Use of compounds targeting HTR2B gene in preparing drugs for treating tumors

Abstract

The present invention provides the use of a compound targeting the HTR2B gene in the preparation of a drug for treating non-functional pituitary adenoma, gastric cancer, uveal melanoma, or renal clear cell carcinoma. The present invention also provides the use of an inhibitor of the HTR2B gene in the preparation of a drug for treating non-functional pituitary adenoma (NFPA), gastric cancer, uveal melanoma, or renal clear cell carcinoma. The inhibitor of the HTR2B gene is PRX‑08066. The present invention finds that the HTR2B gene is highly expressed in NFPA, so it can be used as a target for tumor treatment. At the same time, we found that the use of the HTR2B-specific inhibitor PRX‑08066 can inhibit the in vitro and in vivo growth of non-functional pituitary adenoma alone or in combination with cabergoline; PRX‑08066 can inhibit the growth of primary tumor cells of NFPA, gastric cancer, uveal melanoma, or renal clear cell carcinoma in vitro, and is expected to become a new drug for its treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to HTR2B gene, specifically to the use of a compound targeting HTR2B gene in the preparation of a drug for treating non-functional pituitary adenoma, gastric cancer, uveal melanoma or renal clear cell carcinoma. Background Art

[0002] Pituitary adenomas (pitNETs) are the second most common intracranial tumors, with an annual incidence of 7.5-15 / 100,000 people. Non-functioning pituitary adenomas (NFPAs) are a subtype of pituitary adenomas without excessive secretion of pituitary hormones, accounting for 14%-54% of pituitary adenomas. NFPAs include null cell adenomas and clinically non-functioning pituitary adenomas with hormone expression, especially NFPAs that are positive for transcription factor SF-1. The main clinical manifestations of NFPAs include headaches, visual impairment, and hypopituitarism, and 50% of NFPAs are asymptomatic. Transsphenoidal surgery is the preferred treatment for NFPAs. Experienced neurosurgeons can achieve complete tumor resection in 60-73% of cases. However, 6.9% of patients with completely removed tumors and 40.3%-45.8% of patients with residual tumors experience tumor recurrence after surgery. Postoperative radiotherapy, as an alternative therapy, can increase the 5-year progression-free survival rate from 70% to 95%, while 80% of patients experience hypopituitarism 10-15 years after radiotherapy. Since NFPA expresses dopamine receptors and SSTRs, there are reports of using dopamine receptor agonists (DA) and somatostatin analogs (SSA) to treat recurrent NFPA. A study by Greenman et al. showed that 87% of NFPA patients experienced tumor stabilization or shrinkage after receiving preventive DA treatment after surgery, compared with 46.7% in the control group. In patients with significant residual tumor, this proportion was 58.5%, which was still higher than the control group. Regarding SSA, a study showed that octreotide (a long-term SSA) increased the tumor control rate of SSTR-positive NFPA from 47% to 81%, however, no tumor shrinkage was observed in these patients. Since residual tumors are common after transsphenoidal surgery, new drug therapies are urgently needed to control recurrent NFPA.

[0003] 5-hydroxytryptamine (5-HT), also known as serotonin, is the most widely studied neurotransmitter in the peripheral and central nervous systems. Its receptors have a variety of different receptor subtypes, 13 of which belong to G protein-coupled receptors (GPCRs), which are involved in the regulation of sleep-wake cycles, vomiting, appetite, mood, memory, cognition and many other functions in the central nervous system. In addition, 5-hydroxytryptamine receptors are involved in multiple effects on gastrointestinal motility and cardiac physiology and pathology. As a typical G protein-coupled receptor, serotonin 2B receptor (HTR2B) has been confirmed in past studies to be coupled with Gs and Gi proteins, and can activate the Gβγ-PI3K-Akt pathway and participate in the regulation of cell survival signals. Subsequent studies have found that HTR2B and other 5-HT family receptors are involved in regulating the proliferation of various cells and can promote the occurrence and development of various malignant tumors. However, some studies have also confirmed that it plays an inhibitory role in several human tumors. The expression of HTR2B in different tumors varies greatly, and its effect on pituitary tumors has not been reported. The specific mechanism of regulating cell growth and proliferation needs further study. The HTR2B (5-HT2B) gene is recorded in the NCBI database, see: https: / / www.ncbi.nlm.nih.gov / gene / 3357.

[0004] It has been disclosed that PRX-08066Maleic acid is a selective 5-HT2B receptor antagonist with an IC50 of 3.4nM. It acts on the MCT rat model. In vitro and in vivo studies have shown that the drug can significantly reduce pulmonary artery pressure, inhibit vascular remodeling and improve cardiac function. Summary of the invention

[0005] In view of the above technical problems in the prior art, 1) the present invention provides the use of a compound targeting the HTR2B gene in the preparation of a drug for treating non-functional pituitary adenomas, and the use is to solve the technical problem that the drugs in the prior art are not effective in treating NFPA. 2) The present invention provides the use of a compound targeting the HTR2B gene in the preparation of a drug for treating gastric cancer, uveal melanoma, or renal clear cell carcinoma. 3) The present invention provides the use of an inhibitor of the HTR2B gene in the preparation of a drug for inhibiting the growth of non-functional pituitary adenomas, gastric cancer, uveal melanoma, or renal clear cell carcinoma. Specifically, the HTR2B gene inhibitor is PRX-08066. 4) The present invention provides the use of the HTR2B gene inhibitor PRX-08066 in combination with cabergoline in the preparation of a drug for treating non-functional pituitary adenomas.

[0006] Further, the structural formula of PRX-08066 is:

[0007] The present invention also provides the use of a reagent for detecting HTR2B in preparing a kit for diagnosing non-functional pituitary adenoma, gastric cancer, uveal melanoma, or renal clear cell carcinoma.

[0008] The present invention proves through experiments that the HTR2B gene inhibitor PRX-08066 inhibits the Gaq / PLC / PKC / stat3 signaling pathway through the HTR2B receptor, inhibits the nuclear transfer of pSTAT3 (Tyr705), and thus inhibits cell proliferation.

