Application of m6A demethylase FTO in pituitary endocrine tumors

Abstract

The invention discloses application of m6A demethylase FTO in pituitary endocrine tumors, and belongs to the technical field of biological medicine. Compared with the DGGH, the m6A level in the SGGH is obviously reduced, and the expression of m6A demethylase FTO in the SGGH subtype is increased. Inhibition of FTO in tumor cells significantly reduces hypoxia tolerance, weakens secretion of growth hormone, and enhances sensitivity to octreotide. The FTO-mediated m6A demethylation is unstable through DSP mRNA mediated by an m6A reader FMR1, so that the integrity of a desmosome structure is damaged, and the tumor hypoxia tolerance is enhanced. In the prior art, the targeted FTO-DSP-SSTR2 axis reduces growth hormone secretion, so that tumors are sensitive to octreotide treatment, the invention reveals the key effect of FTO in invasive growth hormone adenoma subtypes, and indicates that FTO may be used as a new treatment target of intractable / persistent SGGH.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of m6A demethylase FTO in pituitary endocrine tumors. Background Art

[0002] Somatotroph adenomas account for approximately 10% of pituitary neuroendocrine tumors (PitNETs) and are one of the most common intracranial tumors in adults. Somatotroph adenomas that secrete growth hormone are associated with an increased risk of severe complications and mortality. Long-term exposure to excess growth hormone can cause deleterious effects on various systems and organs, including the cardiovascular, respiratory, and musculoskeletal systems. In addition, patients with somatotroph adenomas have a significantly increased risk of colorectal, breast, and thyroid cancers. As a result, they generally have a poorer long-term quality of life and a reduced life expectancy of approximately 30% compared with the general population. Somatotroph adenomas belong to the PIT1 spectrum and can be further subdivided into densely granulated (DGGH) and sparsely granulated (SGGH) subtypes. DGGHs typically show a non-invasive growth pattern and have a good prognosis. SGGHs are characterized by aggressive growth, are resistant to traditional treatments, have a poor prognosis, and are classified as refractory / persistent somatotroph adenomas. Despite recent advances in endoscopic techniques, approximately 15% of GH adenomas that invade the cavernous sinus fail to achieve biochemical cure even after optimal postoperative radiotherapy and state-of-the-art medical therapy. Therefore, it is important to explore new drugs for the treatment of refractory / persistent GH adenomas.

[0003] N6-methyladenosine (m6A) modification is the most abundant endogenous RNA modification in eukaryotes. It is involved in various physiological processes including embryonic development, immune system maturation, and neural development. Through the action of RNA methyltransferases and demethylases, m6A recruits specific reader proteins to the target RNA, thereby affecting RNA stability, translation, alternative splicing and other functions. Studies have shown that m6A dysregulation is associated with tumor initiation, invasion, and cell adhesion formation. For example, patent CN113564252A discloses a scheme for inhibiting the methyltransferase complex METTL3 to inhibit somatotroph adenomas. However, the prior art does not disclose the relevant application of m6A demethylase FTO in the treatment of somatotroph adenomas, especially in the treatment of refractory / persistent somatotroph adenomas. Summary of the invention

[0004] [Technical issues]

[0005] The technical problem to be solved by the present invention is to provide relevant applications of m6A demethylase FTO in the treatment of growth hormone adenoma, especially in the treatment of refractory / persistent growth hormone adenoma.

[0006] [Technical solution]

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a growth hormone adenoma subtype diagnostic reagent, which includes a reagent for detecting the expression level of m6A demethylase FTO and / or its encoding gene, and / or a reagent for detecting the expression level of m6A demethylase FTO downstream protein and / or its encoding gene.

[0009] In one embodiment, the downstream proteins of the m6A demethylase FTO include: desmosomal protein DSP and / or desmosomal tissue-related proteins; the desmosomal tissue-related proteins include: PKP3 (destinoside protein 3), DSG2 (desmosomal core glycoprotein 2), DSP (desmosomal core protein), GRHL1 (glutamate acyl cyclase homologous 1), JUP (joining globulin), PKP2 (destinoside protein 2), PRKCA (protein kinase Cα), PERP (p53 effector regulatory protein), SNAI2 (snail family transcription inhibitor 2) at least one of them.

[0010] In one embodiment, the somatotropin adenoma subtypes include densely granulated (DGGH) somatotropin adenoma, and sparsely granulated (SGGH) somatotropin adenoma.

[0011] In a second aspect, the present invention provides use of the diagnostic reagent described in the first aspect in the preparation of a growth hormone adenoma subtype diagnostic product.

[0012] In one embodiment, the product comprises a diagnostic reagent, a diagnostic kit or a diagnostic tool.

[0013] In one embodiment, the method for using the product for diagnosis or auxiliary diagnosis includes: 1) determining the expression level of FTO or its encoding gene in a sample from a subject by using a reagent for detecting the expression level of m6A demethylase FTO or its encoding gene; 2) determining the growth hormone adenoma subtype of the subject based on the difference in expression levels of FTO or its encoding gene and densely granular type (DGGH) growth hormone adenoma in the subject.

[0014] In one embodiment, if the expression level of FTO or its encoding gene is significantly increased compared to the control, the growth hormone adenoma in the subject is determined to be a sparsely granulated (SGGH) growth hormone adenoma.

[0015] In one embodiment, the method for using the product for diagnosis or auxiliary diagnosis includes: 1) determining the expression level of desmosomal protein DSP or its encoding gene in a sample from a subject by using a reagent for detecting the expression level of DSP or its encoding gene; 2) determining the growth hormone adenoma subtype of the subject based on the difference in expression levels of DSP or its encoding gene and densely granular type (DGGH) growth hormone adenoma in the subject.

[0016] In one embodiment, if the expression level of DSP or a gene encoding it is significantly reduced compared to the control, the growth hormone adenoma in the subject is determined to be a sparsely granulated (SGGH) growth hormone adenoma.

[0017] In one embodiment, the method for diagnosis or auxiliary diagnosis using the product includes: 1) determining the expression level of desmosome-associated protein or its encoding gene in a sample from a subject by using a reagent for detecting the expression level of desmosome-associated protein or its encoding gene; 2) determining the growth hormone adenoma subtype of the subject based on the difference in expression levels of desmosome-associated protein or its encoding gene and densely granular type (DGGH) growth hormone adenoma in the subject.

[0018] In one embodiment, if the expression level of desmosome-associated protein or its encoding gene is significantly reduced compared with the control, the growth hormone adenoma in the subject is determined to be a sparsely granulated (SGGH) growth hormone adenoma.

[0019] In a third aspect, the present invention provides any of the following applications of m6A demethylase FTO:

[0020] (A) Application of m6A demethylase FTO inhibitor in the preparation of drugs for adjuvant treatment of growth hormone-producing adenoma;

[0021] (B) Application of m6A demethylase FTO in screening drugs for the treatment of growth hormone adenoma.

