive and non-invasive functional pituitary ACTH adenomas and silent pituitary adenomas; (AC) Expression of POMC, TBX19, and JUN in invasive and non-invasive functional ACTH samples; (D) ROC curves of JUN expression in ACTH and SCA sample sets from the database; ACTH, functional pituitary ACTH adenoma; iACTH, invasive ACTH, invasive functional pituitary ACTH adenoma; noni-ACTH, non-invasive ACTH, non-invasive functional pituitary ACTH adenoma; SCA, silent pituitary adenoma.

[0034] Figure 3 Comparison of JUN protein localization and expression in invasive and non-invasive functional pituitary ACTH adenomas and silent pituitary adenomas; (A) Immunofluorescence images of JUN expression in tissues of invasive and non-invasive functional pituitary ACTH adenomas and silent pituitary adenomas; (B) Statistical graph of immunofluorescence; (C) Statistical analysis of ROC curves of immunofluorescence intensity; (D) Changes in the expression of JUN, POMC, and ACTH after overexpression of JUN in AtT20 cells; (E) Changes in ACTH levels in pituitary ACTH adenoma cells after JUN overexpression; EV, empty vector; JUN, overexpression.

[0035] Figure 4 Effects of JUN overexpression on ACTH secretion in a mouse tumorigenesis model; (AB) Tumor photographs (A) and tumor weight statistics (B) of mice with JUN overexpression; (CD) Body shape photographs (C) and weight statistics (D) of mice without tumors, mice with tumors formed using the empty vector, and mice with tumors formed using JUN overexpression; (E) ACTH levels in mice without tumors, mice with tumors formed using the empty vector, and mice with tumors formed using JUN overexpression. EV, empty vector; JUN, overexpression.

[0036] Figure 51. Interaction and regulation analysis of JUN and POMC in ACTH cells; (AC) JUNCUTTAG analysis of pituitary ACTH tumor tissue: motif (A), enriched gene function (B), gene IGV display (C); (D) Dual-fluorescence quantitative analysis of the binding interaction and regulation of JUN and POMC promoter regions in 293T cells; Renilla, fluorescein; Vector+pGL3-Basic+Renilla, empty vector transfection + fluorescein; Vector+POMC-Promoter+Renilla, empty vector and POMC overexpression + fluorescein; c-JUN+POMC-Promoter+Renilla, cJUN overexpression and POMC overexpression + fluorescein. Detailed Implementation

[0037] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present application in any way.

[0038] In this article, pituitary adrenocorticotropic hormone (ACTH) adenomas include functional pituitary ACTH adenomas (also known as active pituitary ACTH adenomas) and non-functional silent pituitary ACTH adenomas (abbreviated as silent pituitary adenomas, SCAs). Functional ACTH adenomas are adenomas originating from adrenocorticotropic hormone (ACTH) cells in the anterior pituitary gland. They have the autonomous, excessive secretion of ACTH, causing hypercortisolism through continuous stimulation of the adrenal cortex, ultimately leading to the clinical manifestations of Cushing's disease. Non-functional silent pituitary ACTH adenomas are a subtype of pituitary adenomas. Their tumor cells originate from adrenocorticotropic hormone (ACTH) cells in the anterior pituitary gland, and immunohistochemistry shows ACTH positivity. However, the tumor does not clinically cause significant excessive ACTH secretion, and patients typically do not exhibit hypercortisolism or Cushing's disease; they are classified as non-functional pituitary adenomas.

[0039] Clinically, a special type of silent ACTH adenoma (SCA) exists, accounting for approximately 20% of ACTH tumors. Although these tumors express the ACTH lineage transcription factor TPIT, they do not secrete hormones into the bloodstream. Notably, some silent ACTH adenomas can undergo functional transformation, transitioning to an active hormone-secreting state, thus leading to Cushing's disease. After a silent ACTH adenoma transforms into a functional ACTH adenoma, the high hormone levels are more likely to cause serious systemic complications; blocking this mechanism can lower hormone levels. This phenomenon suggests that ACTH adenomas undergo a "silent-to-active" secretory state transition, and elucidating this transition mechanism may lead to the discovery of key molecular switches regulating hormone secretion.