[0009] Compared with the prior art, the present invention has significant technical progress. The present invention found that the HTR2B (5-HT2B) gene is highly expressed in pituitary neuroendocrine adenomas, and therefore can be used as a target for tumor treatment. At the same time, we found that the use of HTR2B (5-HT2B) treatment-specific inhibitor PRX-08066 can inhibit the in vivo and in vitro growth of non-functional pituitary adenomas (cell experiments and animal experiments) and inhibit the in vitro growth of gastric cancer, uveal melanoma, or renal clear cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 :HTR2B is highly expressed in NFPA and its function study (interference or drug inhibition of HTR2B inhibits the proliferation of pituitary tumor cells). (A) Bulk-mRNA sequencing of pitNETs patient tissues and normal pituitary tissues (GTEX database). (B) Immunohistochemistry of clinical PitNETs tissue samples and normal pituitary tissues. (C) Statistical results of immunohistochemistry. (DF) Nude mice were divided into two groups. The control group was subcutaneously injected with GH3 cells on both sides of the nude mice, and 1×10 6 cells (n=5), and the experimental group was injected subcutaneously with GH3-HTR2B cells on both sides of nude mice (n=5). After the tumor was formed, the tumor volume was measured every 72 hours. After the nude mice were killed, the tumors were removed, photographed, and the tumors were weighed. The tumor tissues were taken for immunohistochemical staining. It was found that the volume and weight of tumors overexpressing HTR2B were significantly increased (p<0.001). Immunohistochemical staining of tumors revealed that Ki67 expression in nude mouse tumors in the experimental group was increased. (DE) Plate cloning experiments showed that interfering with HTR2B expression inhibited the proliferation of GH3 and MMQ cells, respectively. (LK) GH3 cells were injected subcutaneously on both sides of nude mice, with 1×10 6cells (n=5). After the tumor was formed, the nude mice were divided into two groups. The experimental group was gavaged with PRX-08066 every 48 hours, and the control group was gavaged with an equal amount of PBS solution every 48 hours. The tumor volume and nude mouse weight were measured every 48 hours. After the nude mice were killed, the tumors were removed, photographed, and the tumors were weighed. Tumor tissues were taken for immunohistochemical staining. It was found that oral administration of PRX-08066 significantly inhibited the growth of GH3 tumors (p<0.01) and had no significant effect on the weight of nude mice. The expression of Ki67 in nude mouse tumors in the PRX-08066-treated group decreased.

[0011] Figure 2: HTR2B upregulates STAT3 phosphorylation level (Tyr705). (A) Bulk-RNA sequencing was performed on the cell lines GH3, MMQ and AtT20 treated with PRX-08066. Two cross-expressed genes, STAT3 and ARRDC4, were found in GH3 and AtT20 cells. (B) RT-qPCR was performed on GH3 and AtT20 cells treated with PRX-08066, and it was found that STAT3 and ARRDC4 were significantly downregulated in GH3 and AtT20 cells treated with PRX-08066. (C) Western Blot verified the inhibitory effect of PRX-08066 on STAT3 phosphorylation in the AtT20 cell line by time gradient and concentration gradient. (D) Western Blot verified the inhibitory effect of PRX-08066 on STAT3 phosphorylation by time gradient and concentration gradient in the GH3 cell line. (E) Western Blot verified the activation effect of HTR2B overexpression on STAT3 phosphorylation in GH3 cell line and AtT20 cell line. (F) Western Blot verified the inhibitory effect of HTR2B intervention on STAT3 phosphorylation in GH3 cell lines and AtT20 cell lines. (G) Take appropriate amount of GH3 tumor tissues from the experimental group and the control group in the nude mouse tumor model PRX-08066 oral administration experiment, add RIPA lysis buffer, grind with a grinder, and then aspirate the supernatant. Western Blot was used to verify the inhibitory effect of PRX-08066 oral administration on STAT3 activation in subcutaneous GH3 tumors of nude mice. (H) Western Blot verified the inhibitory effect of PRX-08066 concentration gradient on STAT3 phosphorylation in PitNETs primary cells.

[0012] Figure 3:PRX-08066 inhibits STAT3 phosphorylation by inhibiting the Gaq / PLC / PKC signaling axis through HTR2B (HTR2B upregulates STAT3 phosphorylation level through the Gαq-PLC-PKC pathway). (A) Western Blot verified the inhibitory effect of Gαq-specific inhibitor YM254890 and Gβγ complex-specific inhibitor gallein on STAT3 phosphorylation under Alpha-ME activation in GH3 cell line. (B) Western Blot verified the inhibitory effect of Gαq-specific inhibitor YM254890 and Gβγ complex-specific inhibitor gallein on STAT3 phosphorylation under Alpha-ME activation in AtT20 cell line. (C) Western Blot verified the inhibitory effect of PLC inhibitor U73122 and PKC inhibitor Ro318220 on STAT3 phosphorylation under Alpha-ME activation in GH3 cell line. (D) Western Blot verified the inhibitory effects of PLC inhibitor U73122 and PKC inhibitor Ro318220 on STAT3 phosphorylation under Alpha-ME activation in AtT20 cell line.

[0013] Figure 4 :CAB up-regulated the phosphorylation level of STAT3 in pituitary tumor cell lines GH3 and AtT20; CAB combined with 5HTR2B inhibitor PRX-08066 can synergistically inhibit the growth of pituitary tumor cells (A) Western Blot verified the level of STAT3 phosphorylation activation in GH3-shCtrl cell line and GH3-shHTRB-548 under the action of CAB. (B) The combined treatment of CAB and PRX-08066 in GH3 cells further inhibited cell viability compared with single drug. (C) Plate cloning experiment verified the inhibitory effect of combined treatment in GH3 cells. (D) Western Blot verified the level of STAT3 phosphorylation activation in AtT20-shCtrl cell line and AtT20-shHTRB-548 under the action of CAB. (E) The combined treatment of CAB and PRX-08066 in AtT20 cells further inhibited cell viability compared with single drug. (F) Plate cloning experiment verified the inhibitory effect of combined treatment in AtT20 cells. (G) Combination treatment of CAB and PRX-08066 in GH3-shCtrl and GH3-shHTR2B-548 cells revealed that interference with HTR2B partially reversed the inhibitory effect of combined treatment on cell viability. (HI) In GH3 cell lines overexpressing HTR2B, the therapeutic effects of CAB and PRX-08066 were significantly reduced. (JN) GH3 cells were subcutaneously injected on both sides of nude mice, with 1×10 6cells (n=5). After the tumor was formed, the nude mice were divided into four groups. The combined treatment group was gavaged with CAB and PRX-08066 every 48 hours, the CAB treatment group was gavaged with an equal amount of CAB every 48 hours, the PRX-08066 treatment group was gavaged with an equal amount of PRX-08066 every 48 hours, and the control group was gavaged with an equal amount of PBS solution every 48 hours. The tumor volume and nude mouse weight were measured every 48 hours. After the nude mice were killed, the tumors were removed, photographed, and weighed. It was confirmed that the combined treatment of CAB and PRX-08066 had a better inhibitory effect on tumor growth than single drugs (p<0.01), and had no significant effect on the weight of nude mice.