[0022] In one embodiment, the somatotropin adenoma comprises a densely granulated (DGGH) somatotropin adenoma, and / or a sparsely granulated (SGGH) somatotropin adenoma.

[0023] In one embodiment, the m6A demethylase FTO inhibitor is capable of inhibiting the expression of m6A demethylase FTO and / or its encoding gene.

[0024] In one embodiment, the inhibitor includes a nucleic acid inhibitor, which has an m6A demethylase FTO encoding gene or its transcript as a target sequence and is an interfering molecule capable of inhibiting the expression of m6A demethylase FTO or its encoding gene. Optionally, the interfering molecule includes: shRNA, siRNA, dsRNA, microRNA, antisense nucleic acid, or a construct or host cell capable of expressing or forming the shRNA, siRNA, dsRNA, microRNA, antisense nucleic acid.

[0025] Preferably, the inhibitor comprises siRNA and / or shRNA.

[0026] In a fourth aspect, the present invention provides a pharmaceutical composition for preventing or treating growth hormone adenoma, wherein the pharmaceutical composition contains an m6A demethylase FTO inhibitor.

[0027] In one embodiment, the pharmaceutical composition further contains a somatostatin analogue. The somatostatin analogue is a class of drugs that inhibit hormone secretion by simulating the effects of endogenous somatostatin; optionally but not limited to, the somatostatin analogue includes octreotide, lanreotide and / or pasireotide.

[0028] In the fifth aspect, the present invention also provides an in vitro method for reducing the secretion of growth hormone in growth hormone adenoma, which method comprises contacting the pharmaceutical composition described in the fourth aspect with the growth hormone adenoma to reduce the secretion of growth hormone in the growth hormone adenoma; the method does not include a method for diagnosing or treating the disease.

[0029] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention experimentally found that compared with DGGH, the level of mA in SGGH was significantly reduced, while the expression of mA demethylase FTO was increased in the SGGH subtype. A series of in vitro and in vivo experiments showed that the inhibition of FTO in tumor cells significantly reduced hypoxia tolerance, weakened growth hormone secretion, and enhanced sensitivity to octreotide. Mechanistically, FTO-mediated mA demethylation leads to impaired desmosome structural integrity and enhanced tumor hypoxia tolerance through mA reader FMR1-mediated DSP mRNA instability. Targeting the FTO-DSP-SSTR2 axis reduces growth hormone secretion, thereby sensitizing tumors to octreotide treatment. The present invention reveals the key role of FTO in the invasive growth hormone adenoma subtype and suggests that FTO may serve as a new therapeutic target for refractory / persistent SGGH. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : Landscape of mA modification between somatotroph adenoma subtypes. a. Schematic diagram of mA-seq workflow comparing DGGH and SGGH samples. 8 samples per group. b. Bar plot of distribution of mA peaks across the genome, along with major mA motifs. c. Distribution of mA sites across mRNA regions. d. Genomic distribution of mA modifications by region. e. Genomic distribution of mA modifications by RNA subtype. f. Distribution of different methylation levels of dysregulated mA peaks within subtypes. g. Differences in global mA methylation levels in DGGH and SGGH as determined by ELISA. h. Scatter plot of distribution of mA modification peaks versus subtype-specific gene expression changes in 16 samples.

[0033] Figure 2:FTO is a key factor contributing to the differences in m6A levels between different somatotroph adenoma subtypes. a. Box plots of m6A-regulated gene expression between DGGH and SGGH. bc. Box plots of FTO (b) and RBM15 (c) levels analyzed by quantitative PCR in a cohort of 69 DGGH and SGGH samples. d. Violin plots of FTO expression in DGGH and SGGH cells in the scRNA-seq cohort. e. Box plots of FTO expression levels in DGGH and SGGH based on the dataset GSE214226. f. Immunohistochemical staining of FTO in DGGH and SGGH tissue samples. g. H-score (histochemical score) of immunohistochemical staining of FTO in DGGH and SGGH tissue samples. h. Box plots of FTO expression in non-invasive and invasive GH adenomas. i. Effect of FTO knockdown on FTO mRNA expression in GH3 cells. j. Effect of FTO overexpression on FTO mRNA expression in GH3 cells. k. Effect of FTO knockdown on FTO mRNA expression in primary tumor cells (primary). l. Western blot analysis demonstrated the effects of FTO knockdown and overexpression in GH3 cells and primary tumor cells. mn. Bar graphs represent global m6A modification levels after FTO manipulation in GH3 cells (m) and primary tumor cells (n).

[0034] Figure 3 : FTO affects the organization of desmosomes in somatotroph adenomas by affecting their organization. a. Top five pathways upregulated and downregulated by GO-BP enrichment analysis in SGGH compared with DGGH. b. Violin plots of desmosome organization pathway activity in DGGH and SGGH cells in the scRNA-seq cohort. c. PCA plots revealing the differences in desmosome organization analysis between DGGH and SGGH. d. ROC curves evaluating the diagnostic performance of desmosome organization-related genes in distinguishing DGGH from SGGH. e. Box plots of DSP, DSG2, PKP3, and PKP2 expression between DGGH and SGGH. f. Schematic diagram showing downregulated genes in desmosome organization in SGGH. g. Changes in the activity of desmosome organization, angiogenesis regulation, and extracellular matrix organization pathways after FTO overexpression (top) and knockdown (bottom). h. Changes in the mRNA levels of Dsp, Dsg2, Pkp2, and Pkp3 after FTO overexpression in GH3 cells. ij. Changes in the mRNA levels of Dsp, Dsg2, Pkp2, and Pkp3 after FTO knockdown in GH3 cells (i) and primary tumor cells (j). k. Immunofluorescence images verify the changes in the protein level and localization of DSG2 after FTO knockdown in primary tumor cells.

[0035] Figure 4: FTO affects desmosome organization in somatotroph adenomas by regulating the m6A level of DSP. a. Venn diagram showing genes that are negatively correlated with FTO and downregulated at the m6A level in SGGH. b. Differences in DSP mRNA levels between DGGH and SGGH samples were determined by qPCR in 69 samples. c. Western blot confirmed that DSP protein levels were higher in DGGH tissues. d. Regression lines show the correlation between FTO and DSP expression, and p-values ​​and R-values ​​were calculated by linear models. e. ROC curves evaluated the diagnostic performance of FTO and DSP in distinguishing DGGH from SGGH. f. Western blot of DSP protein levels after Fto overexpression in GH3 cells. g. Western blot of DSP protein levels after Fto knockdown in GH3 cells (top) and primary tumor cells (bottom). h. Changes in the mRNA level of Dsg2 after Dsp and Fto knockdown in GH3 cells and primary tumor cells, determined by qPCR. i. Western blot showing changes in DSG2 protein expression after Fto and Dsp knockdown in GH3 cells (left) and primary tumor cells (right). j. Immunofluorescence images verifying changes in DSG2 protein levels and localization after FTO and DSP knockdown in primary tumor cells. k. mA levels of DSP in DGGH and SGGH determined by MeRIP-qPCR. l. Graphical representation of differential mA modification peaks on DSP after FTO knockdown by mA-seq. mn. Bar graph showing the effect of FTO knockdown on mA levels of DSP in primary tumor cells (m) and GH3 cells (n), determined by MeRIP-qPCR.

