Application of ENO1 as target in preparation of pharmaceutical composition for treating bladder cancer

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of ENO1 as a target spot in preparation of a pharmaceutical composition for treating bladder cancer. The pharmaceutical composition for treating bladder cancer is a targeted inhibitor of ENO1. The inhibitor is shRNA (short hairpin ribonucleic acid). Experimental results show that ENO1 is a potential therapeutic target for bladder cancer progression, and development of the medicine for inhibiting the expression level of ENO1 is expected to provide a new strategy for bladder cancer treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the use of ENO1 as a target in preparing a pharmaceutical composition for treating bladder cancer. Background Art

[0002] Bladder cancer is a common malignant tumor of the urinary system and is also considered one of the most aggressive malignant tumors. According to statistics in 2023, its incidence rate ranked 8th among malignant tumors worldwide, its incidence rate ranked 4th among men, and its mortality rate ranked 8th. Bladder cancer is mainly divided into non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC) according to pathological type. Surgical treatment is the main intervention method for early bladder cancer, but for advanced bladder cancer, traditional adjuvant therapy has little effect and lacks effective and lasting treatment methods. Therefore, in-depth research on the molecular mechanisms of the occurrence and development of bladder cancer and the development of new therapeutic targets are the top priorities in the current field of bladder cancer research.

[0003] ENO1 (α-enolase), is a glycolytic enzyme that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate during glycolysis. ENO1 is a multifunctional protein that participates in various key intracellular and extracellular biological processes depending on its localization. When located on the cell surface, ENO1 can act as a plasminogen receptor and promote extracellular matrix (ECM) degradation. When intracellular, ENO1 helps organize microtubules and interphase cytoskeleton, and can be bound to exosomes or secreted as soluble proteins, and can also maintain the stability of mitochondrial membranes. Current studies have shown that ENO1 is highly expressed in a variety of tumors and is closely related to the prognosis of tumor patients. In breast cancer, ENO1 can promote tumor cell proliferation and metastasis by regulating the PI3K / AKT pathway. In gastric cancer, knockdown of ENO1 can lead to cell cycle arrest in the G1 phase and promote apoptosis of tumor cells. In lung cancer, ENO1 enhances cell proliferation by accelerating the G1 phase process and upregulating G1 phase cyclin-dependent kinase 6. In liver cancer, ENO1 can promote the translation of YAP1 mRNA and promote tumorigenesis by regulating arachidonic acid metabolism through the YAP1 / PLCB1 / HPGD axis. However, the molecules that ENO1 promotes the occurrence and development of bladder cancer have not been fully elucidated. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention aims to provide a new target for the treatment of bladder cancer and further explore the specific regulatory mechanism of ENO1 on the occurrence and development of bladder cancer.

[0005] The technical solution provided by the present invention is: use of ENO1 as a target in preparing a pharmaceutical composition for treating bladder cancer.

[0006] Preferably, the pharmaceutical composition for treating bladder cancer is a targeted inhibitor of ENO1.

[0007] Preferably, the targeted inhibitor of ENO1 is shRNA.

[0008] Preferably, the nucleotide sequence of the shRNA is as shown in the sequence listing SED ID NO.1 or 2.

[0009] The present invention also provides a pharmaceutical composition for treating bladder cancer, wherein the pharmaceutical composition comprises a targeted inhibitor of ENO1.