[0040] POMC is a precursor gene of ACTH protein. The inventors, through single-cell analysis and bulk RNA-seq, discovered a significant negative correlation between JUN and POMC expression, suggesting that JUN has a negative regulatory effect on hormone secretion. Further pathological studies confirmed that JUN is significantly underexpressed in functional pituitary adrenocorticotropic hormone (ACTH) adenomas, potentially serving as a marker gene for these adenomas. Furthermore, cell and animal experiments showed that ACTH secretion was significantly inhibited after JUN overexpression. This suggests that JUN (gene reference NCBI Gene (Entrez): 3725, protein reference UniProt ID: P05412) is a potential therapeutic target for pituitary ACTH adenomas.

[0041] Therefore, this application proposes the use of reagents for detecting JUN expression levels in the preparation of diagnostic or auxiliary diagnostic kits for functional pituitary adrenocorticotropic hormone adenomas, and proposes a novel use of JUN overexpression in functional pituitary adrenocorticotropic hormone adenomas to inhibit hormone secretion.

[0042] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0043] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0044] Sample collection in the following examples:

[0045] Fresh pituitary adenoma tissues removed via sphenoid sinus surgery at the Department of Neurosurgery, Peking Union Medical College Hospital, from January 2015 to December 2019 were collected. (1) Twelve cases of functional pituitary adrenocorticotropic hormone (ACTH) adenomas (preoperatively diagnosed as Cushing's disease, postoperative pathology and immunohistochemistry showed positive ACTH) and eleven cases of nonfunctional pituitary adenomas (preoperatively without hypersecretion of hormones, postoperative immunohistochemistry showed positive ACTH) were collected for single-cell transcriptome analysis. (2) Six cases of functional pituitary adrenocorticotropic hormone (ACTH) adenomas (preoperatively diagnosed as Cushing's disease, postoperative pathology and immunohistochemistry showed positive ACTH and TPIT) and three cases of silent pituitary adenomas (preoperatively without hypersecretion of hormones, postoperative immunohistochemistry showed positive ACTH and TPIT) were collected and rapidly frozen in liquid nitrogen for bulk RNA-seq. (3) Nineteen cases of functional pituitary adrenocorticotropic hormone (ACTH) adenomas (preoperatively diagnosed as Cushing's disease, postoperative pathology and immunohistochemistry positive for ACTH and TPIT) and nine cases of silent pituitary adenomas (preoperatively without hypersecretion of hormones, postoperative pathology and immunohistochemistry positive for ACTH and TPIT, belonging to the same lineage as ACTH adenomas, a type of non-functional pituitary adenoma) were collected, fixed, embedded, and used for immunological verification. This study was approved by the Ethics Committee of Peking Union Medical College Hospital (approval number: S-K477), and all patients signed informed consent forms.