[0036] Figure 5 : FTO regulates the mRNA stability of DSP by interacting with the m6A reader FMR1. ac. Changes in the mRNA stability of DSP after FTO perturbation after actinomycin D treatment in GH3 cells (ab) and primary tumor cells (c). d. Distribution of predicted scores of m6A modification sites and related RNA binding proteins determined using the SRAMP prediction tool and the RMVar database. e. qPCR results showing the mRNA levels of FMR1 in DGGH and SGGH. f. Bar graph showing the effect of FTO knockdown on the binding of FMR1 to m6A on DSP by RIP-qPCR. g. Changes in the mRNA levels of DSP after FMR1 knockdown in GH3 cells (left) and primary tumor cells (right), quantified by qPCR. h. Western blot of changes in the protein levels of DSP after FMR1 knockdown in GH3 cells (left) and primary tumor cells (right). ij. Changes in DSP mRNA stability under actinomycin D treatment after FMR1 knockdown in GH3 cells (i) and primary tumor cells (j).

[0037] Figure 6:FTO knockdown affects hypoxia tolerance and fibrillar body formation in growth hormone adenoma cells. ac. Cell viability after FTO perturbation was determined by CCK8 in GH3 (ab) and primary tumor cells (c) cells. d. Colony formation after Fto knockdown in GH3 cells. e. Line graph of mouse body weight changes over time. f. Subcutaneous tumor images of xenograft model of GH3 cells injected with Fto knockdown. g. Effect of Fto knockdown on tumor volume. h. Bar graph showing the effect of Fto knockdown on tumor weight. i. The percentage of apoptosis in primary tumor cells and GH3 cells after FTO and DSP knockdown was analyzed by flow cytometry under normal and hypoxic conditions. j. Electron microscopy images showing the difference in fibrillar body fiber density in primary tumor cells after FTO knockdown.

[0038] Figure 7 : Targeting FTO reduces the growth hormone secretion capacity of tumor cells and enhances their sensitivity to somatostatin analogs. a. Bar graph showing the changes in Sstr2 and Gh1 levels after Fto knockdown by RNA-seq analysis. b. Bar graph showing the changes in Sstr2 and Gh1 levels in GH3 cells (left) and primary tumor cells (right) after FTO and DSP knockdown under octreotide (100 nM) stimulation by qPCR analysis. c. Bar graph showing the changes in growth hormone levels in GH3 cells (left) and primary tumor cells (right) after FTO and DSP knockdown under octreotide (100 nM) stimulation. d. Electron microscopy shows a decrease in secretory granules in primary tumor cells after FTO knockdown. e. Bar graph showing the changes in octreotide sensitivity after FTO knockdown in GH3 cells (left) and primary tumor cells (right). f. Bar graph showing changes in octreotide sensitivity after combined treatment with FB23-2 in GH3 cells (left) and primary tumor cells (right). g. After octreotide and FB23-2 treatment in GH3 cells (left) and primary tumor cells (right). i. Subcutaneous tumor images of a xenograft model of GH3 cells after injection of Fto knockdown. j. Effects of Fto knockdown, octreotide, and the combination of the two on tumor volume. k. Bar graph showing the effects of Fto knockdown, octreotide, and the combination of the two on tumor weight. l. Bar graph showing the effects of Fto knockdown, octreotide, and the combination of the two on growth hormone levels in the peripheral blood of animals. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0040] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0041] Example:

[0042] The experimental methods involved in the following embodiments are:

[0043] 1. Sample preparation: A total of 39 DGGH and 30 SGGH were included in this study, and the information of the samples is shown in Table 1. Among them, 8 DGGH and 8 SGGH samples were subjected to high-throughput m6A sequencing, and the information of the cohort samples subjected to m6A sequencing is shown in Table 2. The diagnosis of growth hormone adenoma was confirmed by histopathology and biochemical tests. The subtypes were classified based on fibrosomes. DGGH showed perinuclear keratin expression, while SGGH showed a pattern dominated by fibrosomes (>70%).

[0044] Table 1 Clinical information of the 69 cohorts

[0045]

[0046]

[0047]

[0048]

[0049] Table 2 Clinical information of m6A sequencing samples

[0050]

[0051] 2. High-throughput m6A sequencing: Total RNA was extracted and purified using TRIzol reagent (Invitrogen), and its quantity and purity were determined by NanoDrop ND-1000 instrument. The integrity of RNA was evaluated using Agilent 2100 Bioanalyzer to ensure that the RIN value was greater than 7.0 and confirmed by denaturing agarose gel electrophoresis. After purifying Poly(A) RNA from total RNA using Dynabeads Oligo(dT)25, it was fragmented at 86°C using Magnesium RNA Fragmentation Module (NEB, cat. e6150, USA). The fragmented RNA was incubated in IP buffer of specific antibodies and then incubated with SuperScript TMⅡ reverse transcriptase to transcribe into cDNA. Next, E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP solution (Thermo Fisher, cat.R0133, USA) were used to synthesize U-labeled double-stranded DNA and prepare for linker ligation. The size was selected by AMPureXP beads, treated with thermosensitive UDG enzyme and amplified by PCR. Finally, the cDNA library with an average insert size of 300±50bp was amplified on Illumina NovaSeq TM 2×150 bp paired-end sequencing was performed on 6000.

[0052] 3. MeRIP-seq and RNA-seq data processing: FastQC and fastp were used for quality control and preprocessing of raw sequencing reads. For MeRIP-seq data, MeRIPseqPipe was used for analysis. Cleaned reads were aligned to the hg38 genome using STAR. MACS2 and MetPeak were used to identify m6A enriched peaks. Intersection peaks were retained for subsequent analysis. Multicov was used to calculate the read coverage of IP and Input data for each peak and normalized using the RPKM method. The ratio of IPRPKM (plus 1) and INPUTRPKM (plus 1) was used to represent the methylation level of each m6A peak. FeatureCounts was used to generate gene counts. For differential methylation analysis, the Wilcox test was used to check for significant differences, and DESeq2 was used to identify differentially expressed genes. m6A peak annotation was performed using human annotation files downloaded from the GENCODE database (GENCODE, version 39). HOMER was used to find m6A motifs. For single-cell RNA sequencing (scRNA-seq) data, raw sequencing data were processed using CellRanger (10X Genomics, v3.1.0) with default settings and aligned to the human genome (GRCh38). The matrix files were then processed using CellBender to remove noisy RNA. The resulting clean matrix was analyzed using Seurat (v4.1.0). Specific methods involved in scRNA-seq analysis include preparation of tumor samples and other analyses such as identification of differentially expressed genes (DEGs), cell classification, and pathway enrichment analysis as described in previous studies. For RNA-seq data, cleaned reads were aligned to the Rn6 genome using STAR. Gene expression was quantified using FeatureCounts. DESeq2 was used to identify differentially expressed genes, and clusterProfiler was used for pathway enrichment. Pathway activity was predicted by GSVA and ssGSEA analysis.

