Application of TTPAL protein inhibitors in the preparation of drugs for treating esophageal squamous cell carcinoma

Abstract

The present invention belongs to the field of biomedicine and, more specifically, relates to the use of a TTPAL protein inhibitor in the preparation of a medicament for treating esophageal squamous cell carcinoma. The present invention discovers that elevated TTPAL expression promotes the development of esophageal squamous cell carcinoma and can serve as a diagnostic marker for esophageal squamous cell carcinoma and a poor prognostic indicator. It also demonstrates that TTPAL can serve as a biological target for statins, a cholesterol synthesis inhibitor, in the treatment of esophageal squamous cell carcinoma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and more specifically, relates to the use of a TTPAL protein inhibitor in the preparation of a drug for treating esophageal squamous cell carcinoma. Background Art

[0002] Esophageal cancer primarily includes esophageal squamous cell carcinoma (ESC) and esophageal adenocarcinoma. Most ESC patients are diagnosed in the late stages and generally have a poor prognosis, with a five-year survival rate of approximately 30%. Esophageal SCC is primarily caused by unhealthy dietary habits, such as drinking, smoking, consuming hot beverages, and chewing betel nut. These adverse external stimuli can easily lead to genetic mutations within normal esophageal epithelial cells, including DNA copy number variations and point mutations. DNA copy number variations are one of the main causes of cancer.

[0003] Abnormal activation of cholesterol synthesis promotes the development and progression of tumors. A key gene regulating intracellular cholesterol synthesis levels is sterol regulatory element binding protein 2 (SREBP2). SREBP2's primary function is to regulate the expression of genes or enzymes involved in cholesterol biosynthesis. For example, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) is the rate-limiting enzyme in cholesterol biosynthesis. SREBP2 can bind to the promoter region of the HMGCR gene, promoting HMGCR transcription and initiating de novo cholesterol synthesis.

[0004] The tocopherol α-transfer protein gene, TTPAL, located within this region experiences copy number gain, significantly associated with increased expression levels. TTPAL promotes the development of colorectal cancer by regulating the WNT / β-catenin signaling pathway through TRIP6. In gastric cancer, TTPAL exerts a carcinogenic effect by activating the PI3K / AKT signaling pathway. However, the relationship between the TTPAL gene located in this region and the development and progression of esophageal squamous cell carcinoma, its molecular mechanisms, and its role in the treatment of esophageal squamous cell carcinoma have not been reported. Although clinically targeted inhibitors of cholesterol synthesis have achieved significant results in various tumor cell lines, there has been no breakthrough in inhibiting the development of esophageal squamous cell carcinoma. Summary of the Invention

[0005] The object of the present invention is to provide use of a TTPAL protein inhibitor in the preparation of a medicament for treating esophageal squamous cell carcinoma.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides use of a TTPAL protein inhibitor in preparing a medicine for treating esophageal squamous cell carcinoma.

[0008] The present invention discovered that overexpression of the TTPAL gene significantly accelerates the progression of esophageal squamous cell carcinoma (ESCC) by activating the SREBP2-mediated cholesterol synthesis pathway. Statins, inhibitors of the SREBP2 signaling pathway, were used to significantly suppress the malignant phenotype of ESCC with high TTPAL expression in both in vitro and in vivo models. These findings not only elucidate the key role of the TTPAL-SREBP2 axis in the development and progression of ESCC but also provide a new precision treatment strategy for patients with TTPAL-overexpressing ESCC, potentially serving as a novel treatment option for TTPAL-overexpressing tumors.

[0009] Furthermore, the TTPAL protein inhibitor includes a small molecule compound that inhibits the expression of the TTPAL protein or a preparation that knocks down the TTPAL gene.

[0010] Furthermore, the preparation for knocking down the TTPAL gene includes RNA interference molecules, antisense oligonucleotides or lentiviruses.

[0011] Furthermore, the coding sequence of the RNA interference molecule is CGAGCCATATACTTGACCTTA.

[0012] Furthermore, the drug contains the TTPAL protein inhibitor as the only active ingredient or one of the active ingredients.

[0013] Furthermore, when the drug contains the TTPAL protein inhibitor as one of the active ingredients, the active ingredient also includes statins.

[0014] Furthermore, the statins include simvastatin, atorvastatin, rosuvastatin and pitavastatin.

[0015] The present invention also provides a medicine for treating esophageal squamous cell carcinoma, comprising the TTPAL protein inhibitor.