[0014] Figure 5 :GLO-ATP cell viability assay in vitro demonstrated that the combination of PRX-08066 and CAB can inhibit the growth of primary human pituitary tumor cells.

[0015] Figure 6 : TCGA database shows that high expression of HTR2B indicates poor prognosis in gastric cancer, skin and uveal melanoma, and bladder urothelial carcinoma (A). GLO-ATP activity assay in vitro proved that PRX-08066 inhibited the growth of gastric cancer (AGS, HGC27, and MGC-803), uveal melanoma (MuM-2B), and renal clear cell carcinoma (786-O) cells (B); in vitro protein assay proved that PRX-08066 inhibited the growth of gastric cancer (AGS, HGC27, and MGC-803), uveal melanoma (MuM-2B), and renal clear cell carcinoma (786-O) cells; (C, E, and F), but failed to inhibit the growth of skin melanoma cells (B16 and A375, D). DETAILED DESCRIPTION

[0016] Example 1. RNA-seq detection of HTR2B expression levels in human pitNETs

[0017] Experimental materials: pitNETs specimens, Trizol (Thermo Fisher USA)

[0018] Experimental methods:

[0019] (I) Primary cell preparation

[0020] 1. The pitNET specimens removed during surgery are placed in serum-free culture medium and sent to the laboratory.

[0021] 2. Wash three times with Hank's solution and remove fat, connective tissue, blood and other debris.

[0022] 3. Use surgical scissors to cut the tissue into small pieces (1mm 3 ), then washed three times with Hank's solution and transferred to a small penicillin bottle.

[0023] 4. Add 5-6 times the volume of 0.25% trypsin solution depending on the amount of tissue blocks, digest at 37°C for 20-40 minutes, shake once every 5 minutes, or blow once with a pipette to separate the cells.

[0024] 5. Add 3-5 ml of culture medium to terminate trypsin digestion (or add trypsin inhibitor).

[0025] 6. Let stand for 5-10 minutes to allow the undispersed tissue pieces to sink, and then add the suspension to a centrifuge tube.

[0026] 7. Centrifuge at 1000 rpm for 10 minutes and discard the supernatant.

[0027] 8. Add 5 ml of Hank's solution to disperse the cells, centrifuge again, and discard the supernatant.

[0028] 9. Add 1-2 ml of DMEM culture medium and resuspend to obtain a single cell suspension.

[0029] (II) RNA-seq and data analysis

[0030] 1. Extraction of total RNA (see Example 5)

[0031] 2. RNA-seq library preparation and sequencing

[0032] (1) Removal of rRNA: 2 μg of total RNA is required for each sample and Ribo-ZeroTM Magnetic

[0033] Kit and remove rRNA according to the instructions.

[0034] (2) RNA fragmentation: Use NEBNext First Strand Synthesis Reaction Buffer to fragment total RNA that does not contain rRNA

[0035] (3) First Strand cDNA Synthesis: The fragmented RNA obtained in the previous step was reverse transcribed and synthesized into first cDNA using the NEBNext Multiplex Oligos for Illumina Kit.

[0036] (4) 2nd Strand cDNA Synthesis: The fragmented RNA obtained above was reverse transcribed using the NEBNext Multiplex Oligos for Illumina kit to synthesize 2nd cDNA

[0037] (5) Tail end repair: Use the NEBNext Multiplex Oligos for Illumina kit to repair the ds cDNA obtained in the previous step and add an A tail

[0038] (6) Sequencing adapter ligation: The product obtained in the previous step is ligated to the sequencing adapter

[0039] (7) PCR amplification: PCR amplification of the cDNA that has been connected to the adapter for sequencing

[0040] (8) Sequencing: The amplified library was quality checked using a 2100 Bioanalyzer chip. Once the quality check was qualified, sequencing was started using an Illumina HiSeq sequencer according to the operating procedures.

[0041] 3. RNA-seq information analysis

[0042] The raw data in Fastq format were processed by FastQC and NGSQC was used to remove low expression values.

[0043] Quality reads were integrated into the mouse genome (GRCm38 / mm10 version) by Tophat2, and differential expression

[0044] Genes were analyzed using limma and edgeR in R language.

[0045] 4. Experimental results: The analysis found that the expression of HTR2B gene in each lineage of pituitary tumor (PIT1, TPIT, SF1 and Null cell lineage) was higher than that in normal pituitary in the GTEX database ( Figure 1 A) and its statistical analysis ( Figure 1 A down).

[0046] Example 2. Tumor immunohistochemistry detection of HTR2B expression in human PitNETs

[0047] Experimental materials: paraffin tissue chip

[0048] Experimental methods:

[0049] (I) Preparation of paraffin sections

[0050] 1. Get materials

[0051] Tumor specimen collection from surgical patients

[0052] 2. Fixation

[0053] The obtained tissue was washed with physiological saline and immediately placed in neutral formalin fixative for 30-50 minutes.

[0054] 3. Washing

[0055] After the material is fixed, rinse it with running water for several hours or overnight.

[0056] 4. Dehydration

[0057] The materials were dehydrated in 70%, 80%, and 90% ethanol solutions for 30 min each, and then placed in 95% and 100% ethanol solutions twice, each for 20 min.

[0058] 5. Transparency

[0059] Mix equal amounts of pure alcohol and xylene for 15 minutes, xylene I for 15 minutes, and xylene II for 15 minutes (until transparent).

[0060] 6. Waxing

[0061] Put it into a mixture of half xylene and half paraffin for 15 minutes, then put in paraffin I and paraffin II for 50-60 minutes each.

[0062] 7. Embedding

[0063] When embedding, use tweezers to pick up the paraffin mold (metal material) and heat it slightly over an alcohol lamp, then place it on a flat table. Take out the wax cup containing pure paraffin from the incubator and pour a little paraffin into it. Then heat the tweezers slightly over an alcohol lamp, pick up the material and put it into the wax mold with the cut surface facing down, arranging them neatly. Then put the embedding box on it and gently pour in the molten wax.

[0064] 8. Slicing

[0065] ① Place the fixed and trimmed paraffin block on the slicer table.