[0053] 4. Transient transfection of siRNA: Use Lipofectamine 3000 transfection reagent for cell transfection experiments. Plant the cells in a six-well plate the day before transfection to ensure that the cell density is about 50% to 60% during transfection. Prepare two centrifuge tubes and add 125μl / well of Opti-MEM reduced serum medium to each centrifuge tube. Add the required amount of siRNA to the first centrifuge tube and add an appropriate amount of Lipofectamine 3000 reagent to the second centrifuge tube. Mix the solutions in the two centrifuge tubes thoroughly and let stand at room temperature for 15 minutes to form a transfection complex. Add the transfection complex evenly to the six-well plate and start subsequent functional experiments 48h after transfection.

[0054] 5. Construction of stable cell lines: When performing virus packaging, first culture 293T cells in a culture dish to a cell density of 70% for transfection. Mix the target plasmid with the packaging plasmids PSPAX2 and pMD2.G in a ratio of 4:3:1 and transfect them into 293T cells. Collect the virus solution 48 hours and 72 hours after transfection, and filter with a 0.22μm filter to remove cell debris. When the target cell density is about 70%, replace the culture medium containing the virus and 0.001% polybrene to increase the infection efficiency. After 24 hours, replace the new virus culture medium and continue to culture. After 48 hours, use a culture medium containing 2μg / ml puromycin to screen positive cells, verify the lentiviral infection and expression efficiency, and ensure the accuracy of the experimental results.

[0055] 6. RNA stability assay: FTO or FMR1 knockdown cells were treated with actinomycin D at a final concentration of 2 μM for different time points. Subsequently, total RNA was extracted using TRIzol reagent. Expression levels were evaluated by RT-qPCR, and the half-life of mRNA was calculated.

[0056] 7. Cell lines and cell culture: Human growth hormone adenoma cells were derived from primary cells isolated from patient tumors and cultured in DMEM / F12 medium (Gibco, New York, USA) supplemented with 20% FBS. The rat growth hormone adenoma cell line GH3, which is preferred as an in vitro model of SGGH, was obtained from the Cell Resource Center of the Basic Medical College, Peking Union Medical College, Institute of Basic Medicine, Chinese Academy of Medical Sciences. GH3 cells were cultured in high glucose DMEM containing 10% fetal bovine serum (FBS) (Gibco, USA), 100 U / mL penicillin and 100 μg / mL streptomycin. All cells were cultured in a humidified environment at 37°C and 5% CO2. All cell lines used in this study were tested and confirmed to be free of mycoplasma contamination.

[0057] 8. RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR): Total RNA was extracted from tissues and cells using TRIzol (Invitrogen, USA) following the manufacturer's protocol. Subsequently, total RNA was reverse transcribed into complementary DNA (cDNA) using a reverse transcription kit (Takara BIOINC, Kusatsu, Shiga, Japan). For real-time quantitative PCR, cDNA was used as a template and amplified using specific primers and SYBR Premix Ex Taq RNAse H kit (Takara Bio, Tokyo, Japan) with the Roche LightCycler 480 II detection system. The amplification protocol included an initial incubation at 95 °C for 5 min, followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C. Each experiment was repeated at least three times. The resulting data were analyzed using the formula: 2-(Ct target-Ct reference) to calculate the relative expression level, which was then compared with the control sample. The primers used are listed in Table S3.

[0058] 9. m6A immunoprecipitation (MeRIP): First, adjust the RNA concentration to about 1 μg / μL, take 18 μL of total RNA (about 18 μg) for fragmentation, mix it with 2 μL of 10x RNA fragmentation buffer and divide it into 200 μL PCR tubes. According to experimental requirements, you can choose to heat at 94°C for 3 minutes to obtain RNA fragments of 100-150 bp, or heat at 70°C for 7 minutes to obtain fragments of about 200 bp. Immediately after fragmentation, terminate the reaction with 2 μL of 0.5M EDTA and cool it on ice. Subsequently, the reaction volume was filled to 180 μL, 18 μL of 3M sodium acetate and 1 μL of glycogen were added for precipitation, and then 600 μL of anhydrous ethanol was used for precipitation at -20°C overnight. After centrifugation at 4°C and 12,000 × g for 30 minutes, wash twice with 800 μL of 75% ethanol, dry and dissolve the RNA with 50 μL of nuclease-free water. Next, the anti-m6A magnetic beads were prepared by mixing 25 μL of magnetic beads A / G with 250 μL of 1×IP buffer and washing, followed by adding 5 μg of anti-m6A antibody and rotating at room temperature for 30 minutes. The magnetic beads were washed twice with 500 μL of 1×IP buffer in preparation for subsequent immunoprecipitation experiments. In the immunoprecipitation step, 1 / 10 of the fragmented RNA was separated and stored as the input group, and the remaining RNA was mixed with the pretreated magnetic beads, rotated and mixed at 4°C for 2 hours, and then washed three times to remove unbound RNA. Finally, the bound RNA was eluted by adding elution buffer to the magnetic beads and shaking at 4°C for 1 hour. The elution steps were repeated and the eluates were combined. RNA was recovered using the Magen Hipure Serum / plasma miRNA Kit, and finally RNA was eluted with 30 μL of nuclease-free water, and the samples were stored at -80°C for subsequent analysis.

[0059] 10. m6A ELISA: Use the enzyme-linked EpiQuik m6A RNA methylation quantification kit to detect the m6A RNA methylation level in RNA. First, prepare RNA, control samples and related reagents according to the instructions of the kit, add binding solution to each well, then add control samples and test samples in turn, seal with sealing film, and incubate at 37°C for 90 minutes to complete RNA binding. After RNA binding, wash three times with the Wash Buffer in the kit, add capture antibody solution, and incubate at room temperature for 1 hour. Then add detection antibody solution after washing, incubate at room temperature for 30 minutes, and wash as described above to complete RNA capture. Finally, add developer and incubate at room temperature for 10 minutes. When the color of the positive control well turns to moderate blue, add stop solution to each well in turn, and use a microplate reader to read the absorbance at 450nm. m6A% = (Sample OD value-NC OD value) ÷ RNA input amount / (positive control OD value-negative control OD value) ÷ positive control input amount.

[0060] 11. RNA-binding protein immunoprecipitation (RIP): Wash the freshly excised whole tissue three times with pre-cooled PBS. Collect tumor cells using the primary tumor cell isolation method described above. Collect cells by centrifugation at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Subsequently, use Magna RIP TM RIP experiments were performed using the RNA Binding Protein Immunoprecipitation Kit (Millipore, catalog number 17-700). After immunoprecipitation, RNA enrichment was analyzed using qPCR.