[0010] The experimental results of the present invention found that ENO1 has a positive regulatory effect on the proliferation ability of bladder cancer cells. After knocking down ENO1 in bladder cancer cells, the proliferation rate of bladder cancer cells decreased significantly, and ENO1 mainly affected the proliferation of bladder cancer cells by regulating the ubiquitination modification level of PCNA K48. The research results of the present invention suggest that ENO1 is a potential therapeutic target for the progression of bladder cancer, and the development of drugs that inhibit the expression level of ENO1 is expected to provide a new strategy for the treatment of bladder cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The expression level of ENO1 in bladder cancer tissue and its correlation with the prognosis of bladder cancer, including: a. ENO1 mRNA expression level diagram analyzed in TCGA bladder cancer dataset; b. ENO1 protein expression diagram in bladder cancer tissue detected by immunohistochemistry; c. ENO1 correlation analysis diagram with the prognosis of bladder cancer patients; Figure 2 After constructing ENO1 knockdown stable expression strain using lentiviral vector in T24 cells, a. RT-qPCR was used to detect the mRNA expression level of ENO1; b. Western blotting was used to detect the protein expression level of ENO1; Figure 3 The proliferation rate of bladder cancer cells decreased significantly after knocking down ENO1, where: a. CCK-8 experiment was used to detect the proliferation rate of bladder cancer cells; b. Cell clone experiment was used to detect the proliferation rate of bladder cancer cells; c. Edu experiment was used to detect the proliferation rate of bladder cancer cells; Figure 4 The effects of ENO1 on PCNA expression were detected, including: a. Immunoprecipitation-mass spectrometry experiments were used to screen whether ENO1 could affect bladder cancer cell proliferation through PCNA; b. Immunoprecipitation experiments were used to show that ENO1 protein interacted with PCNA protein; c. RT-qPCR was used to detect the expression level of PCNA mRNA in ENO1 stable knockdown cells; d. Western blotting was used to detect the expression level of PCNA protein in ENO1 stable knockdown cells; Figure 5The method is to detect the effect of ENO1 on PCNA expression by regulating PCNA ubiquitination modification, including: a. western blotting to detect changes in PCNA protein stability in ENO1 stable knockdown cells; b. western blotting to detect changes in PCNA ubiquitination levels in ENO1 stable knockdown cells; c. western blotting to detect changes in PCNA K48 ubiquitination levels in ENO1 stable knockdown cells. 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. Experimental methods:

[0014] 1. Main materials

[0015] Two lentiviral knockdown vectors were constructed by Shanghai GeneCare Gene.

[0016] 2. Cell culture

[0017] The immortalized human bladder cancer cell lines used in this study, including T24, were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. All cells were cultured under a strict sterile environment and were qualified by mycoplasma detection and STR identification. When reviving cells, the frozen cells were taken out of the liquid nitrogen tank and quickly transferred to a 37°C water bath, and gently shaken to thaw the cells quickly. After thawing, the cells were quickly transferred to a cell ultra-clean bench sterilized by ultraviolet light. The cell suspension was transferred into a 15 mL centrifuge tube, an appropriate amount of complete medium was added, and the mixture was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cell pellet was then resuspended with fresh complete medium, and the cells were transferred to a cell culture flask and cultured in a 37°C, 5% CO2 incubator. The next day, the growth of the cells was observed under a microscope, and the medium was changed or passaged as needed.

[0018] 3. Lentiviral vector construction and transfection

[0019] Bladder cancer cells were transfected with lentivirus to construct ENO1 stable knockdown bladder cancer cells (shENO1#1, shENO1#2), the specific sequences are as follows: shENO1#1: 5'-GGACTTCAAGTCTCCCGATGA-3' (sequence list SED ID NO.1), shENO1#2: 5'-GCGGTTCTCATGCTGGCAACA-3' (sequence list SED ID NO.2). The ENO1 knockdown lentivirus used in the experiment was constructed by Shanghai Gene Gene Co., Ltd.

[0020] The specific steps for lentiviral transfection of cells are as follows: (1) Prepare 2 ml of cell suspension with a density of 3-5×104 cells / ml using complete culture medium, and inoculate 500 ul of the cell suspension into a 24-well culture plate. Culture at 37°C for 16-24 hours until the cell confluence reaches 20-30%; (2) Take the virus out of the refrigerator and slowly thaw it on ice. Use complete culture medium to dilute the virus according to the experimental requirements; (3) According to the cell MOI and virus titer, calculate the formula: virus volume = (MOI × number of cells) / virus titer. Aspirate the supernatant in the well, add the corresponding volume of virus and the corresponding infection enhancement solution, mix well, and continue to culture; (4) Culture at 37°C for 16 hours, replace with complete culture medium, and continue culture. Observe cell morphology during infection. If changes occur, change the medium 8 hours in advance to maintain normal cell growth; (5) The medium can be changed according to the growth status of the cells to maintain cell activity; (6) About 72 hours after infection, when the fluorescence expression is high, observe under a microscope. The group with an infection efficiency of about 80% and good cell growth is passaged and cultured; (7) When the cells grow well, add puromycin to the cell culture medium and continue culturing; (8) Regularly observe the growth status and morphological changes of cells under a microscope. Non-transfected cells gradually die due to inability to tolerate puromycin, while transfected cells can survive and continue to grow.