[0066] ②Fix the slicing knife on the knife holder with the blade facing upwards.

[0067] ③ Shake and push the spiral to bring the paraffin block close to the blade, but not beyond the blade.

[0068] ④ Adjust the angle and position between the paraffin block and the knife edge, so that the blade and the paraffin slice are about 15 degrees.

[0069] ⑤ Adjust the thickness regulator to the required slice thickness, generally 4-10 microns.

[0070] ⑥After everything is adjusted, you can start slicing. At this time, shake the wheel with your right hand to cut the wax block into wax strips, and hold the brush with your left hand to lift the wax strip. The shaking speed should not be too fast, usually 40-50r / min is appropriate.

[0071] ⑦ When the cut wax tape is 20-30cm long, use another brush in your right hand to gently pick up the wax tape to prevent it from curling, and pull it into a tape, and lay it flat on the wax tape box with the smoother side close to the knife facing down, and the more wrinkled side facing up.

[0072] ⑧ Use a single-sided blade to cut a small section of wax film, place it on a slide, add a drop of water, and observe under a magnifying glass or microscope to see if the section is good.

[0073] ⑨ After the slicing work is completed, the slicer should be removed and the paraffin on the knife should be wiped off with chloroform, and the slicer should be wiped clean and properly stored.

[0074] 9. Spreading film, patch

[0075] Turn on the water bath to maintain the water temperature at 40-45°C and prepare 30% ethanol solution.

[0076] ① When slicing, place a bowl of 30% ethanol solution on the table next to the slicer.

[0077] ② Use small tweezers to pick up the wax strip that has been cut with a blade in advance, and place it on the surface of the ethanol solution to unfold the slice.

[0078] ③ Use small tweezers to gently separate the connected slices, and use a glass slide to put the intact and unfolded slices into warm water to fully unfold them.

[0079] ④ Take another clean glass slide, pick up the unfolded slice, place it at 1 / 3 of the slice, mark or label the other end on the grinded surface, and place it on the slice rack.

[0080] 10. Dewaxing and rehydration

[0081] Adjust the temperature of the water bath to 60°C. When the water temperature is controlled at 60°C, place the slices and the slice rack into a dry staining jar, put it into the water bath, cover it with a lid (can be sealed), and leave it for 30 minutes until the wax melts.

[0082] Afterwards, the paraffin sections were dewaxed with xylene I and II for 5 min each, then placed in 100%, 95%, 90%, 80%, and 70% alcohol solutions for 3-5 min each, and then placed in distilled water for 3 min.

[0083] 11. Dyeing

[0084] The sections were stained in hematoxylin for about 10-30 min.

[0085] 12. Washing

[0086] Rinse with running water for about 15 minutes to make the slices turn blue to prevent them from falling off.

[0087] 13. Differentiation

[0088] Place the slices in 1% hydrochloric acid ethanol solution to fade for about 2 seconds to tens of seconds. When the slices turn red and the color becomes lighter, it is done.

[0089] 14. Rinse

[0090] The slices were then placed in running tap water to restore their blue color.

[0091] 15. Dehydration I

[0092] The slices were immersed in 50% ethanol → 70% ethanol → 80% ethanol for 3-5 min each.

[0093] 16. Counterstaining

[0094] Contrast staining was performed with 0.5% eosin ethanol solution for 1-3 min.

[0095] 17. Dehydration II

[0096] Wash the slices in 95% ethanol to remove excess red, then place them in anhydrous ethanol for 3-5 minutes. Finally, use absorbent paper to absorb excess ethanol.

[0097] 18. Transparency

[0098] The slices were placed in xylene I and II for 3-5 min each.

[0099] 19. Sealing: Neutral gum sealing

[0100] (II) Immunohistochemistry

[0101] 1. Place the paraffin sections in a 60°C constant temperature oven and bake for 120 minutes;

[0102] 2. Dewaxing and hydration: xylene (10 min) → xylene (10 min) → anhydrous ethanol (5 min × 2 times) → 95% ethanol (5 min

[0103] ×2)→90% (5 min)→85% ethanol (5 min)→80% ethanol (5 min)→75% ethanol (5 min);

[0104] 3. Rinse with distilled water twice for 5 minutes;

[0105] 4. Add 3% H2O2 and incubate for 30 min (room temperature);

[0106] 5. Wash twice with distilled water;

[0107] 6. Antigen repair: high pressure repair or microwave repair | Citric acid repair solution: 0.4g citric acid and 3g sodium citrate to make 1000ml

[0108] High pressure repair: high pressure 2 minutes; microwave repair: preheat 5 minutes, high fire 4 minutes, medium fire 5 minutes;

[0109] 7. Wash with PBS 3 times for 5 minutes each, and block for 30 minutes;

[0110] 8. Add primary antibody and incubate at 4°C overnight or at 37°C for 2-3 hours;

[0111] 9. Rinse with PBS 3 times for 5 minutes each;

[0112] 10. Add secondary antibody and incubate at 37°C for 30-60 min;

[0113] 11. Wash with PBS 3 times for 5 min each;

[0114] 12. DAB color development, rinse thoroughly with tap water;

[0115] 13. Re-stain with hematoxylin and rinse thoroughly with tap water; 14. Dehydrate and make transparent for 5 to 10 minutes each;

[0116] 14. Sealing: Neutral resin, cover with glass slide (do not leave small bubbles in the tissue area), and let it dry naturally.

[0117] (III) Data Statistics

[0118] All surgical pathology samples were reviewed independently by two pathologists. Histochemistry score (H-SCORE) is a histological scoring method for processing immunohistochemistry results. It converts the number of positive cells and their staining intensity in each section into corresponding values ​​to achieve the purpose of semi-quantitative tissue staining.

[0119] For example, H-score = Σpi(i+1) is used, where pi represents the percentage of positive cells to all cells in the slice; i represents the staining intensity.

[0120] Experimental results: HTR2B is highly expressed in the SF1 lineage (NFPA) ( Figure 1 B and C)

[0121] Example 3. Confirmation of the promoting effect of overexpression of HTR2B on tumor growth and the inhibitory effect of PRX-08066 on tumor growth in mice

[0122] Experimental Materials:

[0123] Human renal epithelial cell line HEK293T cells, rat pituitary tumor cell lines GH3 and MMQ cell lines, and mouse pituitary tumor cell line AtT20 cell line were purchased from ATCC, USA. pLVshRNA-EGFP-Puro (shRNA-HTR2B) and pLV-EGFP-Puro (pLV-HTR2B) were purchased from General Biotechnology (Anhui) Co., Ltd. Experimental methods:

[0124] (I) Transfection of pLV-HTR2B plasmid:

[0125] The cells were suspended in regular culture medium and the cell density was adjusted to 5 × 10 5 Cells / well were seeded in 12-well culture plates.