[0061] 12. Immunohistochemistry: The collected tumor tissues were embedded in paraffin and cut into slices of about 5 μm thickness and pasted on siliconized slides. Then, the slices were placed in a 60°C constant temperature oven and baked overnight to fix. When dewaxing, the tissue clearing solution was used for 15 minutes of immersion, repeated twice. Then, gradient washing was performed through 100%, 95%, 70%, and 50% ethanol, each concentration lasting 5 minutes, and finally the hydration process was completed with deionized water. In the antigen retrieval stage, citric acid buffer was added to immerse the slices, microwaved on high for 5 minutes, heated on medium and low for 15 minutes, and then cooled to room temperature. In order to block endogenous peroxidase, the slices were treated with 3% hydrogen peroxide solution for 20 minutes. Subsequently, the slices were washed 3 times with PBS for 5 minutes each time, blocked with 10% goat serum to reduce nonspecific staining background, and incubated at room temperature for 1 hour. The primary antibody was added directly to the blocked slices, ensuring that the entire tissue was covered, and incubated overnight at 4°C in a humidified box. The next day, wash the sections three times with phosphate-buffered saline for 10 minutes each time. Then add horseradish peroxidase-labeled secondary antibody to cover the entire section and incubate at room temperature for 1 hour. Then wash it three more times with PBST for 5 minutes each time. Add DAB colorimetric solution and observe under a microscope until the section turns yellow-brown. The DAB treatment time for each section is the same. After incubation for 30 seconds to 1 minute, rinse with water immediately to stop the color reaction. Then counterstain the nucleus with hematoxylin staining solution for 30 seconds, and then quickly wash with water to stop the reaction. Finally, dehydrate and seal the sections. Dehydrate the sections in 95% and 100% ethanol in turn, wash for 1 minute each time, and dry them in a fume hood. Use neutral gum to seal the sections for easy observation under the microscope.

[0062] 13. Western blot analysis: discard the cell supernatant culture medium and wash three times with PBS. Add an appropriate amount of RIPA lysis buffer, blow and mix well and fully lyse. Centrifuge at 4℃, 12000×g for 15 minutes, and collect the supernatant as the protein solution. Use the BCA protein quantification kit to measure the protein concentration. Before loading, use the PAGE gel rapid preparation kit to make gel. First, mix 4ml of the lower gel solution and 4ml of the lower gel buffer evenly, then add 80μl of the coagulant, mix quickly and pour immediately into the center of the glass plate for making gel. Then cover the lower gel with anhydrous ethanol for sealing. After the lower gel solidifies, pour out the anhydrous ethanol and dry it. Then mix 1ml of the upper gel solution and 1ml of the upper gel buffer evenly and add 20μl of the coagulant, mix and quickly pour into the glass plate, and immediately insert the hole comb. Wait for the colloid to completely solidify. Prepare the same amount of protein sample and use 1× loading buffer to make the same volume. After loading, run the sample at 100V for 25 minutes, followed by 120V for about 60 minutes. After the electrophoresis, activate the PVDF membrane by soaking it in methanol. First, make sure the black side of the transfer chuck is facing down and place a wet sponge on the black side of the transfer chuck. The sponge needs to be completely wet to ensure that the current flows evenly. Place a wet white filter paper on the sponge. The filter paper should be as wet as the sponge to ensure good current transfer. Carefully place the completed gel on the filter paper. Make sure the gel is flat and not folded or damaged. Place the PVDF membrane on the gel. Make sure the membrane is in good contact with the gel and there are no air gaps. Place another wet filter paper and sponge on the PVDF membrane. Finally, cover the white side of the transfer chuck, remove bubbles and ensure that the chuck is properly closed. Transfer the membrane at 0.25A for 2 hours. Place the transfer tank in an ice box or in a cold storage during the transfer process. After the transfer is completed, the PVDF membrane is washed once with TBST solution to remove residual substances on the surface, and then blocked with 5% BSA for 2 hours to prevent nonspecific binding. Use 3% BSA solution as the primary antibody diluent, and incubate the primary antibody at 4°C overnight. The primary antibody is recovered the next day, washed 3 times with 1×TBST, 10 minutes each time; incubate the secondary antibody at room temperature for 1 hour; wash 3 times with TBST, 10 minutes each time. After washing the membrane, mix the developer and evenly add it to the membrane, and place it in a chemiluminescence instrument to detect the signal.

[0063] 14. Flow cytometry: GH3 and primary cells were cultured in normal cell culture oxygen concentration and hypoxic environment (1% O2) before and after knocking down FTO and DSP genes. The cells were removed, the supernatant culture medium was aspirated into the flow tube, trypsin was added to digest and collect the cells, centrifuged at 1000 rpm for 5 minutes, the culture medium was discarded, PBS was added to resuspend, and centrifuged at 1000 rpm for 3 minutes, and repeated once. Subsequently, the staining operation was performed according to the Annexin V AlexaFluor 647 / 7AAD staining kit, negative and positive controls were set, and the stained cells were analyzed by Beckman flow cytometer (Beckman Coulter, Miami, FL, USA).

[0064] 15. Cell viability assay: Cell viability was assessed by using CCK-8 assay. After transfection, GH3 and PDFS cells were seeded in 96-well plates at a density of 1×103 cells / well, respectively. On days 0, 1, 2, 3, and 4, 10 μl of CCK-8 solution was added to each well in sequence and incubated for 2 hours. The absorbance at 450 nm was then measured using a microplate reader.

[0065] To investigate the effects of FB23-2 and OCT on GH adenoma cell viability, GH3 and primary tumor cells were seeded into 96-well plates at a density of 8 × 103 cells / well. After 24 h, the cell culture medium was changed to medium containing different concentrations of FB23-2 or OCT or their combination. Three days later, the absorbance was measured as described previously.

[0066] 16. Clone formation assay: Add 2 ml of cell suspension and 1,000 cells to each well of a six-well plate, shake evenly, and place in an incubator for 10-14 days. After the incubation, remove the six-well plate and remove the culture medium, add PBS to wash 3 times, add 1 ml of methanol to each well for 20 minutes; remove the methanol after fixation and wash with PBS. Then add 1 ml of crystal violet staining solution for 20 minutes. After staining, absorb the crystal violet solution, soak and wash in water until the background is transparent; let it stand and dry, take pictures, and count the number of clones in each well.

[0067] 17. Growth hormone detection: The concentration of growth hormone in GH3 cell culture medium and serum was evaluated using an ELISA kit from Millipore (EZRMGH) according to the protocol provided by the manufacturer. Similarly, another set of ELISA kits from Elabscience (E-EL-H0177) was used to determine the concentration of growth hormone in primary tumor cell and organoid culture medium. The growth hormone levels were then normalized to the results of the control cell and organoid viability tests, as well as the weight of the tumor.