[0046] Example 1: Single-cell transcriptome sequencing

[0047] Library Construction: Twelve freshly resected functional pituitary adrenocorticotropic hormone-secreting adenoma (ACTH-PA) tissue samples and eleven non-functional pituitary adenoma (NF-PA) control tissue samples were obtained and transported to the laboratory on ice in centrifuge tubes containing tissue preservation solution. The tissue samples were placed in sterile culture dishes and washed three times with pre-cooled phosphate-buffered saline to remove surface blood and impurities. Subsequently, the tissues were minced using sterile surgical scissors into small pieces approximately 1-2 mm³. The shredded tissue pieces were transferred to a tissue dissociation enzyme mixture containing collagenase I, collagenase IV, hyaluronidase, and DNase I. Digestion was performed at 37°C in a water bath or a constant-temperature shaker for 30-60 minutes, with gentle pipetting every 10 minutes. After digestion, the reaction was terminated by adding culture medium containing fetal bovine serum. The filtrate was collected by filtering through 70μm and 40μm cell sieves, centrifuged at 300×g for 5 minutes at 4°C, and the supernatant was discarded. The cell pellet was resuspended in PBS. If red blood cell contamination was severe, red blood cell lysis buffer was added and incubated on ice for 5 minutes, followed by centrifugation and washing. Cell viability and concentration were assessed using trypan blue staining, with a viability ≥85%. The single-cell suspension was diluted to an appropriate concentration (e.g., 700-1200 cells / μL), and a microfluidic single-cell capture system was used to encapsulate single cells with gel beads carrying cell and molecular tags in nanoliter droplets. Cell lysis and mRNA release are completed within the droplet. First-strand cDNA is synthesized via reverse transcription using oligo(dT) primers (incubation at 42°C for 90 minutes, followed by inactivation at 72°C for 5 minutes). After demulsification, the cDNA product is recovered and purified by silica gel centrifugation column. Then, 12-16 cycles of PCR amplification are performed to enrich the cDNA. The amplified cDNA is fragmented to 200-500 bp using an ultrasonic fragmentation device or enzyme digestion. After end repair and A-tailing, sequencing adapters containing P5 / P7 sequences and sample indexes are ligated. Fragments of the ligation products are enriched by PCR amplification. Finally, AMPure XP magnetic beads are used for purification and fragment size screening to obtain a single-cell transcriptome sequencing library. Preferably, Tn5 transposase can be used to directly fragment and add adapters to double-stranded cDNA, eliminating the need for PCR pre-amplification and reducing library bias.

[0048] High-throughput sequencing: Quality control was performed on the constructed libraries. Qubit was used to detect library concentration, and Agilent Bioanalyzer was used to detect the distribution of library fragment sizes. After mixing equal amounts of libraries that met the quality requirements, paired-end sequencing was performed using a next-generation sequencing platform with a read length of 150 bp.

[0049] Bioinformatics analysis: Cell Ranger software was used to perform quality control, alignment, and multiple alignment removal on the raw sequencing data. Single-cell gene expression matrices were generated based on cell and molecular tags. Low-quality cells were removed based on the expression profile characteristics of each cell. Seurat software was used to normalize, reduce dimensionality, and perform cell clustering analysis on the expression matrix. Cell populations were annotated based on known cell marker genes to identify different cell types in pituitary adenomas, such as tumor cells, immune cells, and stromal cells. Finally, downstream bioinformatics analyses, such as differentially expressed gene analysis and pathway enrichment analysis, were performed.

[0050] To assess the relationship between non-secretory, invasive, and benign secretory cells, we projected these epithelial cells onto different branches based on their pseudo-time ordering using trajectory analysis. The results showed that cortical gonadotropin-producing cells were enriched in the state 1 branch; non-secretory epithelial cells from NF-PA were enriched in the anterior branch and state 2 branch; while non-secretory epithelial cells from ACTH-PA were enriched in the anterior branch and the pre-state 1 branch. Figure 1 AC). Furthermore, we found significant differences between NF-PA and ACTH-PA, and between invasive and non-invasive ACTH-PAs. Figure 1 We further analyzed gene expression patterns in the epithelial cell developmental trajectory and identified 6014 genes with dynamic expression changes. We categorized these genes into one benign expression pattern (P1) and three invasive growth patterns (P2-P4), with each cluster exhibiting specific expression characteristics. Figure 1 D). Functional enrichment analysis showed that genes with glandular cell functions (such as hormone secretion, Wnt signaling pathway, and cation transport) were expressed at higher levels in highly differentiated cortisol-secreting cells, while their expression was downregulated in poorly differentiated, invasive cells. When we identified activating genes in poorly differentiated cells, we found that these cells were enriched in multiple known oncogenic pathways, including cellular respiration, DNA damage repair, and the cell cycle. In particular, a large number of genes responsible for cellular respiration and electron transport were significantly activated in the undifferentiated stage of tumor cells, while they were silenced in the terminal differentiation stage. Figure 1 D). These findings are also supported by the expression of 52 cell state-specific marker genes, including the oncogenic transcription factor JUN (pre-carcinogenesis: oncogene), the mitochondrial cellular respiration regulatory gene NDUFA4 (pre-state 1: cellular respiration regulatory gene), and the hypothalamic-pituitary-adrenal axis regulatory gene GAL (state 1: hormone secretion regulatory gene). Figure 1 E). Intersection with transcription factors obtained from SENIC analysis revealed that JUN, as a transcription factor, showed significant differences in the transition from quiescent to active secretion. Figure 1F), and the expression of JUN is in the opposite direction to that of POMC (F). Figure 1 G). ROC analysis revealed that JUN distinguished ACTH from NF with an AUC of 0.7833, a sensitivity of 91.67% (95% CI: 64.61% – 99.57%), and a specificity of 80.00% (95% CI: 49.02% – 96.45%), p = 0.0250 (G). Figure 1 (H) This indicates that JUN has good clinical diagnostic value in differentiating between functional pituitary adrenocorticotropic hormone adenomas and non-functional pituitary adenomas.