[0126] 1. Change the culture medium of cells one day before transfection

[0127] 2. Prepare DNA-Lipofectamine 2000 complex as follows:

[0128] a. Dilute DNA (0.8 g) with 50 ul serum-free medium and mix gently;

[0129] b. Before using Lipofectamine 2000, mix gently, dilute 2ulLipofectamine 2000 with 48ul serum-free medium, mix gently, and incubate at room temperature for 5 minutes;

[0130] c. Add liquid a+b together, mix gently, and incubate at room temperature for 20 minutes to allow the DNA-Lipofectamine2000 complex to form;

[0131] 3. Add 100ul DNA-Lipofectamine 2000 complex to the culture plate and shake gently back and forth;

[0132] 4. After culturing in a 37-degree incubator for 24 to 48 hours, subculture at a ratio of 1:10 and add puromycin for selection (GH3, 2ug / ml; MMQ 1ug / ml; AtT20, 1ug / ml).

[0133] 5. Wait for the cells to grow full under the action of G418 and freeze them for seed preservation. Re-cloning culture: After digesting the cells, count 50 cells, add 22ml of complete medium, mix well, and dispense into 96-well plates (200ul / well). The next day, under a microscope, find the wells with single cells and mark them. After the marked wells are full, digest and transfer them to 4-well or 6-well culture plates.

[0134] (II) Establishment of nude mouse tumor-bearing animal model

[0135] 1. Generally, 4-6 weeks old nude mice are selected

[0136] 2. Collect the logarithmically growing pLV-HTR2B overexpressing or control GH3 cells, add 10 ml of serum-free matrix, and incubate at 1500

[0137] rpm, centrifuged for 3 min, washed three times in a row, and finally mixed with serum-free matrix. The number of tumor cells was counted using a hemocytometer (average cell count in 5 squares × 10 4 After calculating the total number of tumor cells, the tumor cell density was adjusted to 10 7 Each mouse was injected subcutaneously into the thigh muscle with 100ul (i.e. 1×

[0138] 10 6 Tumor cells), small tumor nodules can be palpated in about 2 weeks.

[0139] 3. For the experiment to prove that PRX-08066 inhibits tumors, when the tumor volume grew to about 100 mm3, the animals were divided into DMSO and PRX-08066 groups, and DMSO and PRX-08066 were intraperitoneally injected once every two days. The vertical and horizontal sizes of the tumors (unit: mm) were measured with a vernier caliper.

[0140] 4. After 21 days of administration, the nude mice were killed, the tumors were removed, weighed, photographed, and cut into pieces and stored in cryopreservation tubes and 4% formalin.

[0141] 5. Perform tumor immunohistochemical staining (Ki67 staining) according to the steps in Example 2.

[0142] Experimental results: In in vivo animal experiments, overexpression of HTR2B promoted the growth of PitNETs cell lines GH3 and Att20 tumors ( Figure 1 D, E, F), while PRX-08066 can inhibit tumor cell growth ( Figure 1 I, J, K).

[0143] Example 4. The clone formation test confirmed that PRX-08066 inhibited the growth of GH3 cells, and the inhibitory effect of PRX-08066 was weakened after intervening in HTR2B expression.

[0144] Experimental equipment: Rat pituitary tumor cell line GH3 cells were purchased from ATCC, USA. PRX-08066 (purchased from MedChemExpress, USA)

[0145] Experimental methods:

[0146] 1. Digest the GH3 cells of the control group or shHTR2B in the logarithmic growth phase with trypsin, resuspend them in complete medium (basal medium + 10% fetal bovine serum) into a cell suspension, and count them;

[0147] 2. Cell inoculation: Inoculate 400-1000 cells / well in each experimental group in a 6-well culture plate (determined according to cell growth, generally 700 cells / well; if it is a 12-well plate, the system is halved); add PRX-08066 at the concentration shown in the figure

[0148] 3. Continue culturing until 14 days or until the number of cells in most single clones is greater than 50, changing the medium every 3 days and observing the cell status;

[0149] 4. After cloning is completed, take pictures of the cells under a microscope, then wash once with PBS, add 1 mL of 4% paraformaldehyde to each well for fixation for 30-60 minutes, and wash once with PBS;

[0150] 5. Add 1 ml of crystal violet stain to each well and stain the cells for 20 minutes;

[0151] 6. Wash the cells twice with 1 ml of PBS, dry them, and take pictures with a digital camera (take pictures of the entire six-well plate and each well separately).

[0152] Experimental results: PRX-08066 inhibited GH3 cell clone formation, and the inhibitory effect of PRX-08066 was weakened after intervening in the expression of HTR2B ( Figure 1 G,H).

[0153] Example 5. RNA-seq screening of PRX-08066-affected genes STAT3 and ARRDC4

[0154] Experimental materials: Rat pituitary tumor cell line GH3 cells were purchased from ATCC, USA. PRX-08066 (purchased from MedChemExpress, USA).

[0155] Experimental methods:

[0156] (I) Sample preparation

[0157] 1. Inoculate 10 6 cells / well, the culture medium volume was 2 ml / well, and PRX-08066 was added to make the concentration 20 μM

[0158] 2. Culture in an incubator at 37°C and 5% CO2 for 24 hours.

[0159] (ii) RNA-seq (see Example 1)

[0160] (III) qRT-PCR

[0161] (1) RNA extraction and RT-PCR

[0162]

On Ice

[0163] 1. Collect cells and keep 1×10 cells in each tube 7 Cells

[0164] 2. Centrifuge at 3600 rpm for 3 min and discard the supernatant.

[0165] 3. Add 1 ml Tripure reagent to each sample (10 6 -10 7 1 ml), vortex vigorously to shake off the cells and completely lyse them.

[0166] 4. Leave at room temperature for 5 minutes, or store at 4 degrees.

[0167] 5. Add 1 / 5 volume of trisol chloroform (chloroform) to each tube, vortex vigorously until it becomes milky, (stand at room temperature for 5 minutes)

[0168] 6. Centrifuge at 12000 rpm, 4°C for 15 min (the sample is divided into three layers: the bottom layer is a yellow organic phase, the upper layer is a colorless aqueous phase and an intermediate layer. The RNA is mainly in the aqueous phase, and the volume of the aqueous phase is about 60% of the Tripure reagent used).