[0016] Furthermore, the medicine also includes statins, and the statins include simvastatin, atorvastatin, rosuvastatin and pitavastatin.

[0017] The present invention also provides use of the TTPAL protein detection reagent in preparing a product for the prognosis of esophageal squamous cell carcinoma.

[0018] Beneficial effects:

[0019] The present invention found that high expression of TTPAL promotes the development of esophageal squamous cell carcinoma and can be used as an indicator for the diagnosis of esophageal squamous cell carcinoma and poor prognosis. It also proves that TTPAL can be used as a biological target for statins, a cholesterol synthesis inhibitor, to treat esophageal squamous cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 3 is the expression level of TTPAL in esophageal squamous cell carcinoma, where A is the expression of TTPAL in tumor tissue and adjacent tissue of esophageal squamous cell carcinoma patients; B is the relationship between TTPAL copy number and mRNA expression in tumor tissue of esophageal squamous cell carcinoma patients; C is the mRNA expression of TTPAL in tumor tissue and adjacent tissue of esophageal squamous cell carcinoma patients; D is the relationship between TTPAL copy number variation and mRNA expression in tumor tissue of esophageal squamous cell carcinoma patients; E is the immunoblot of TTPAL in tumor tissue and adjacent tissue of esophageal squamous cell carcinoma patients, N is adjacent tissue, and T is tumor tissue; F is the immunohistochemistry of TTPAL in tumor tissue and adjacent tissue of esophageal squamous cell carcinoma patients; G is the analysis of the immunohistochemistry results of TTPAL in tumor tissue and adjacent tissue of esophageal squamous cell carcinoma patients; H is a representative diagram of TTPAL expression in tumor tissue; I is the analysis of TTPAL expression and the survival curve of patients with esophageal squamous cell carcinoma.

[0021] Figure 2 The figure shows that TTPAL promotes the proliferation and clone formation of esophageal squamous cell carcinoma. Among them, A is the immunofluorescence image of TTPAL expression protein knockdown in the cell line; B is the proliferation statistics of KYSE180 cells after TTPAL knockdown; C is the proliferation statistics of ECA109 cells after TTPAL knockdown; D is the clone formation experiment; E is the statistical graph of KYSE180 cell clone formation experiment; F is the statistical graph of ECA109 cell clone formation experiment.

[0022] Figure 3 Figure 1 shows that knockdown of TTPAL inhibits the in vivo tumorigenicity of esophageal squamous cell carcinoma. A shows tumor formation in nude mice following subcutaneous inoculation. B shows the statistical volume of tumors formed by KYSE180 cells in nude mice following subcutaneous inoculation. C shows the statistical volume of tumors formed by ECA109 cells in nude mice following subcutaneous inoculation.

[0023] Figure 4 Figure 3 is an analysis diagram of the inhibition of cellular cholesterol synthesis pathway by knockdown of TTPAL, where A is a bubble diagram of the enrichment analysis of differentially expressed genes in the signal pathway by transcriptome sequencing; B is an immunoblot diagram of SREBP2 and its downstream enzymes related to the cholesterol synthesis pathway in KYSE180 and ECA109 cell lines with TTPAL knockdown; C is an analysis diagram of the RT-PCR results of SREBP2 and its downstream enzymes related to the cholesterol synthesis pathway in KYSE180 cell lines with TTPAL knockdown; D is an analysis diagram of the RT-PCR results of SREBP2 and its downstream enzymes related to the cholesterol synthesis pathway in ECA109 cell lines with TTPAL knockdown; E is a diagram showing the relationship between the expression of SREBP2, a key transcription factor in cholesterol synthesis, and TTPAL in tumor tissues of TCGA-ESCC patients; F is a diagram showing the relationship between the expression of HMGCR and TTPAL in tumor tissues of TCGA-ESCC patients.

[0024] Figure 5 The figures are analysis graphs showing the inhibition of cellular cholesterol content by knocking down TTPAL, wherein: A is the cholesterol content analysis graph of TTPAL in KYSE180 cells; B is the cholesterol content analysis graph of TTPAL in ECA109 cells; C is the philipin staining graph; D is the statistical graph of philipin staining in KYSE180 cells; E is the statistical graph of philipin staining in ECA109 cells; F is the proliferation ability analysis graph of KYSE180 cells that recovered from TTPAL knockdown; G is the proliferation ability analysis graph of ECA109 cells that recovered from TTPAL knockdown.