[0051] Table 1 shows the clinical characteristics of patients in ACTH-PA and NF-PA samples used for scRNA-seq (single-cell transcriptome sequencing) analysis.

[0052] Table 1

[0053]

[0054]

[0055] Table 2 shows the markers used to identify different cell types in this embodiment.

[0056] Table 2

[0057]

[0058] Table 3 shows the statistical cell proportions of each subtype in ACTH-PA and NF-PA.

[0059] Table 3

[0060]

[0061] Example 2: Bulk RNA-seq sequencing

[0062] Library construction: First, total RNA was extracted from tumor tissue. Poly(A) tailed mRNA was further enriched using Oligo(dT) magnetic beads for poly(A) selection. The enriched RNA was then fragmented and reverse transcribed into more stable complementary DNA (cDNA). Next, the cDNA underwent end repair, A-tailing was added, and sequencing adapters containing P5 / P7 sequences and a sample index were ligated. Finally, the adapter-ligated DNA fragments were enriched by PCR amplification, purified using magnetic beads, and screened for fragment size to obtain a sequencing library.

[0063] High-throughput sequencing: After library construction, quality control is required. This typically involves using Qubit for initial library quantification and Agilent Bioanalyzer to detect fragment size distribution, ensuring the library quality meets requirements. Subsequently, equal volumes of quality-compliant libraries are mixed and loaded onto next-generation sequencing (NGS) platforms (such as Illumina NovaSeq, NextSeq, etc.) for sequencing. Sequencing can be performed using either single-end or paired-end sequencing. Paired-end sequencing captures both ends of the RNA molecule, facilitating more accurate sequence alignment, discovery of new transcripts, and identification of PCR repeats, and is therefore widely recommended for RNA sequencing. Sequencing read lengths are typically 150 bp (i.e., 2 × 150 bp paired-end sequencing).

[0064] Bioinformatics Analysis: After sequencing, the raw data (FASTQ format) is first quality-controlled to remove low-quality sequences and sequencing adapters (Table 4-6). Then, alignment software (such as STAR, HISAT2) is used to align high-quality sequencing reads to a reference genome or transcriptome. After alignment, each gene or transcript is quantified to calculate its expression level. Commonly used quantitative indicators include Counts, FPKM, and TPM. After obtaining the gene expression matrix, downstream analysis can be performed, mainly including differential expression analysis using software such as DESeq2 and edgeR to screen for genes that show significant changes under different conditions.

[0065] Results: Compared with silent, non-functional pituitary adenomas, POMC expression was significantly increased in invasive and non-invasive functional ACTH-secreting adenomas. Figure 2 A) JUN expression was significantly reduced ( Figure 2 C), while TBX19 did not show a significant trend of difference between SCA and aggressive functional ACTH tumors (C). Figure 2 B).

[0066] Table 4. RNA sequencing data volume of non-invasive functional pituitary ACTH adenomas

[0067]

[0068] Table 5. RNA sequencing data volume of invasive functional pituitary ACTH adenomas

[0069]

[0070] Table 6. RNA sequencing data volume of silent, non-functional pituitary ACTH adenomas

[0071]

[0072] Furthermore, the inventors downloaded FPKM data from a database and performed ROC statistics. The data source was Zhang et al. (https: / / doi.org / 10.6084 / m9.figshare.21340161 (v4); n = 44, 19 cases of ACTH, 25 cases of SCA). The results showed that JUN expression in the ACTH group (median 4.986) was significantly lower than that in the SCA group (median 7.151) (P<0.0001, Cliff's δ = -0.866); ROC curve analysis showed that JUN distinguished ACTH from SCA with an AUC of 0.933, P<0.0001, a sensitivity of 84.21% (95% CI: 62.43% – 94.48%), and a specificity of 96.00% (95% CI: 80.46% – 99.79%). Figure 2 D). This study confirms that JUN has excellent clinical diagnostic value in differentiating functional pituitary adrenocorticotropic hormone adenomas from silent pituitary adenomas.