[0068] 18. Organoid culture and drug response assay: Tumor samples were washed, minced, then dissociated and cultured into organoids using specific kits and culture media. After centrifugation, the cells were mixed with Matrigel, placed in culture plates and incubated. The success of organoid formation was monitored by morphology and viability. Organoids larger than 100 μm were used for drug screening and passaging. Recurrent organoids were dissociated, collected and cultured again. Drug screening was performed on first-generation PDOs using modified culture media without Y-27632. Organoids were dissociated, mixed with Matrigel / modified culture media, and seeded into laminin-coated 384-well plates. After 48 hours, culture media containing FB23-2 (4 μM) or OCT (100 nM) or their combination was added. After 4 days, viability was assessed using CellTiter-Glo 2.0, and relative luminescence units were normalized to the DMSO control group (100% viability) as previously reported. All organoids used in this study were tested and confirmed to be free of mycoplasma contamination.

[0069] 19. Animal experiments: When using female BALB / c nude mice aged 4 to 6 weeks for subcutaneous tumor formation experiments, the mice were first randomly divided into several groups, each containing 5 mice. 2×106 cells were inoculated subcutaneously on the back of the nude mice, and the volume of the cell suspension inoculated each time was 0.1 ml. When the tumor was visible to the naked eye, the mouse status was monitored regularly and the mouse weight and tumor volume were recorded. If the effect of the drug on the tumor was observed, intraperitoneal injections were started every three days. The formula for calculating the tumor volume is 0.5×length×width2. This method can quantitatively evaluate the growth rate of the tumor and the effect of knocking down the FTO gene on tumor growth. The tumor volume reached 2000mm 3 When the experiment is about 10%, the experiment is terminated to avoid causing excessive suffering or unnecessary death to the animals.

[0070] 20. Electron microscopy observation: First, collect the cell or bacterial precipitate by centrifugation, then remove the culture medium, add electron microscopy fixative at 4°C, mix and fix for 2-4 hours, and store and transport the fixed samples at 4°C. Next, perform an agar pre-embedding step, that is, centrifuge the cells or bacteria, discard the supernatant, add 0.1M phosphate buffer (pH7.4) for washing, and then use a pre-dissolved 1% agarose solution for embedding. After that, post-fix with 1% osmic acid in 0.1M phosphate buffer PB (pH7.4), protect from light and fix at room temperature for 2 hours, and then rinse three times. The room temperature dehydration process includes dehydration with different concentrations of alcohol in sequence, followed by dehydration twice with 100% acetone. Acetone and 812 embedding agent were used for infiltration embedding, and then the sample was placed in the embedding plate and baked at 37°C overnight. The embedding plate was then placed in an oven at 60°C for polymerization for 48 hours, and the resin block was taken out for ultrathin sectioning, and 60-80nm sections were made using an ultrathin slicer. Finally, staining was performed, and after staining, a transmission electron microscope was used for observation, and images were collected for analysis.

[0071] The primers and their sequences involved in the following examples are shown in Table 3.

[0072] Table 3 qPCR primers and their sequence information

[0073]

[0074]

[0075] Table 4 si / shRNA and its sequences used in the following examples

[0076]

[0077] Example 1: Analysis of m6A level differences between growth hormone adenoma subtypes and determination of key genes

[0078] Experimental methods: 16 tumor samples from 16 patients with growth hormone adenomas (including 8 SGGH and 8 DGGH) were collected for MeRIP-seq and RNA-seq. The experimental process is as follows: Figure 1 As shown in a.

[0079] Experimental results: After merging the m6A peaks of different subtypes, 48,528 m6A peaks were obtained for further analysis. The data showed that most genes exhibited a single m6A peak, and the identified m6A peaks were mainly enriched in the classic GGACH motif ( Figure 1 b). These m6A peaks are mainly located in the coding sequence (CDS) region and the region near the stop codon, and mRNA is the most enriched fragment ( Figure 1Differential methylation analysis showed that there were 8,660 (60.94%) hypomethylated m6A peaks and 5,551 (39.06%) hypermethylated m6A peaks in SGGH compared with DGGH, indicating that the overall m6A modification of SGGH was reduced ( Figure 1 f). Subsequent quantitative analysis of overall m6A levels using an ELISA assay confirmed the low levels of m6A modification in SGGH ( Figure 1 g). Through comprehensive analysis of m6A modification levels and gene expression profiles, it was observed that there was a positive correlation between m6A modification levels and gene expression in growth hormone adenomas ( Figure 1 h). The results of this example reveal that there are different m6A modification landscapes between SGGH and DGGH, characterized by a significant downregulation of m6A levels in SGGH. This change in m6A modification may affect gene expression regulation and promote the pathogenesis and phenotypic characteristics of the growth hormone adenoma subtype.

[0080] Next, this example investigated which methylase or demethylase may lead to the difference in m6A levels between the two isoforms. Based on the sequencing data, FTO and RBM15 were significantly upregulated in SGGH ( Figure 2 a). However, in the growth hormone adenoma patient cohort (DGGH=39, SGGH=30), a public dataset (DGGH=11, SGGH=10) (GEO database, accession number: GSE214226) and a single-cell RNA sequencing dataset (DGGH=2, SGGH=2) (NGDC database, accession number: PRJCA009690) ( Figure 2 In the GH group, only FTO was confirmed to be consistently upregulated. We then used immunohistochemical staining analysis in a cohort of patients with growth hormone adenomas (43 samples from a partial cohort of 69 patients, DGGH = 21, SGGH = 22) and also found that FTO was upregulated in SGGH ( Figure 2 fg). In addition, according to the literature (Cytodifferentiation of pituitary tumors influences pathogenesis and cavernous sinus invasion, PMID: 37119095), Knosp 1-2 was defined as non-invasive, and Knosp 3-4 was defined as invasive. The information of the cohort samples that underwent m6A sequencing is shown in Table 2. Compared with non-invasive tumors, FTO was significantly overexpressed in invasive growth hormone adenomas ( Figure 2h). Then, FTO interference was performed in GH3 cell lines and primary tumor cells (shFto-RAT#1, shFto-RAT#2, siFTO-human#1, and siFTO-human#2 were used to inhibit FTO expression, and the shRNA sequences used are shown in Table 4; pCDH plasmid was used to overexpress FTO, and the sequence information of the FTO encoding gene is shown in NCBI Accession No. 291905). The results are shown in Figure 2 il. Overexpression of FTO resulted in a decrease in the overall m6A level, whereas knockdown of FTO resulted in an upregulation of m6A levels ( Figure 2 These results suggest that FTO is the major demethylase regulating m6A modification in different somatotroph adenoma subtypes.

[0081] Example 2: FTO promotes the invasive phenotype of SGGH by regulating desmosome organization

[0082] Experimental methods: Differential analysis and pathway enrichment were performed on 8 SGGHs and 8 DGGHs; desmosome organization pathway activity was predicted by single-cell sequencing of a cohort from the literature (the Pudmed accession number of the literature: PMID: 36754052, and the dataset in the literature is included in the NGDC database, accession number: PRJCA009690); PCA analysis and ROC analysis based on desmosome organization genes.