[0025] Figure 6 These are in vitro experimental analysis diagrams, where A is the activity analysis diagram of simvastatin-treated control and TTPAL-knockdown KYSE180 cells; B is the IC50 statistical diagram of simvastatin-treated control and TTPAL-knockdown KYSE180 cells; C is the in vitro clone formation ability experiment diagram; and D is the statistical diagram of the in vitro clone formation ability experiment.

[0026] Figure 7 These are experimental diagrams of the PDX mouse model, where A is the protein immunoblot of TTPAL and SREBP2; B is the tumor formation diagram of PDX mice; C is the statistical diagram of PDX1 tumor volume, D is the statistical diagram of PDX2 tumor volume; E is the statistical diagram of PDX1 tumor weight; and F is the statistical diagram of PDX2 tumor weight. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0028] Example 1: Expression level of TTPAL in esophageal squamous cell carcinoma.

[0029] 1. Immunoblotting experiment.

[0030] 1. Prepare protein samples: Collect cells with a cell scraper and centrifuge at 1500 rpm / min for 5 minutes to obtain a cell pellet. Add cell lysis buffer to the cell pellet, lyse on ice for 30 minutes, and boil in a water bath for 10 minutes to obtain a protein sample.

[0031] 2. Polyacrylamide gel electrophoresis: prepare the separation gel. After the separation gel solidifies, prepare the top layer of the stacking gel and add the protein sample and start electrophoresis to obtain the polyacrylamide gel containing the protein.

[0032] 3. Transfer and Block: Transfer the protein-containing polyacrylamide gel to a PVDF membrane at a constant voltage of 80 V for 90 minutes. Block the PVDF membrane in a 5% skim milk solution for 60 minutes.

[0033] 4. Antigen-antibody immune reaction: Prepare antibody diluent. Incubate at room temperature for 2 h. Wash the blocked PVDF membrane three times with PBST for 10 min each time. Use horseradish peroxidase-conjugated goat anti-rabbit IgG and horseradish peroxidase-conjugated goat anti-mouse IgG as secondary antibodies. Dilute the two secondary antibodies with PBS at a volume ratio of 1:5000 to prepare the secondary antibody working solution. Incubate the PVDF membrane with the secondary antibody working solution at room temperature for 2 h. Wash the PVDF membrane three times with PBST and develop the results.

[0034] 2. Immunohistochemistry.

[0035] 1. Sample preparation: Tumor and adjacent tissue specimens from esophageal cancer patients were directly fixed and sectioned using paraffin.

[0036] 2. Antigen retrieval: Place the slices in citric acid antigen retrieval solution and heat in a microwave for antigen retrieval. After returning to room temperature, wash in PBS solution three times, each time for 10 minutes.

[0037] 3. Endogenous peroxidase blocking: Tumor and adjacent tissue sections of esophageal cancer patients were placed in a 3% H2O2 solution, kept in the dark at room temperature for 25 minutes, and then washed three times in PBS solution.

[0038] 4. Serum blocking: Shake dry the slices, draw a circle around the tumor tissue with an immunohistochemistry pen, add 3% BSA solution into the circle and block at room temperature for 30 minutes.

[0039] 5. Antibody incubation: Prepare antibody diluent according to the instructions and incubate at 4°C overnight. Prepare label antibody diluent according to the instructions and incubate at room temperature for 1 hour.

[0040] 6. DAB color development: After washing with PBS, add DAB color development solution and wash to terminate color development. After sealing with neutral gum, examine under a microscope, collect images, analyze, and process the images.

[0041] 3. Experimental results.

[0042] The expression of TTPAL in tumor tissues and normal tissues and its relationship with the prognosis of ESCC patients were evaluated by immunoblotting and immunohistochemistry results of clinical samples and the TCGA database. Figure 1 The results from Figures A to H show that the increase in TTAPL copy number promotes the expression of TTPAL, and the expression of TTPAL in esophageal squamous cell carcinoma tumor tissue is higher than that in adjacent tissues. Figure 1Figure I shows that patients with esophageal squamous cell carcinoma with high expression of TTPAL have a worse prognosis.

[0043] Example 2: TTPAL promotes the proliferation and clone formation of esophageal squamous cell carcinoma.

[0044] 1. Construction of an esophageal cancer cell line with TTPAL knockdown.