[0021] 4. RT-qPCR experiment

[0022] After constructing a stable knockdown cell line of ENO1 in T24 cells, RT-qPCR was used to detect the expression levels of ENO1 and PCNA mRNA. According to the experimental plan, cells were inoculated in a well plate in advance (taking a 24-well plate as an example). Add 1 / 5 volume of chloroform (about 100 μL) to the EP tube containing the lysate, shake vigorously until fully mixed, and place it on ice for 10 minutes after ensuring that there is no stratification. After standing, centrifuge at 12000 rpm for 15 minutes at 4°C. At this time, stratification will appear in the EP tube, proteins and phenolic substances will be located in the inorganic phase (the white film in the lower layer and the middle layer), and RNA will be located in the uppermost aqueous phase. Carefully aspirate the colorless and transparent liquid part of the upper layer and transfer it to a new 1.5 mL enzyme-inactivated EP tube. Add an equal volume of isopropanol to the above EP tube, mix it upside down, place it on ice for 10 minutes, and then centrifuge it at 4°C and 12000 rpm for 20 minutes. Discard the supernatant, add 1 mL of 75% ethanol to wash the precipitate, and centrifuge at 4°C, 12,000 rpm for 5 minutes. Repeat this step once. After discarding the supernatant, open the EP tube and place it in a clean bench to dry for 15-30 minutes until the residual ethanol is completely evaporated. Add 20-30 μL RNase-free water to the precipitate, mix well, and store at -80°C or proceed to the next concentration determination. RT-qPCR reaction was performed on a real-time fluorescence quantitative PCR instrument (LightCycler480Ⅱ, Mannheim, Germany) using the KODSYBR qPCR (TOYOBO Co., Ltd, Osaka, Japan) kit, and each gene primer was synthesized at Suzhou Jikai Company.

[0023] 5. Western blotting

[0024] After constructing ENO1 stable knockdown cell lines in T24 cells, an appropriate amount of cell lysis buffer (containing protease inhibitors) was added according to the amount of cells in the well plate. After lysis, the cells were collected into EP tubes and sonicated, followed by centrifugation at 12000rpm for 5 minutes. After the protein concentration was determined by the BCA method, it was heated in a 95℃ metal bath for 5 minutes for denaturation, and 35ug of protein sample was loaded into each well and separated by 10% SDS PAGE. Transfer to PVDF membrane (Millipore, USA). After blocking with 5% BSA for 1 hour, the PVDF membrane was incubated with primary antibodies (ENO1, PCNA, Ub, K48, K63, GAPDH) at 4℃ overnight. The next day, the membrane was washed 3 times with PBST and then the secondary antibody working solution was added. It was incubated at room temperature for 1 hour, and the bound protein was detected by ECL chemiluminescence.

[0025] 6. Protein stability test

[0026] After constructing ENO1 stable knockdown cell line in T24 cells, bladder cancer cells were plated in 12-well plates and cultured to 70-80% density. 10 μg / ml protein synthesis inhibitor cycloheximide (CHX) was added to the cell culture medium, and cells were collected in 1.5 ml centrifuge tubes at different time points (0, 2, 4, 6 hours). Western blotting experiments were performed to detect the expression levels of related proteins.