[0083] Experimental results:

[0084] Gene expression analysis of 16 patients with growth hormone adenomas (including 8 SGGH and 8 DGGH) showed that multiple pathways related to cell-cell junctions were downregulated in SGGH ( Figure 3 a), especially the pathways of desmosomes and cell-cell junctions. Desmosome organization is a subclass of cell-cell junction organization. Single-cell RNA sequencing data from the PMID:36754052 cohort showed a significant downregulation of desmosome organization in SGGH ( Figure 3 b). Based on genes related to desmosome organization, DGGH and SGGH were clearly clustered and separated by PCA analysis ( Figure 3 c). ROC analysis showed that most genes associated with desmosome organization had strong discriminatory power to distinguish DGGH from SGGH ( Figure 3 d). Genes that play a key role in desmosome organization, including DSP, DSG2, PKP2, and PKP3, which are essential for cell junctions, were significantly downregulated in SGGH compared with DGGH ( Figure 3 ef).

[0085] Furthermore, this example tested whether FTO affects the regulation of desmosome organization.

[0086] Experimental method: RNA sequencing of GH3 cells with perturbation of FTO expression (the method for perturbation of FTO expression refers to Example 1) was followed by prediction of ssGSEA pathway activity. After perturbation of FTO expression in GH3 cells and primary tumor cells, qPCR and immunofluorescence analysis were performed to detect changes in key genes of desmosome tissue. For qPCR primers, see Table 3.

[0087] Experimental results: RNA sequencing and ssGSEA analysis were performed on GH3 cells with perturbation of FTO expression. The results showed that the activity of the desmosome pathway was decreased after FTO overexpression and increased after FTO knockdown ( Figure 3 g). Consistently, FTO overexpression and knockdown down-regulated and up-regulated desmosome-related genes such as DSP, DSG2, PKP2, and PKP3, respectively ( Figure 3 hj). In order to further investigate the functional changes in desmosome organization, desmosome proteins (DSG) were defined as markers of desmosomes. The DSG protein family is an important component of desmosome organization. As cell surface transmembrane proteins, it consists of four members: DSG1, DSG2, DSG3, and DSG4. They are connected to DSP to form desmosomes or hemidesmosomes. Among these members, DSG2 is the only DSG protein involved in desmosome organization and is a key component of desmosomes. Cell immunofluorescence showed that cell surface DSG2 expression increased after FTO knockdown ( Figure 3 k). These data further confirm a role for FTO in regulating the desmosome organization dysfunction observed in somatotroph adenoma subtypes.

[0088] Example 3: FTO affects desmosome organization in growth hormone adenoma by regulating the m6A level of DSP

[0089] This example verifies the specific mechanism by which FTO affects desmosome organization, namely, FTO affects the desmosome organization of growth hormone adenoma by regulating the m6A level of DSP.

[0090] Since the overall m6A level in SGGH is lower than that in DGGH, we mainly focused on genes with downregulated m6A levels. By strictly intersecting the differentially expressed genes after FTO interference, the differentially expressed genes between growth hormone adenoma subtypes, and the genes with downregulated m6A levels in SGGH, 19 genes were found to be negatively correlated with FTO expression ( Figure 4 a) Among these genes, DSP was selected for further analysis because it is a key component of desmosome organization.

[0091] The present invention found that DSP mRNA and protein levels in SGGH were significantly reduced ( Figure 4 bc). Consistently, the levels of FTO and DSP were negatively correlated in two independent datasets ( Figure 4d). ROC analysis showed that FTO and DSP had good discrimination efficiency in distinguishing DGGH from SGGH ( Figure 4 e).

[0092] Furthermore, the effects of FTO knockdown and overexpression were verified by cell experiments. FTO overexpression and knockdown models were constructed in GH3 cell lines, and FTO knockdown models were constructed in primary tumor cells. The results showed that in cell lines, FTO knockdown and overexpression led to upregulation and downregulation of DSP protein levels, respectively ( Figure 4 fg).

[0093] In order to clarify the regulatory effect of FTO on DSP-mediated desmosome organization, FTO knockdown, DSP knockdown and FTO knockdown + DSP knockdown models were constructed in GH3 cell lines and primary tumor cells (methods refer to Example 1, siRNA is si-RAT-Dsp#1, si-RAT-Dsp#2, si-Human-DSP#1, si-Human-DSP#2, sequence see Table 4), and qRT-PCR, protein blotting and immunofluorescence assay were performed. The data showed that the mRNA and protein levels of DSG2 decreased after knocking down DSP, while knocking down FTO partially restored the mRNA and protein levels of DSG2 ( Figure 4 hi). Immunofluorescence assay showed that the expression of DSG2 on the cell surface was reduced after knocking down DSP, while knocking down FTO partially restored the expression of DSG2 on the cell surface ( Figure 4 j). MeRIP-qPCR further confirmed that the m6A level of DSP in DGGH was significantly higher than that in SGGH ( Figure 4 k). m6A sequencing and MeRIP-qPCR further verified the significant dysregulation of m6A after FTO knockdown ( Figure 4 ln). These results suggest that FTO regulates DSP expression through m6A modification and affects the desmosome function of growth hormone adenomas.

[0094] Example 4: FTO regulates the stability of DSP mRNA by interacting with the m6A reader protein FMR1

[0095] This example explores how FTO-mediated m6A modification affects the level of DSP mRNA. Since DSP mRNA expression is positively correlated with FTO mRNA expression and its m6A modification level is lower in SGGH ( Figure 4 a), therefore, we hypothesized that m6A modification might affect the stability of DSP mRNA.

[0096] Actinomycin D (which inhibits new RNA synthesis) treatment increased the stability of DSP mRNA after FTO knockdown, while FTO overexpression decreased its stability ( Figure 5ac). This suggests that FTO affects the stability of DSP mRNA through m6A. The RMVar database (https: / / rmvar.renlab.org / ) was used to find RNA binding proteins at these sites. A total of five high-confidence sites and one medium-confidence site were found. Among them, site 485 was associated with three RNA binding proteins, namely FMR1, HNRNPC, and SND1. Site 1038 was associated with two RNA binding proteins, namely NUDT21 and ACIN1 ( Figure 5 d). In addition, the expression level of FMR1 in SGGH tends to be lower than that in DGGH ( Figure 5 e). Subsequently, the binding of FMR1 to the 485 site was verified using RIP-qPCR. FTO knockdown significantly reduced the binding of FMR1 to the m6A site on DSP ( Figure 5 f). Importantly, the present invention found that after knocking down FMR1 (using shFmr1-Rat#1, shFmr1-Rat#2, si-Human-FMR1#1, si-Human-FMR1#2 to inhibit FMR1 expression, the shRNA and siRNA sequences used are shown in Table 4), the mRNA, protein levels and mRNA stability of DSP were reduced ( Figure 5 These results suggest that FMR1, as a reader protein of m6A modification, regulates the stability of DSP mRNA.

[0097] Example 5: FTO knockdown inhibits hypoxia tolerance and fibrosome formation in somatotroph adenomas

[0098] To further elucidate the clinical relevance of FTO in regulating invasive growth hormone adenomas, this example conducted a series of functional experiments using cell lines, primary tumor cells and animal models, as follows.

