Application of NOTCH3 as target in preparation of pharmaceutical composition for treating bladder cancer

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of NOTCH3 serving as a target spot to preparation of a pharmaceutical composition for treating bladder cancer. The invention relates to a targeted inhibitor of NOTCH3, which is shRNA (short hairpin ribonucleic acid). Experimental results show that NOTCH3 is a key oncogene of bladder cancer and can be used as a therapeutic target of bladder cancer, and an inhibitor of NOTCH3 can be used for preparing a pharmaceutical composition for treating bladder cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the use of NOTCH3 as a target in preparing a pharmaceutical composition for treating bladder cancer. Background Art

[0002] Bladder cancer is a global health problem. It is the tenth most common cause of cancer and the thirteenth most common cause of cancer death worldwide. According to statistics, there are more than 570,000 new cases of bladder cancer and about 210,000 deaths worldwide each year, which imposes a heavy burden on the medical and health system. According to the invasiveness and pathological characteristics, bladder cancer is usually divided into non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). Although standard treatments such as radiotherapy, chemotherapy and surgery have significantly prolonged the survival of bladder cancer patients, the high recurrence rate of NMIBC and the high invasiveness of MIBC have not been completely solved. Therefore, in-depth research on the pathogenic mechanism of bladder cancer and its key driving genes, and the development of more accurate and efficient treatment methods have become an urgent need to improve patients' prognosis and quality of life.

[0003] The Notch signaling pathway is a highly conserved signal transduction mechanism that involves transmembrane receptors encoded by the Notch gene. It widely regulates important biological processes such as cell fate determination, embryonic development, tissue repair, and organ formation. The Notch family includes four receptors (Notch1-4). Studies have shown that abnormal regulation of the Notch signaling pathway is closely related to the occurrence and progression of a variety of solid tumors, including breast cancer, hepatocellular carcinoma, colorectal cancer, and gastric cancer. Although the function of Notch signaling in a variety of cancers has been widely studied, its specific role in the malignant progression of bladder cancer remains unclear. Studies have found that the expression of Notch1 and Notch2 is closely related to the prognosis of bladder cancer patients, but there are still few reports on the role and potential mechanisms of Notch3 in bladder cancer, which urgently need further exploration. Summary of the invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to explore the regulatory role of NOTCH3 in bladder cancer and provide new targets and new ideas for the treatment of bladder cancer.

[0005] The technical solution provided by the present invention is: use of NOTCH3 as a target in preparing a pharmaceutical composition for treating bladder cancer.

[0006] Preferably, the pharmaceutical composition is a targeted inhibitor of NOTCH3.

[0007] The present invention also provides a targeted inhibitor of NOTCH3, wherein the targeted inhibitor of NOTCH3 is shRNA.

[0008] Furthermore, the nucleotide sequence of shRNA is shown in the sequence listing SED ID NO.1.

[0009] The present invention also provides a pharmaceutical composition for treating bladder cancer, wherein the composition comprises the NOTCH3 targeted inhibitor.

[0010] The experimental results of the present invention found that NOTCH3 expression was elevated in bladder cancer tissue. After targeted inhibition of NOTCH3, the proliferation, invasion and migration abilities of bladder cancer cells decreased. In addition, by targeted inhibition of NOTCH3 in vivo, the growth and metastasis of mouse bladder tumors were inhibited, indicating that NOTCH3 is a potential therapeutic target for bladder cancer, and inhibitors that target NOTCH3 inhibition are potential new drugs for the treatment of bladder cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a high expression diagram of NOTCH3 in bladder cancer; (A) is the analysis of the expression of NOTCH3 in the bladder cancer datasets GSE13507 and GSE37815; (B) is the relationship between NOTCH3 expression and patient survival; (C)-(E) are the expression of NOTCH3 in tumor tissues and paired adjacent cancer tissues detected at the protein and mRNA levels respectively; Figure 2 The expression diagram of NOTCH3 in bladder cancer after silencing. (A) and (B) are the transfection efficiency of NOTCH3 detected at the protein and mRNA levels, respectively. Figure 3 The proliferation, invasion and migration of bladder cancer cells were inhibited after NOTCH3 was silenced; the proliferation ability of bladder cancer cells after NOTCH3 was silenced was detected by CCK8 (A) and plate cloning assay (B); the migration ability of bladder cancer cells after NOTCH3 was silenced was detected by scratch assay (C); the migration and invasion ability of bladder cancer cells after NOTCH3 was silenced was detected by Transwell assay (D); Figure 4 The growth and metastasis of mouse tumors were inhibited after targeted inhibition of NOTCH3; (A) is the growth of tumors in mice after targeted inhibition of NOTCH3; (B) is the lung metastasis of mouse tumors after targeted inhibition of NOTCH3. DETAILED DESCRIPTION