[0027] 7. CCK8 Experiment

[0028] After constructing ENO1 stable knockdown cell line in T24 cells, bladder cancer cells in logarithmic growth phase were digested with trypsin, then centrifuged and resuspended, cell counting was performed, 2000 cells were quantitatively inoculated in each well in a 96-well plate, and 100ul culture medium was added to each well. At the same time, a zeroing well (culture medium only) was set, and 5 replicates were set for each group. The observation growth time points were set to 24h, 48h, 72h, 96h, and 120h. After reaching each time point, 10μL CCK8 reagent was added to each well and cultured for 3-4 hours. After the culture was completed, after sufficient shaking and dissolution, the absorbance of each well at a wavelength of 450nm was measured using an enzyme plate reader. According to the measurement results, a cell growth curve was drawn to compare the growth trend differences between the experimental group and the control group.

[0029] 8. Cell clone formation experiment

[0030] After constructing a stable knockdown cell line of ENO1 in T24 cells, bladder cancer cells in the logarithmic growth phase were taken, digested with trypsin, centrifuged and resuspended, and then the cells were counted. The cells were inoculated into 6-well plates at a density of 500 cells per well and cultured for 7-10 days. The culture medium was replaced every 3 days, and the growth status of the cells was observed. After the culture was completed, the cells were fixed with 4% paraformaldehyde for 15-30 minutes and then washed three times with PBS. Then, crystal violet stain was added for staining for 10-20 minutes, and then washed three times with PBS. Finally, the clone formation was observed and photographed under a microscope, and the number of clones formed was calculated.

[0031] 9. Edu Experiment

[0032] After constructing a stable ENO1 knockdown cell line in T24 cells, the Edu kit (C0071S, Beyotime USA) was used to detect changes in the proliferation rate of bladder cancer. The specific steps are as follows: (1) Take bladder cancer cells in the logarithmic growth phase, digest them with trypsin, centrifuge and resuspend them, and then place an appropriate amount of cells in a 6-well plate. Culture the cells in an incubator overnight, and then treat them with drugs after they have recovered to a normal state; (2) Prepare 2X EdU working solution: dilute EdU (10 mM) with cell culture medium at a ratio of 1:500 to obtain 20 μM 2X EdU working solution; (3) Add equal volumes of 2X EdU working solution (20 μM) preheated to 37°C into a 6-well plate to make the final concentration of EdU reach 1X; (4) Continue incubating the cells for 2 hours; (5) After EdU labeling, remove the culture medium, add 4% paraformaldehyde, and fix at room temperature for 15 minutes; (6) After removing the fixative, add 1 ml of washing solution to each well and wash the cells three times, each time for 3-5 minutes; (7) After removing the washing solution, add 1 ml of PBS permeabilization solution containing 0.3% Triton X-100 to each well and incubate at room temperature for 10-15 minutes; (8) After removing the permeabilization solution, wash the cells 1-2 times with 1 ml of washing solution per well, each time for 3-5 minutes; (9) Prepare Click Additive Solution: Dissolve one tube of Click Additive in 1.3 ml of deionized water and mix thoroughly until completely dissolved. (10) Prepare the Click reaction solution. Note that the Click reaction solution must be used within 15 minutes after preparation. (11) After removing the wash solution, add 0.5 ml of Click reaction solution to each well and gently shake the culture plate to ensure that the reaction mixture can evenly cover the sample; (12) After incubation at room temperature in the dark for 30 minutes, remove the Click reaction solution and wash three times with washing solution, each time for 3-5 minutes; (13) Prepare 1X Hoechst 33342 solution: dilute Hoechst 33342 (1000X) with PBS at a ratio of 1:1000; (14) After removing the washing solution, add 1 ml of 1X Hoechst 33342 solution to each well and incubate at room temperature in the dark for 10 minutes. (15) Remove the 1X Hoechst 33342 solution. Wash with washing solution three times, 3-5 minutes each time. Then perform fluorescence detection. The maximum excitation wavelength of Azide 488 is 495nm, and the maximum emission wavelength is 519nm; Hoechst 33342 is blue fluorescence, with a maximum excitation wavelength of 346nm and a maximum emission wavelength of 460nm.