[0169] 7. Add 400ul of isopropanol (melting point -88.5℃, will not solidify if pre-cooled) to a new EP tube (equal volume of isopropanol).

[0170] 8. Carefully aspirate the supernatant.

[0171] 9. Add 400ul to pre-cooled isopropanol and gently invert (let stand at room temperature for 20min), or leave at -20℃ overnight.

[0172] Prepare 70% ethanol using DEPC water and anhydrous ethanol.

[0173] 10. Centrifuge at 12000 rpm, 4°C for 15 min and discard the supernatant.

[0174] 11. Add 0.8 ml of 70% ethanol, shake gently until the precipitate floats, and centrifuge at 10,000 rpm and 4°C for 10 min. Discard the supernatant, and centrifuge at 1,000 rpm for 1 min. Aspirate the remaining supernatant, invert on filter paper, and dry the RNA precipitate in air.

[0175] 12. Dissolve RNA and add 10-50 μl DEPC water.

[0176] 13.Nanodrop measures RNA concentration and purity.

[0177] (2) Reverse transcription

[0178] Reverse transcription (RT)-takara kit [on ice]

[0179] ① Loading samples into eight tubes

[0180] Example: 20μl system

[0181] Reagent usage

[0182] 5×ELV reverse transcription premix 4μl

[0183] Total RNA 1000ng

[0184] RNase Free dH2O to make up to 20 μl

[0185] Mix gently and then perform reverse transcription

[0186] ②Thermocycler reverse transcription

[0187] 37℃15min (reverse transcription)

[0188] 85℃5sec (inactivation reaction of reverse transcriptase)

[0189] 4℃ insulation

[0190] After completion, the cDNA was stored at 4°C

[0191] (3)PCR

[0192] ① 96-well dedicated sample loading plate

[0193] Real Time PCR system——10μl system or 20μl [operate on ice]

[0194] Example: 10μl system

[0195] Reagent usage

[0196] SYBR@Green Premix (containing rox) (2×) 5μl

[0197] PCR Forward Primer(10μM)0.4μl

[0198] PCR Reverse Primer(10μM)0.4μl

[0199] cDNA solution (this solution is the cDNA solution after 1000ng mRNA reverse transcription, diluted 30 times) 3μl

[0200] DEPC or ddH20 (sterile distilled water) 1.2 μl

[0201] ②After adding everything, stick the qPCR-specific transparent film first.

[0202] ③ Centrifuge at 1000rpm for 1min

[0203] ④Get on the machine

[0204] Experimental results: RNA-seq and qPCR successfully screened and verified that PRX-08066 downregulated the expression of gene STAT3 in GH3 and Att20 cell lines (Figure 2A, B).

[0205] Example 6. Western Blot demonstrated that overexpression of HTR2B could upregulate the phosphorylation level of STAT3 in GH3 and AtT20, and interference with 5HTR-2b or use of PRX-08066 could downregulate the phosphorylation level of STAT3.

[0206] Experimental Materials:

[0207] Human renal epithelial cell line HEK293T cells, rat pituitary tumor cell line GH3 cells, and mouse pituitary tumor cell line AtT20 cells were purchased from ATCC, USA.

[0208] Plasmids: pLVshRNA-EGFP-Puro (shRNA-HTR2B), pLV-EGFP-Puro (pLV-HTR2B) were purchased from General Biotechnology (Anhui) Co., Ltd.

[0209] Antibodies: STAT3 primary antibody (Cell Signaling Technology, USA), p-STAT3 primary antibody (Cell Signaling Technology, USA), HTR2B primary antibody (ABclonal, Wuhan, China), β-actin primary antibody (Sevier, Wuhan, China), goat anti-rabbit (mouse) secondary antibody (Cell Signaling Technology, USA), protein marker (ThermoFisher USA)

[0210] Experimental methods:

[0211] (I) pLV-HTR2B overexpression and shHTR2B knockdown plasmid transfection: Same as Example 3

[0212] (II) Sample preparation

[0213] 1. Inoculate 10 6cells / well (the seeded cells were GH3 or AtT20), the culture medium volume was 2 ml / well, PRX-08066 was added as shown in the figure (the concentration gradient experiment was as shown in the figure, and the concentration of the time gradient experiment was 20 μM), and an equal volume of PBS was added to the control group.

[0214] Routine culture was performed in an incubator at 2.37°C and 5% CO2. The culture time was as shown in the figure for the time gradient experiment. The concentration gradient time was 24h.

[0215] (III) Extraction of total cell protein and Western Blot.

[0216] (1) Preparation of cell culture lysate

[0217] 1. Place cells on ice and wash cells with ice-cold PBS.

[0218] 2. Aspirate PBS and add ice-cold lysis buffer (10 7 cells / 100mm culture dish / 150cm2 flask, add 1mL; every 5x10 6 cells / 60mm dish / 75cm2 flask plus 0.5mL).

[0219] 3. Use a pre-chilled plastic cell scraper to scrape the adherent cells from the culture dish, and then gently transfer the cell suspension to a pre-chilled centrifuge tube. Alternatively, digest the cells with trypsin and wash the cells with PBS, then resuspend the cells in lysis buffer in a small centrifuge tube.

[0220] Continuously shake at 4.4°C for 30 minutes.

[0221] 5. Place in a microcentrifuge and centrifuge at 4°C and 12,000 rpm for 20 minutes.

[0222] 6. Gently remove the centrifuge tube from the centrifuge and place it on ice. Aspirate the supernatant and transfer it to a new tube pre-chilled on ice, and discard the pellet.

[0223] (2) Sample preparation

[0224] 1. Take a small amount of lysate for protein quantitative analysis. Determine the protein concentration of each cell lysate.

[0225] 2. Determine the amount of protein to be loaded and add an equal volume of 2X diluted Laemmli sample buffer. We recommend using

[0226] The following methods reduce and denature samples unless the online antibody data sheet indicates that non-reducing and non-denaturing conditions should be used.

[0227] 3. When reducing and denaturing the sample, boil the cell lysate in the sample buffer at 100°C for 5 minutes. The lysate can be aliquoted and stored at -20°C for later use.