[0012] In order to facilitate the understanding of the present invention, the present invention is described in more detail below in conjunction with the accompanying drawings and specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0013] 1. Methods 1. Cell culture The immortalized human bladder cancer cell line T24 was cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (Hyclone, USA) and 1% penicillin / streptomycin (Invitrogen, USA) at 37°C in a humidified atmosphere with 5% CO2.

[0014] 2. Cell transfection The required cells were plated one day before transfection. The cell growth density was observed under a microscope. The ideal state was reached when the density reached about 50%. The transfection instructions were followed, the virus MOI = 20, and fresh complete medium was replaced 16 hours after transfection. Subsequent experiments were performed 48 hours after transfection. shRNA targeting NOTCH3 (CCACGAGGATGCTATCTGT, Gene Chem, Shanghai, China, as shown in the sequence list SED ID NO.1) was used to downregulate the expression of NOTCH3.

[0015] 3. RT-qPCR Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, USA). RNA concentration was measured using NanoDrop ND-2000 (USA). Subsequently, total RNA was reverse transcribed into cDNA using PrimeScript™ RT Reagent Kit (Perfect RealTime) (Takara, Japan). RT-qPCR was performed on a Roche LightCycler 480II Real-TimePCR Detection System (Roche, Basel, Switzerland), and relative mRNA expression levels were calculated using the 2−ΔΔCt method normalized to GAPDH levels.

[0016] 4. Western blot (WB) Total protein was obtained from bladder cancer tissue or bladder cancer cells. Total protein was separated by 10% SDS-PAGE electrophoresis, and then the protein was transferred to a PVDF membrane. After blocking with 5% skim milk powder, the membrane was incubated with the primary antibody at 4°C overnight, and then incubated with the enzyme-labeled secondary antibody at room temperature for 1 hour. Immunoreactivity was detected by chemiluminescence. The results were analyzed by ImageJ software to obtain the optical density ratio of the target protein relative to β-Actin.

[0017] 5. Immunohistochemistry After dewaxing and hydration, the sections were heated in a citrate buffer for antigen retrieval by microwave heating. After rinsing with PBS, endogenous enzyme activity was eliminated with 3% hydrogen peroxide, and nonspecific sites were blocked with 3% BSA. Subsequently, the sections were incubated with the primary antibody (NOTCH3) in a 4°C wet box overnight, washed with PBS, and incubated with the secondary antibody at room temperature for 2 hours. During the color development process, DAB was added dropwise until brown-yellow color appeared. After hematoxylin staining of the nucleus, it was dehydrated with gradient ethanol and transparentized with xylene, and finally sealed with neutral gum to complete the experiment.

[0018] 6. CCK8 Experiment Bladder cancer cells in logarithmic growth phase were digested with trypsin, centrifuged, resuspended, and counted. 2000 cells / well were quantitatively inoculated into 96-well plates. 100ul culture system was set in each well. Zero well (culture medium only) was set at the same time. Five replicate wells were set in each group and then placed in an incubator. 100 μl CCK8 reagent was added to each well at the specified time point (0h, 24h, 48h, 72h), and the absorbance was measured at 450nm after incubation at 37°C for 2h. The growth curve of the cells was drawn according to the results to compare the growth trend differences between the experimental group and the control group.