[0073] Example 3 Immunofluorescence Validation

[0074] To verify the expression differences between single-cell transcriptome and bulk RNA-seq, immunofluorescence staining was performed on 28 clinical samples (including 19 functional pituitary adrenocorticotropic hormone adenoma tissues (ACTH) and 9 non-functional pituitary adenoma tissues (SCA), all from Peking Union Medical College Hospital) for localization and semi-quantitative analysis.

[0075] SCA and ACTH tumor tissues were collected and fixed in formalin solution at 4°C for 48 h. Tissue blocks were prepared by paraffin embedding (using graded ethanol and xylene for clearing, followed by embedding after paraffin embedding). The tissues were cut into 4 μm thick sections using a paraffin microtome, mounted on detachable glass slides, baked at 60°C for 2 h, dewaxed with xylene, and rehydrated with graded ethanol. Antigen retrieval was performed using high-pressure heat retrieval with sodium citrate buffer (pH 6.0). After cooling, the slides were blocked with 5% goat serum at room temperature for 1 h. Rabbit anti-JUN monoclonal antibody (CST, 9165, dilution 1:200) was added and incubated overnight at 4°C in a humidified chamber. The next day, after washing with PBS, Alexa Fluor® 488-labeled goat anti-rabbit fluorescent secondary antibody (dilution 1:500) was added and incubated at room temperature in the dark for 1 h. After washing again with PBS, the slides were mounted with a DAPI-containing anti-fluorescence quenching mounting medium. Images were acquired using a 3D-Histec instrument and software under the same exposure parameters, and the mean fluorescence intensity (MFI) of green fluorescence (JUN) was quantitatively analyzed and normalized with DAPI nuclear staining. The differences in fluorescence intensity of JUN protein expression between the experimental group and the control group were compared to assess its expression level changes in different tissues. Independent samples t-tests were used for comparisons between the two groups, and P < 0.05 was considered statistically significant.

[0076] Experimental Results: Compared with non-functional pituitary adenomas, JUN expression was significantly reduced in functional pituitary adrenocorticotropic hormone adenomas (SCA vs iACTH -25.27 ± 7.898 P value 0.0047, SCA vs noniACTH difference -29.56 ± 7.814 P value 0.0014). ROC curve analysis showed that JUN distinguished ACTH from SCA with an AUC of 0.9524, a sensitivity of 85.71% (95% CI: 65.36% – 95.02%), and a specificity of 100.00% (95% CI: 72.25% – 100.00%), P < 0.0001. Figure 3 (AC) indicates that JUN has excellent clinical diagnostic value in differentiating between functional pituitary adrenocorticotropic hormone adenomas and silent pituitary adenomas.

[0077] Example 4: Overexpression of JUN in a functional pituitary ACTH tumor cell line

[0078] Mouse pituitary tumor cells (AtT-20, ATCC® CCL-89™) were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and penicillin / streptomycin, and routinely passaged at 37 ℃ in a 5% CO2 incubator. Cells in the logarithmic growth phase were transiently transfected using Lipofectamine 3000 (Invitrogen) with the JUN overexpression plasmid (OriGene, MG227043) and the corresponding empty vector control, according to the manufacturer's instructions. Cells and culture supernatant were collected 48 h post-transfection. Total protein was extracted after cell lysis and quantified using the BCA method. Equal amounts of protein were subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. Western blot analysis was performed using anti-POMC antibody (Abcam, ab32820) and anti-ACTH antibody (Abcam, ab233869), with GAPDH as an internal control. Chemiluminescence imaging and band grayscale were used to compare the expression levels of POMC precursor and mature ACTH proteins between the two groups. Simultaneously, the supernatant of transfected cell culture was collected, and after centrifugation to remove particulate matter, the ACTH secretion concentration in the supernatant was calculated using a mouse ACTH ELISA kit (Abcam, ab325629) according to the manufacturer's instructions, and a standard curve was used to calculate the ACTH secretion concentration in the supernatant. Each experiment was performed in triplicate, with independent replicates three times. Results are expressed as mean ± standard deviation. Independent samples t-tests were used to compare two groups, and P < 0.05 was considered statistically significant. All procedures were performed under aseptic conditions to ensure the reliability of the experimental results.