[0228] (3) Glue preparation

[0229] 1. Wash the glass plate, sample comb, and spacer with detergent, rinse with ddH2O several times, wipe with ethanol, and dry;

[0230] 2. Place a spacer between two clean glass plates and install the glass plates according to the instructions in the Bio-Rad MiniⅡ / Ⅲ manual;

[0231] 3. Prepare 8.0 ml of 10% separation gel according to the following volume and mix well: 3.0 ml of ddH2O; 2.1 ml of 1.0 mol / L Tris-HCl pH = 8.8

[0232] ml; 30% Acr-Bis 2.8ml; 10% SDS 80ul; 10% AP 56ul; TEMED 6ul.

[0233] 4. Pour separation gel between the glass plates and immediately cover with a layer of heavy distilled water. The gel will polymerize after about 20 minutes.

[0234] 5. Prepare 6% concentrated gel 3.0ml according to the following volume and mix well: ddH2O 1.0mol / LTris-HCl pH=6.830% Acr-Bis; 2.0ml 400ul 600ul; 10% SDS 10% AP TEMED; 36ul 24ul 4ul.

[0235] 6. Pour off the upper layer of heavy distilled water, absorb dryness with filter paper, prepare concentrated gel, and insert the sample comb.

[0236] (4) Sample loading and gel running

[0237] 1. Load equal amounts of protein and molecular weight markers into the wells of SDS-PAGE gel. The total protein loading amount of cell lysate or tissue homogenate is 20-30 μg, and the loading amount of purified protein is 10-100 ng.

[0238] 2. Run the gel at 100V for about 1-2 hours.

[0239] The gel percentage depends on the size of the target protein.

[0240] Protein size Gel percentage 4–40 kDa 20％ 12–45 kDa 15％ 10-70 kDa 12.5％ 15-100 kDa 10％ 25-100 kDa 8％

[0241] (5) Protein transfer from gel to membrane

[0242] Choose PVDF membrane. Activate PVDF with methanol for 1 minute and rinse PVDF with transfer buffer before preparing the transfer layer. Transfer time and voltage may need to be optimized. We recommend following the manufacturer's instructions. Protein transfer can be checked by Ponceau S staining before the blocking step.

[0243] (6) Antibody staining

[0244] 1. Block the membrane with blocking buffer for 1 hour at room temperature or overnight at 4°C.

[0245] 2. Incubate the membrane with appropriately diluted primary antibodies (p-STAT3, STAT3, HTR2B, β-actin) in blocking buffer. We recommend overnight incubation at 4°C; other conditions can be optimized.

[0246] 3. Wash the membrane three times with TBST, 5 minutes each time.

[0247] 4. Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer for 1 hour at room temperature.

[0248] 5. Wash the membrane three times with TBST, 5 minutes each time.

[0249] 6. When the signal is generated, follow the kit manufacturer's recommendations. Remove excess reagents and cover the membrane with clear plastic film.

[0250] 7. Use darkroom development techniques to capture chemiluminescent images, or use conventional image scanning methods to capture colorimetric detection images.

[0251] Experimental results:

[0252] Overexpression of HTR2B upregulated the phosphorylation level of STAT3 in GH3 and AtT20 ( Figure 2E ), and interference with HTR2B or use of PRX-08066 downregulated the phosphorylation level of STAT3 in GH3, AtT20, and human primary PitNETs cells ( Figure 2C, D, F, G, H ).

[0253] Example 7. PRX-08066 inhibits STAT3 phosphorylation by inhibiting the Gαq / PLC / PKC signaling axis via HTR2B.

[0254] Experimental materials and methods:

[0255] Rat-derived pituitary tumor cell line GH3 cells, mouse-derived pituitary tumor cell line AtT20 cells (ATCC). α-Methyl-5-hydroxytryptamine maleate (Alpha-ME, Tocris Bioscience, UK), Ro-31-8220 (Biyuntian, Shanghai, China), U73122 (MedChemExpress, USA), YM-254890 (MedChemExpress, USA), PRX-08066 (MedChemExpress, USA), Gallein (MedChemExpress, USA) Experimental results: PRX-08066 inhibits the Gaq / PLC / PKC signaling axis through HTR2B, thereby inhibiting STAT3 phosphorylation.

[0256] ( Figure 3 AD)

[0257] Example 8. Western Blot demonstrates that cabergoline (CAB) upregulates STAT3 phosphorylation levels in GH3 and AtT20 and is dependent on HTR2B

[0258] Experimental materials: rat pituitary tumor cell line GH3 cells, mouse pituitary tumor cell line AtT20 cells (ATCC). Cabergoline (Sigma, USA), PRX-08066 (MedChemExpress, USA); plasmids: pLVshRNA-EGFP-Puro (shRNA-HTR2B), pLV-EGFP-Puro (pLV-HTR2B) were purchased from General Biotechnology (Anhui) Co., Ltd.

[0259] Experimental methods:

[0260] (I) shHTR2B knockdown plasmid transfection: Same as Example 3

[0261] (I) Sample preparation

[0262] 1. Inoculate 10 6 cells / well, medium volume 2ml / well, add CAB and PRX-08066 according to the concentration shown in the figure

[0263] 2.37℃, 5% CO2 incubator for 24 hours

[0264] (ii) Western Blot: Same as Example 5

[0265] Experimental results: Cabergoline (CAB) up-regulated the phosphorylation level of STAT3 in GH3 and AtT20, and this effect disappeared after interfering with shHTR2B ( Figure 4 A, D).

[0266] Example 9. GLO-ATP cell viability assay demonstrated in vitro that PRX-08066 and CAB can jointly inhibit the growth of GH3, AtT20 cell line and human PitNETs primary cells; overexpression of HTR2B can lead to GH3 resistance to CAB.

[0267] Experimental Materials:

[0268] As in Example 8, primary cells were extracted from NFPA surgical specimens

[0269] Experimental methods:

[0270] (I) Transfection of GFP-5HTR-2b overexpression and shHTR2B knockdown plasmids: see Example 3

[0271] (II) Primary cell preparation and culture

[0272] 1. During surgery, the human pituitary neuroendocrine adenoma specimen is removed and placed in serum-free culture medium and sent to the laboratory.

[0273] 2. Wash three times with Hank's solution and remove fat, connective tissue, blood and other debris.

[0274] 3. Use surgical scissors to cut the tissue into small pieces (1mm 3 ), then washed three times with Hank's solution and transferred to a small penicillin bottle.