[0019] 7. Plate cloning experiment Take the bladder cancer cells in the logarithmic growth phase, digest them with trypsin, centrifuge, resuspend them, and count the cells. Quantify the number of 500 cells / well and inoculate them into a 6-well plate. After adding complete culture medium, continue to culture for 10-14 days, observe the cell status and cell clones every 3 days, and change the cell medium. After observing that at least 50 cells are formed in each cell clone, discard the culture medium, wash with PBS 3 times, and then add 1ml4% paraformaldehyde to each well for 15 minutes. Then wash with PBS again, add an appropriate amount of crystal violet staining solution to each well, continue to stain the cells for 30 minutes, wash with PBS once, dry in the fume hood, and take pictures with a camera.

[0020] 8. Scratch test After digesting and centrifuging the cells, spread them on a 6-well plate, shake them thoroughly to allow the cells to adhere evenly, and supplement the culture medium and culture them in an incubator. When the growth density reaches about 80%, take out the 6-well plate and use a 20μL pipette tip to vertically scratch the surface of the well plate from one side of the well plate to the other side. The pipette tip cannot be tilted. After the scratch is completed, rinse with PBS to remove the detached cells. Replace the serum-free culture medium and continue to culture in the incubator. Take pictures under a microscope at 0h and 24h and 48h after culture, record the scratch width and take pictures to save the data. Use Image J software for subsequent analysis of the scratch area.

[0021] 9. Transwell experiment (1) Transwell assay to detect migration ability Digest the cells, centrifuge, resuspend, count, adjust the cell density, and add 100 μL (adjusted to contain about 50,000 cells) of cell suspension to each chamber. Add complete culture medium to the lower chamber and culture for 12-24 hours. After the culture, gently rinse the chamber with PBS, then add 4% paraformaldehyde to fix for 20 minutes, recover the paraformaldehyde and rinse with PBS three times, add 0.1% crystal violet to stain for 20 minutes, and rinse three times. Finally, wipe the cells on the inner surface of the upper chamber with a cotton swab and rinse with PBS. Observe under a microscope, take pictures, and save the data.

[0022] (2) Transwell assay to detect invasion ability First, dilute Matrigel matrix gel with serum-free cell culture medium at a ratio of 1:8 (can be placed on ice), and take a Transwell special 24-well plate with a chamber. Add 100μL of diluted gel to each chamber, put it in the incubator for incubation for 2h, then take it out, aspirate the upper layer of liquid, add 100μL of serum-free culture medium to each well, and place it in the incubator for 30min to hydrate the basement membrane. At the same time, the different groups of cells that have been intervened are digested, centrifuged, resuspended, counted, and the cell density is adjusted. 100μL (adjusted to contain about 50,000 cells) of cell suspension is added to each chamber. Complete culture medium is added to the lower chamber and cultured for 12-24 hours. After the culture is completed, the chamber is gently rinsed with PBS, and then 4% paraformaldehyde is added for fixation for 20min. After recovering the paraformaldehyde, it is rinsed with PBS three times, and 0.1% crystal violet is added for staining for 20min, and rinsed three times. Finally, the cells on the inner surface of the upper chamber are wiped off with a cotton swab and rinsed with PBS. Observe under a microscope, take pictures, and save the data.

[0023] 10. Animal Experimentation (1) Construction of subcutaneous xenograft model in mice: Take T24 cells in the logarithmic growth phase, resuspend them in 1:1 PBS and Matrigel matrix gel, and adjust the cell density to 1×10 7 / mL. 500μL of cell suspension was injected subcutaneously into 4-5 week old female BALB / c-nude mice to form skin papules. The mice were randomly divided into a control group and an experimental group, with 6 mice in each group. About 7 days after inoculation, the tumor gradually became visible. The long diameter and wide diameter of the tumor were measured with a vernier caliper every 2 days, the tumor volume was calculated (formula: volume = long diameter × wide diameter² / 2), and the tumor growth curve was drawn. After 4 weeks, the mice were euthanized, the tumor tissue was separated, the volume was measured, weighed, and photographed for record, and the relevant data were saved for further analysis.