[0079] Experimental results: After JUN overexpression, the expression of POMC and ACTH was significantly reduced. Figure 3 D), cellular ACTH hormone secretion was suppressed (EV vs. JUN -0.9455 ± 0.1670, p value 0.0001) Figure 3 (E) demonstrates the regulatory effect of JUN on ACTH hormone secretion from functional pituitary adrenocorticotropic hormone cells.

[0080] Example 5: Effects of JUN overexpression on tumors and mouse ACTH hormones in a functional pituitary ACTH tumor animal model

[0081] Using AtT-20 mouse pituitary tumor cells as the research object, cells were infected with lentivirus carrying the mouse Jun gene (LV-JUN) and empty vector control (LV-NC), respectively. After screening with puromycin, a cell line stably overexpressing JUN (AtT-20 / JUN) and an empty vector control (AtT-20 / NC) were established, and the overexpression efficiency of JUN protein was verified by Western blot. Two groups of cells were mixed with Matrigel and subcutaneously inoculated into the axillae of 4-6 week old female BALB / c nude mice (n=8 per group). Starting from day 7 post-inoculation, tumor length and short diameters were measured every 3 days, and volume was calculated. Body weight and general condition were also recorded. At the experimental endpoint (day 28 or when the tumor reached 1500 mm³), blood was collected via the orbital rim to separate serum. Serum adrenocorticotropic hormone (ACTH) levels were measured using a mouse ACTH ELISA kit (Abcam, ab325629). Concentrations were calculated using a standard curve. Independent samples t-tests were used for comparisons between the two groups. Repeated measures ANOVA was performed on the tumor growth curves. A p-value < 0.05 was considered statistically significant. The entire experiment was conducted using an SPF-grade barrier system and was approved by the animal ethics committee.

[0082] Results: Tumor growth was not significantly affected by JUN overexpression. Figure 4 A&B), the reduction of body size and weight in mice after tumor formation ( ) Figure 4 C&D), hormone secretion is significantly suppressed ( Figure 4 E) demonstrates that JUN overexpression has a significant inhibitory effect on ACTH hormone secretion from functional pituitary adrenocorticotropic hormone cells.

[0083] Example 6: Detection of the binding and interaction between JUN and POMC in functional pituitary ACTH tumor tissue

[0084] Using one collected ACTH tumor tissue as material, the binding sites of JUN proteins were analyzed at the whole-genome level using CUT&Tag (Cleavage Under Targets and Tagmentation) technology. Specifically, the fresh tissue was minced and cross-linked to prepare a nuclear suspension, which was then incubated with rabbit anti-JUN monoclonal antibody (CST, 9165) and Protein A / G-Tn5 fusion protein. Targeted cleavage was performed using the Tn5 enzyme, and the recovered DNA was amplified by PCR to construct a sequencing library. Paired-end sequencing (PE150) was performed using the Illumina NovaSeq platform. After the raw data underwent FASTP quality control, Bowtie2 alignment was performed against a reference genome (mm10 or hg38), and peak calling was performed using MACS2 (q<0.05) to obtain the whole-genome binding regions of JUN. De novo motif scanning of the peak regions was performed using HOMER software to identify characteristic JUN binding motifs. Gene annotation of the peaks was performed using ChIPseeker, and GO and KEGG pathway enrichment analysis was combined to reveal the potential biological functions regulated by JUN. JUN binding signals at POMC gene loci were visualized in the IGV browser to determine whether JUN directly binds to POMC gene regions (including promoters and enhancers). To further verify the regulatory relationship, a dual-luciferase reporter system was constructed in HEK-293T cells: the promoter region of the mouse Pomc gene (from -2000 bp to +100 bp upstream of the transcription start site) was cloned into the pGL3-Basic luciferase reporter vector, and simultaneously co-transfected with the JUN overexpression plasmid (OriGene, MG227043) or an empty vector control, with the pRL-TK Renja luciferase plasmid as an internal control. Forty-eight hours after transfection, the ratio of fluorescence activity between firefly and Renja luciferase was measured using a dual-luciferase assay kit (Promega), and the changes in relative fluorescence intensity between the JUN overexpression group and the control group were compared to clarify the regulatory role (activation or inhibition) of JUN on POMC transcriptional activity. The luciferase reporter gene experiment was performed in triplicate. Data are expressed as mean ± standard deviation. The t-test was used for comparison between the two groups, and P < 0.05 was considered statistically significant.