[0275] 4. Add 5-6 times the volume of 0.25% trypsin solution depending on the amount of tissue blocks, digest at 37°C for 20-40 minutes, shake once every 5 minutes, or blow once with a pipette to separate the cells.

[0276] 5. Add 3-5 ml of culture medium to terminate trypsin digestion (or add trypsin inhibitor).

[0277] 6. Let stand for 5-10 minutes to allow the undispersed tissue pieces to sink, and then add the suspension to a centrifuge tube.

[0278] 7. Centrifuge at 1000 rpm for 10 minutes and discard the supernatant.

[0279] 8. Add 5 ml of Hank's solution to disperse the cells, centrifuge again, and discard the supernatant.

[0280] 9. Add 1-2 ml of DMEM culture medium and count on a hemocytometer.

[0281] 10. Adjust the cells to 5×10 5 / ml, transferred to a 25ml cell culture flask and cultured at 37℃.

[0282] (III) GLO-ATP cell viability assay

[0283] 1. The cells were suspended in regular culture medium and the cell density was adjusted to 1×10 4 Cells / well were seeded in 96-well culture plates. Drug-containing medium was added with PRX-08066 and CAB at concentrations shown in the figure. The blank control group contained 0.3% DMSO.

[0284] 2. The cells after administration were cultured at 37°C and 5% CO2 for 24 hours.

[0285] 3. Add CellTiter-GLO solution to each well, incubate on a shaker for 2 minutes, and incubate at room temperature in the dark for 15 minutes.

[0286] 4. Detect chemiluminescence using an enzyme-labeled instrument.

[0287] Experimental results: PRX-08066 and CAB can jointly inhibit the growth of GH3, AtT20 cell lines and human PitNETs primary cells ( Figure 4 B, E; Figure 5 ), when HTR2B is overexpressed or knocked down, the inhibitory effect of CAB on GH3 is weakened or enhanced ( Figure 4 H, I, G).

[0288] Example 10. In vitro clone formation experiments demonstrated that PRX-08066 and CAB can jointly inhibit the growth of GH3 and AtT20 cell lines. Experimental materials: Cabergoline (Sigma, USA), PRX-08066 (MedChemExpress, USA), and the rest are the same as in Example 3.

[0289] Experimental method: The dosing method is shown in the figure, and the rest is the same as in Example 4

[0290] Experimental results: PRX-08066 and CAB can jointly inhibit the growth of GH3 and Att20 cell lines in vitro ( Figure 4 C, F)

[0291] Example 11. Nude mouse xenograft tumor model demonstrated in vivo that PRX-08066 and CAB can jointly inhibit GH3 and AtT20 tumor growth.

[0292] Experimental materials: Cabergoline (Sigma, USA), PRX-08066 (MedChemExpress, USA); the rest is the same as in Example 3

[0293] Experimental method: Cabergoline injection method: intraperitoneal injection, the control group was injected with an equal volume of PBS, and the rest was the same as in Example 3. Experimental results: PRX-08066 and CAB can jointly inhibit the growth of GH3 and AtT20 tumors in vivo ( Figure 4 J, K, L, M, N).

[0294] Example 12. TCGA Pan-Cancer Atlas and Survival Analysis

[0295] Experimental materials: TCGA Pan-Cancer Atlas

[0296] Experimental methods: RNA-seq gene expression and clinical characteristics of the TCGA Pan-Cancer Atlas were downloaded from UCSC Xena (https: / / xenabrowser.net / ) using the R package UCSCXenaTools. TCGA PanCanAtlas data including 9,875 primary tumor samples of 33 tumor types were evaluated. The expression level of genes was evaluated using FPKM. We performed survival analysis for each TCGA cohort, generated survival curves using the Kaplan-Meier method, and used the log-rank test to determine statistical significance. We estimated median values ​​based on the association between gene expression and patient overall survival in each independent TCGA cohort using the R package survminer (https: / / CRAN.R-project.org / package=survminer). For univariate analysis, we calculated hazard ratios using the Cox proportional hazards regression model using the R package survival (https: / / CRAN.R-project.org / package=survival). Experimental results: TCGA suggests that high expression of HTR2B in gastric cancer (STAD), skin (SKCM) and uveal melanoma (UVM), and bladder urothelial carcinoma (BLCA) indicates a poor prognosis ( Figure 6 A).

[0297] Example 13. GLO-ATP activity assay In vitro demonstrated that PRX-08066 inhibited the growth of gastric cancer (AGS, HCG27 and MGC-803), uveal melanoma (MuM-2B) and renal clear cell carcinoma (786-O) cells.

[0298] Experimental materials: PRX-08066 (MedChemExpress, USA), gastric cancer cell lines (AGS, HCG27 and MGC-803), uveal melanoma (MuM-2B), renal clear cell carcinoma (786-O) and skin melanoma cell lines (B16 and A375). Experimental methods: The rest is the same as in Example 4.

[0299] Experimental results: PRX-08066 inhibited gastric cancer cell lines (AGS, HCG27 and MGC-803) in vitro.

[0300] Cell growth of uveal melanoma (MuM-2B) and renal clear cell carcinoma (786-O) Figure 6 B).

[0301] Example 14. PRX-08066 inhibits STAT3 phosphorylation in gastric cancer, uveal melanoma and renal clear cell carcinoma cell lines. Experimental materials: PRX-08066 (MedChemExpress, USA), gastric cancer cell lines (AGS, HCG27 and MGC-803), uveal melanoma (MuM-2B), renal clear cell carcinoma (786-O) and skin melanoma cell lines (B16 and A375).

[0302] Experimental method: The rest is the same as Example 6.

[0303] Experimental results: In vitro protein experiments demonstrated that PRX-08066 inhibited the growth of gastric cancer (AGS, HGC27 and MGC-803), uveal melanoma (MuM-2B) and renal clear cell carcinoma (786-O) cells; Figure 6 -C, E and F), but failed to inhibit the growth of skin melanoma cells (B16 and A375, Figure 6 -D).

Claims

1. Use of a compound targeting the HTR2B gene in the preparation of a drug for treating pituitary adenoma, wherein the compound is PRX-08066, and the structural formula is: .

2. The use of HTR2B gene inhibitor PRX-08066 in combination with cabergoline in the preparation of a drug for treating pituitary adenoma, wherein the structural formula of PRX-08066 is: .

3. Use of a reagent for detecting HTR2B in the preparation of a kit for diagnosing pituitary adenoma, wherein the reagent for detecting HTR2B is PRX-08066, and the structural formula is: .

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