[0024] (2) Construction of mouse lung metastasis model: Take luciferase-labeled T24 cells in the logarithmic growth phase, resuspend them in PBS and adjust the cell density to 1×10 6100 μL of cell suspension was injected into 4-5 week old female BALB / c-nude mice via tail vein. During injection, attention was paid to disinfection and slow operation to avoid bubbles. Two months later, the mice were euthanized, lung tissues were collected, fixed with 4% paraformaldehyde and stained with H&E to observe metastatic nodules and record relevant data.

[0025] 11. Statistical analysis Data processing was performed using GraphPad Prism 8.0 software. Independent sample t-test was used for comparison between the two groups. All experimental data are expressed as mean ± standard error (SEM) and repeated at least 3 times. P < 0.05 was considered statistically significant.

[0026] 2. Results 1. NOTCH3 is highly expressed in bladder cancer In the bladder cancer datasets GSE13507 and GSE37815, it was found that NOTCH3 was significantly overexpressed in bladder cancer compared with normal bladder tissue and was associated with poor prognosis of patients. Figure 1 As shown in (A) and (B).

[0027] 2. NOTCH3 is highly expressed in bladder cancer tissues and cells The present invention collected tumor tissues from patients who underwent radical cystectomy and their paired normal bladder tissues, and found by immunohistochemistry, Western blot and RT-qPCR that NOTCH3 was significantly overexpressed at both mRNA and protein levels compared with normal tissues or cells. Figure 1 Shown in (CE).

[0028] 3. Silencing NOTCH3 inhibits the expression of NOTCH3 In order to explore the role of NOTCH3 in the malignant progression of bladder cancer, the present invention silenced NOTCH3 in T24 cells and detected its transfection efficiency by RT-qPCR and Western blot. Figure 2 The results shown in the figure show that compared with the control group (sh-NC), the expression of NOTCH3 in the experimental group (sh-NOTCH3) was significantly downregulated at both the mRNA and protein levels, indicating that NOTCH3 was successfully silenced and the next experiment could be carried out.

[0029] 4. Silencing NOTCH3 inhibits the proliferation, migration and invasion of bladder cancer cells In order to explore whether silencing NOTCH3 can inhibit the malignant progression of bladder cancer, after silencing NOTCH3 in T24 cells, CCK8 assay, plate cloning assay, scratch assay, and Transwell assay were used to detect cell proliferation, migration, and invasion. Figure 3As shown, the experimental results showed that the proliferation, migration and invasion abilities of cells in the experimental group (sh-NOTCH3) were significantly reduced compared with those in the control group (sh-NC).

[0030] 5. Targeted inhibition of NOTCH3 can inhibit the growth and metastasis of mouse bladder tumors To explore whether targeted inhibition of NOTCH3 could inhibit the growth and metastasis of bladder cancer, NOTCH3-silenced T24 cells were injected into mice subcutaneously and through the tail vein to observe the growth and metastasis of tumors. Figure 4 The results shown in the figure indicate that targeted inhibition of NOTCH3 significantly suppressed the growth and metastasis of bladder tumors in mice.

[0031] NOTCH3 is highly expressed in bladder cancer and is associated with poor prognosis in patients. After silencing NOTCH3, the proliferation, migration and invasion of bladder cancer cells were significantly inhibited. In addition, after targeted inhibition of NOTCH3, the growth and metastasis of bladder tumors in mice were also significantly inhibited, suggesting that NOTCH3 is a potential therapeutic target for bladder cancer.

Claims

1. Use of NOTCH3 as a target in the preparation of a pharmaceutical composition for the treatment of bladder cancer.

2. The use according to claim 1, characterized in that: The pharmaceutical composition for treating bladder cancer is a targeted inhibitor of NOTCH3.

3. A targeted inhibitor of NOTCH3, characterized in that The inhibitor is shRNA.

4. The targeted inhibitor of NOTCH3 according to claim 3, characterized in that Its nucleotide sequence is shown in the sequence listing SED ID NO.

1.

5. A pharmaceutical composition for treating bladder cancer, characterized in that: A targeted inhibitor of NOTCH3 comprising the NOTCH3 of claim 3 or 4.

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