[0045] 1. Lentiviral Particle Packaging: shTTPAL-1 and shTTPAL-2 were separately constructed into the pLKO.1-Puromycin plasmid and mixed with the PsPAX2 and pMD2.G packaging plasmids. PEI transfection reagent was added according to the manufacturer's instructions. The cells were allowed to stand for 20 minutes. Transfection was initiated when the cell density reached 80%. 48 hours after transfection, the supernatant was collected and filtered to obtain the lentivirus. The shCon group served as a control group transfected with an empty plasmid. The shTTPAL-1 group was transfected with a plasmid containing shTTPAL-1, and the shTTPAL-2 group was transfected with a plasmid containing shTTPAL-2.

[0046] shTTPAL-1: 5'-CGAGCCATATACTTGACCTTA-3'.

[0047] shTTPAL-2: 5'-GCCAGTGAGAACTACTTGTAT-3'.

[0048] 2. The filtered lentivirus was used to infect the esophageal squamous cell carcinoma cell line. After 48 hours, 1 μg / mL puromycin was added and screened for 1 week to obtain an esophageal squamous cell carcinoma cell line with stable knockdown of TTPAL.

[0049] 2. Clone formation experiment.

[0050] Digest and count cells in the logarithmic growth phase. Plate 1,000 cells in a 6-well plate and continue culturing for 2 weeks, changing the medium every 3 days and observing cell growth. Fix the cells with 1 mL of 4% paraformaldehyde for 20 minutes, then rinse with PBS. Add 1 mL of crystal violet stain, stain for 15 minutes, then rinse with PBS and photograph each well.

[0051] 3. CCK-8 experiment.

[0052] Place 1000 cells in the logarithmic growth phase into each well of a 96-well plate and incubate the plate in an incubator for 1 week. Add 10 μg / mL CCK8 solution and incubate at 37°C for 1 hour. Detect and count the cells using a microplate reader.

[0053] 4. Experimental results.

[0054] The effects of TTPAL knockdown on the growth of esophageal squamous cell carcinoma were examined by clone formation and CCK-8 assays. Figure 2 As shown in Figure A, the expression of TTPAL protein in ESCC cell lines KYSE180 and ECA109 was knocked down using lentivirus containing shTTPAL-1 and shTTPAL-2; Figure 2 As shown in Figures B and C, the proliferation capacity of ESCC cell lines KYSE180 and ECA109 was reduced after TTPAL knockdown. Figure 2 As shown in Figures D, E, and F, knockdown of TTPAL reduced the clonogenic capacity of ESCC cell lines KYSE180 and ECA109.

[0055] Example 3: Knockdown of TTPAL inhibited the tumorigenicity of esophageal squamous cell carcinoma in vivo.

[0056] 1. Nude mouse tumor formation experiment.

[0057] Control and knockdown TTPAL cells were cultured to the logarithmic phase. After digestion and cell counting, a cell-to-Matrigel mixture was prepared at a 2:1 volume ratio. Six-week-old female nude mice were subcutaneously inoculated with these cells. Tumor growth and size were measured on day 21 after inoculation.

[0058] 2. Experimental results.

[0059] The nude mouse subcutaneous tumor formation experiment was used to examine the effect of knocking down TTPAL on the tumorigenicity of esophageal squamous cell carcinoma in vivo. Figure 3 As shown, knockdown of TTPAL inhibited the growth of tumor volume and weight in ESCC cell lines KYSE180 and ECA109.

[0060] Example 4: Analysis of the inhibition of cellular cholesterol synthesis pathway by knockdown of TTPAL.

[0061] 1. Transcriptome sequencing.

[0062] Total RNA was extracted from control and TTPAL knockdown esophageal squamous cell carcinoma cell lines and converted into sequence-ready cDNA libraries. Sequencing was performed using the Illumina sequencing platform.

[0063] 2. Experimental results.

[0064] like Figure 4 As shown in A, transcriptome sequencing revealed that differential gene signaling pathways were enriched in the cholesterol synthesis pathway after knocking down TTPAL. Figure 4 As shown in Figures E and F, the TCGA database found that TTPAL and SREBP2 and their target gene HMGCR expressions were positively correlated. Figure 4As shown in Figures B, C, and D, quantitative PCR and immunoblotting experiments demonstrated that knockdown of TTPAL reduced the expression of the transcription factor SREBP2 and its downstream target genes. TTPAL inhibits the cholesterol signaling pathway in esophageal squamous cell carcinoma by reducing the expression of the transcription factor SREBP2 and its downstream target genes.