[0033] 10. Statistical analysis

[0034] Data processing and statistical analysis were mainly performed using GraphPad Prism 8.0 software. For comparisons between groups, independent sample t-test or one-way ANOVA was used. If the data did not conform to a normal distribution, Wilcoxon rank sum test or Kruskal-Wallis test was used for comparisons between groups. All experimental data are presented as mean ± standard deviation (SD), and each experiment was repeated at least 3 times. P values ​​less than 0.05 were considered statistically significant.

[0035] 2. Experimental Results

[0036] 1. ENO1 is highly expressed in bladder cancer tissue and is closely related to prognosis

[0037] By analyzing the TCGA bladder cancer dataset, it was found that the mRNA level of ENO1 was significantly upregulated in bladder cancer tissues compared with normal tissues, such as Figure 1 As shown in a. Immunohistochemistry experiments confirmed that the expression level of ENO1 protein was also significantly upregulated in bladder cancer tissues, such as Figure 1 As shown in b and c. Survival analysis showed that in the TCGA bladder cancer dataset, high expression of ENO1 was closely associated with poor prognosis of bladder cancer patients, such as Figure 1 As shown in middle d, it suggests that ENO1 can be used as a marker related to the prognosis of bladder cancer.

[0038] 2. Knockdown of ENO1 significantly inhibited the proliferation of bladder cancer cells

[0039] Lentivirus was used to construct a cell line with stable knockdown of ENO1. RT-qPCR and western blotting results showed that the expression level of ENO1 in the shENO1 group was significantly downregulated compared with the shNC group. Figure 2 As shown in a and b. CCK-8 experiments, cell cloning experiments and Edu experiments found that knocking down ENO1 significantly inhibited the proliferation of bladder cancer cells, as shown in Figure 3 As shown in ac.

[0040] 3. ENO1 regulates PCNA protein expression

[0041] To further explore the mechanism by which ENO1 regulates the proliferation of bladder cancer cells, the present invention found through immunoprecipitation-mass spectrometry experiments that ENO1 may regulate PCNA expression and participate in the proliferation of bladder cancer cells. Figure 4 As shown in Figure a, PCNA, as a proliferation-related marker, has been confirmed to be involved in the proliferation of various tumor cells. Immunoprecipitation confirmed that ENO1 protein can interact with PCNA protein, such as Figure 4As shown in b. In the bladder cancer cell line with stable knockdown of ENO1, the mRNA expression level of PCNA did not change significantly, while the PCNA protein level was significantly downregulated, as shown in Figure 4 As shown in c and d.

[0042] 4. ENO1 promotes PCNA expression by regulating PCNA ubiquitination level

[0043] The above research results suggest that ENO1 only affects the expression level of PCNA protein, while protein stability plays an important regulatory role in protein expression. Therefore, the present invention further detected the changes in PCNA protein stability in bladder cancer cells with ENO1 knockdown. Protein stability experiments showed that PCNA protein stability decreased significantly after ENO1 knockdown. Figure 5 In addition, previous literature has shown that protein ubiquitination modification plays an important role in regulating protein stability. The present invention found that after knocking down ENO1, the ubiquitination level of PCNA was significantly increased, as shown in Figure 5 Further experiments found that knocking down ENO1 only affected the ubiquitination level of PCNA K48 chain, but had no significant effect on the ubiquitination level of PCNA K63 chain, as shown in Figure 2. Figure 5 As shown in c. The above research results suggest that ENO1 can enhance the stability of PCNA protein and promote PCNA expression by inhibiting the ubiquitination level of PCNA K48 chain.

Claims

1. Use of ENO1 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 ENO1.

3. A targeted inhibitor of ENO1, characterized in that: The inhibitor is shRNA.

4. The targeted inhibitor of ENO1 according to claim 3, characterized in that: The nucleotide sequence of shRNA is shown in the sequence listing SED ID NO.1 or 2.

5. A pharmaceutical composition for treating bladder cancer, characterized in that: The pharmaceutical composition comprises the targeted inhibitor of ENO1 according to claim 3 or 4.

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