[0099] Experimental methods: CCK8 proliferation function analysis was performed on GH3 cells and primary tumor cells with FTO expression perturbation; clone formation analysis was performed on GH3 cells with FTO expression perturbation; subcutaneous tumor experimental analysis was performed on GH3 cells with FTO expression perturbation; apoptosis analysis was performed on GH3 cells and primary tumor cells with FTO and DSP expression perturbation under normoxic and hypoxic conditions; electron microscopy was performed on primary tumor cells with FTO knockdown. The experimental method of FTO and DSP expression perturbation is referred to Example 3.

[0100] Experimental results: FTO did not affect the proliferation or colony formation of cell lines and primary tumor cells ( Figure 6 ad). In addition, FTO did not affect the tumor volume and weight in subcutaneous tumor formation ( Figure 6eh). Hypoxic conditions may play an important role in pituitary tumorigenesis, and desmosome dysfunction may affect the hypoxic tolerance of growth hormone adenoma cells. The present invention tested the importance of FTO on cell viability under hypoxic conditions (1% oxygen). The results of flow cytometry analysis showed that FTO knockdown did not affect cell apoptosis under normal oxygen conditions, but significantly increased the apoptosis rate under hypoxic conditions. In addition, DSP knockdown increased the apoptosis rate under normal oxygen conditions, but reduced the apoptosis rate under hypoxic conditions ( Figure 6 i). On the other hand, there are differences between DGGH and SGGH in the pathology of fibrillar bodies. The destruction of desmosome organization has been shown to be related to the formation of fibrillar bodies. Electron microscopy showed that after FTO knockdown, the filaments in the fibrillar bodies of primary tumor cells were more sparsely distributed ( Figure 6 j).

[0101] Example 6: Targeting FTO reduces the growth hormone secretion capacity of tumor cells and enhances their sensitivity to somatostatin analogs

[0102] The main treatment for patients with somatotroph adenomas is somatostatin analogs, such as octreotide. However, SGGH patients usually show resistance to these drugs. Therefore, the present invention further evaluated the effect of FTO on the growth hormone secretion capacity of somatotroph adenoma cells and the sensitivity to somatostatin analogs. Octreotide treats somatotroph adenomas mainly by targeting somatostatin receptors, reducing the transcription level of growth hormone, thereby inhibiting its secretion and, to a certain extent, inhibiting the growth of somatotroph adenomas. The reduction of somatostatin receptor 2 (SSTR2) and the increase of somatostatin receptor 5 (SSTR5) are the key features of refractory / persistent somatotroph adenomas. Therefore, the present invention detected the effect of FTO on the transcription and protein levels of SSTR2, SSTR5 and growth hormone.

[0103] Experimental methods: RNA sequencing of GH3 cells with perturbed FTO expression was used to analyze the changes in mRNA levels of Sstr2 and Gh1; qPCR and Western-blot analysis were performed on GH3 cells and primary tumor cells with perturbed FTO and DSP expression; electron microscopy was performed on primary tumor cells with perturbed FTO expression; CCK8 cell viability analysis and growth hormone secretion level analysis were performed on GH3 cells, primary tumor cells and organoids with perturbed FTO expression after combined octreotide and targeted FTO. Subcutaneous tumor experiments were performed on GH3 cells with perturbed FTO expression to analyze changes in octreotide sensitivity.

[0104] RNA sequencing results showed that after FTO knockdown, the level of SSTR2 was significantly upregulated, while the level of the growth hormone encoding gene Gh1 was significantly downregulated ( Figure 7 a). These findings were subsequently validated using qPCR, Western blot, and growth hormone ELISA experiments ( Figure 7 bc). Electron microscopy showed that in primary tumor cells, the secretory granules decreased after FTO knockdown ( Figure 7 d). Regarding sensitivity to octreotide, FTO knockdown enhanced the sensitivity of GH3 and primary tumor cells to octreotide ( Figure 7 e). Treatment with FB23-2, a methyltransferase inhibitor of FTO, enhanced the sensitivity of somatotroph adenoma organoids and cells to octreotide and inhibited the secretion of growth hormone ( Figure 7 fh). In subcutaneous tumors, we found that octreotide more effectively inhibited the growth of FTO knockdown cells and further reduced the secretion of growth hormone ( Figure 7 il). Taken together our results, targeting FTO reduced tolerance in the hypoxic microenvironment, inhibited GH secretion function, and enhanced the sensitivity of GH adenomas to octreotide.

[0105] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A diagnostic reagent for growth hormone adenoma subtype, characterized in that: The diagnostic reagent includes a reagent for detecting the expression level of the m6A demethylase FTO and / or its encoding gene, and / or a reagent for detecting the expression level of the downstream protein of the m6A demethylase FTO and / or its encoding gene.

2. The diagnostic reagent according to claim 1, characterized in that The downstream proteins of the m6A demethylase FTO include: desmosome protein DSP and / or desmosome tissue-related proteins; the desmosome tissue-related proteins include: at least one of PKP3, DSG2, DSP, GRHL1, JUP, PKP2, PRKCA, PERP, and SNAI2.

3. Use of the diagnostic reagent according to claim 1 or 2 in the preparation of a growth hormone adenoma subtype diagnostic product.

4. The use according to claim 3, characterized in that: The product includes a diagnostic reagent, a diagnostic kit or a diagnostic tool.

5. Any of the following applications of m6A demethylase FTO: (A) Application of m6A demethylase FTO inhibitor in the preparation of drugs for adjuvant treatment of growth hormone-producing adenoma; (B) Application of m6A demethylase FTO in screening drugs for the treatment of growth hormone adenoma.

6. The use according to claim 5, characterized in that: The somatotropin adenoma includes densely granulated DGGH somatotropin adenoma and / or sparsely granulated SGGH somatotropin adenoma.

7. The use according to claim 5, characterized in that: The inhibitor includes a nucleic acid inhibitor, which has an m6A demethylase FTO encoding gene or its transcript as a target sequence and is an interfering molecule capable of inhibiting the expression of m6A demethylase FTO or its encoding gene; preferably, the interfering molecule includes: shRNA, siRNA, dsRNA, microRNA, antisense nucleic acid, or a construct or host cell capable of expressing or forming the shRNA, siRNA, dsRNA, microRNA, antisense nucleic acid; preferably, the inhibitor includes siRNA and / or shRNA.

8. A pharmaceutical composition for preventing or treating growth hormone adenoma, characterized in that: The pharmaceutical composition contains an m6A demethylase FTO inhibitor.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition also contains a somatostatin analog.

10. A method for reducing growth hormone secretion in a growth hormone-secreting adenoma in vitro, characterized in that: The method comprises contacting the pharmaceutical composition of claim 8 or 9 with a growth hormone adenoma to reduce the secretion of growth hormone by the growth hormone adenoma; the method does not include a method for diagnosing or treating the disease.

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