[0065] Example 5: Knockdown of TTPAL inhibits cellular cholesterol content analysis.

[0066] 1. Cholesterol content determination.

[0067] Cells in the logarithmic growth phase were digested and washed to obtain a cell pellet. The cells were ultrasonically disrupted and set aside. Following the instructions (Total Cholesterol Assay Kit, BB-47435, Beibo Biotech), the reagents were added. Mix well and incubate at 37°C for 10 minutes. The absorbance at 510 nm was measured using a microplate reader. The shCon+cholesterol group and the shTTPAL+cholesterol group were set up. Specifically, 2.5 μg / mL of exogenous cholesterol was added to 200 μL of culture medium and the treatment was continued until day 6.

[0068] 2. Filipin staining experiment.

[0069] The cells were seeded into a 24-well plate with a coverslip and cultured overnight. After washing three times with PBS, the cells were fixed with paraformaldehyde for 15 minutes. The cells were incubated with 0.1 mg / mL philipidin III at room temperature for 30 minutes. The cells were photographed and counted under a fluorescence microscope.

[0070] 3. Experimental results.

[0071] like Figure 5 As shown in Figure A, the cholesterol content measurement experiment found that the cholesterol content in esophageal squamous cell carcinoma cells with TTPAL knockdown was reduced. Figure 5 As shown in Figures C, D, and E, philipin staining and immunofluorescence staining experiments demonstrated that the cholesterol content in esophageal squamous cell carcinoma cells decreased after TTPAL knockdown; Figure 5 Figures F and G show that CCK-8 experiments found that cholesterol supplementation could restore the proliferation capacity of TTPAL knockdown esophageal squamous cell lines. This suggests that TTPAL inhibits the proliferation capacity of esophageal squamous cell cells by reducing the cholesterol content in them.

[0072] Example 6: In vitro experimental analysis.

[0073] 1. Experimental methods

[0074] The control group and the shTTPAL-1 group of the TTPAL knockdown esophageal squamous cell carcinoma cell line were treated with 50 μM simvastatin, and the difference in sensitivity of the control and TTPAL knockdown esophageal squamous cell carcinoma cell lines to simvastatin was detected using the CCK-8 and clone formation assays described in Example 1.

[0075] 2. Experimental results.

[0076] Test results such as Figure 6 As shown in A and B, the IC50 of simvastatin in esophageal squamous cell carcinoma cell lines with TTPAL knockdown was significantly reduced compared with the control group; Figure 6 Figures C and D show that esophageal squamous cell carcinoma cells with TTPAL knockdown were more sensitive than the control, and their clone-forming ability was significantly lower than that of the control group.

[0077] Example 7: PDX mouse model experiment.

[0078] 1. Establishment of PDX model.

[0079] Fresh tumor tissue specimens from patients with esophageal squamous cell carcinoma were obtained and divided into three groups: PDX1 group, PDX2 group, and PDX3 group. Protein immunoblotting was performed. Fresh tumor tissues from the PDX1 and PDX2 groups were cut into 10 mm pieces under sterile conditions. 3 Small tissue pieces of about 500 mm were implanted into immunodeficient mice. 3 The control group received intraperitoneal injection of 3 mg / kg (mouse weight) of dimethyl sulfoxide, and the simvastatin group received intraperitoneal injection of 3 mg / kg (mouse weight). The two groups were injected once every two days for 5 weeks.

[0080] 2. Experimental results.

[0081] The experimental results are as follows Figure 7 As shown in Figure 5A, Western blotting of the three tumor tissues showed that SREBP2 expression was also reduced in tumor tissues with low TTPAL expression. Figure 7 As shown in Figures B, C, D, E, and F, simvastatin significantly inhibited tumors with high TTPAL expression, but had no significant therapeutic effect on tumors with low TTPAL expression. Statins, cholesterol synthesis inhibitors, can effectively inhibit the tumorigenesis of esophageal squamous cell carcinoma cells with high TTPAL expression.

[0082] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0083] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0084] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Use of an inhibitor of TTPAL protein in the preparation of a drug for treating esophageal squamous cell carcinoma, characterized in that: The inhibitor is an RNA interference molecule, and the coding sequence of the RNA interference molecule is CGAGCCATATACTTGACCTTA.

2. The use according to claim 1, characterized in that The drug contains the inhibitor as the only active ingredient or one of the active ingredients.

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