[0085] Research results: Quality control analysis yielded the motif of JUN (…). Figure 5 A), and enrichment analysis was performed on the bound genes ( Figure 5 B), and it was also found that POMC and JUN have significant binding and interaction (B). Figure 5 C). Experiments using dual-fluorescent reporter genes confirmed a direct interaction between JUN and POMC, and that JUN significantly inhibited POMC gene expression. Figure 5 D).

[0086] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

[0087] Although this application has been described in detail above with general descriptions and specific embodiments, some modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of this application are all within the scope of protection claimed in this application.

Claims

1. Application of JUN in the preparation of drugs for the treatment of excessive secretion of functional pituitary adrenocorticotropic hormone (ACTH) adenoma.

2. Application of JUN in the preparation of drugs for the prevention or treatment of diseases caused by excessive secretion of functional pituitary adrenocorticotropic hormone (ACTH) adenomas; Optionally, the relevant disease is characterized by hypercortisolemia; Optionally, the relevant disease is Cushing's disease.

3. The application as described in claim 1 or 2, characterized in that, Overexpression of JUN can be used to treat functional pituitary adrenocorticotropic hormone (ACTH) over-secretion in adenomas.

4. A drug for treating excessive secretion of functional pituitary adrenocorticotropic hormone (ACTH) from adenomas, characterized in that, The drug includes a substance that overexpresses JUN.

5. The drug as described in claim 4, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. Application of reagents for detecting JUN expression levels in the preparation of products for the identification or diagnosis of functional pituitary adrenocorticotropic hormone adenomas.

7. The application as described in claim 6, characterized in that, JUN expression is low in patients with functional pituitary adrenocorticotropic hormone adenomas compared to non-functional pituitary adenomas.

8. The application as described in claim 6 or 7, characterized in that, The detection reagents are selected from one or more of the following methods: sequencing, PCR, liquid chromatography, mass spectrometry, and immunoassay. Optionally, the immunoassay includes one or more of the following: Western blot analysis, radioimmunoassay, immunofluorescence assay, immunoprecipitation, immunodiffusion, electrochemiluminescence immunoassay, ELISA assay, and immunopolymerase chain reaction. Optionally, the detection reagent contains a specific antibody against JUN or a functional fragment thereof.

9. The application according to claim 6 or 7, characterized in that, The products include one or more of the following: chips, reagent kits, reagents, test strips, and devices.

10. A diagnostic or screening system or device for functional pituitary adrenocorticotropic hormone-secreting adenomas, characterized in that, include: The result determination unit is used to analyze the level of markers in the detected samples and output the results, wherein the markers include JUN; Optionally, the sample is selected from tissue or blood; Optionally, it also includes a detection unit for detecting the level of a marker in the sample; Optionally, the result determination unit includes an input section for inputting the level of the marker; Optionally, the result determination unit further includes an analysis section, used to analyze the results based on the level of the marker to determine whether the subject has a functional pituitary adrenocorticotropic hormone adenoma or a non-functional pituitary adenoma; Optionally, the result determination unit further includes an output section for outputting the analysis results of the analysis section.

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