Application of TTPAL protein inhibitor in preparation of medicine for treating esophageal squamous cell carcinoma

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of a TTPAL protein inhibitor in preparation of a medicine for treating esophageal squamous cell carcinoma. The invention finds that the high expression of TTPAL promotes the development of esophageal squamous-cell carcinoma, and TTPAL can be used as a judgment index for diagnosis and poor prognosis of esophageal squamous-cell carcinoma. Meanwhile, the TTPAL is proved to be capable of serving as a biological target for treating esophageal squamous cell carcinoma as a target cholesterol synthesis inhibitor statin drug.

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 mainly includes esophageal squamous cell carcinoma and esophageal adenocarcinoma. Most patients with esophageal squamous cell carcinoma are diagnosed in the late stage, and the prognosis is generally poor, with a five-year survival rate of about 30%. Esophageal squamous cell carcinoma is mainly caused by bad eating habits such as drinking, smoking, hot drinks, and chewing betel nuts. These adverse external stimuli can easily cause gene mutations in normal esophageal epithelial cells, including DNA copy number variation and point mutations. Among them, DNA copy number variation is one of the main causes of cancer.

[0003] Abnormal activation of cholesterol synthesis promotes the occurrence and development of tumors, and the important gene that regulates the level of cholesterol synthesis in cells is: sterol regulatory element binding protein 2 or SREBP2. The main function of SREBP2 is to regulate the expression of genes or enzymes related to cholesterol biosynthesis. For example, 3-hydroxy 3-methylglutaryl coenzyme A reductase HMGCR is the rate-limiting enzyme in cholesterol biosynthesis, and SREBP2 can bind to the promoter region of the HMGCR gene, promote HMGCR transcription, and initiate de novo synthesis of cholesterol.

[0004] The copy number of the tocopherol α transfer protein gene TTPAL located in this region increases, and is significantly correlated with the increase in its expression level. TTPAL promotes the occurrence of colorectal cancer by regulating the WNT / β-catenin signaling pathway through TRIP6. TTPAL plays a carcinogenic role in gastric cancer by activating the PI3K / AKT signaling pathway. However, there are no reports on the relationship between the TTPAL gene located in this region and the occurrence and development of esophageal squamous cell carcinoma and its molecular mechanism, as well as its relationship with the treatment of esophageal squamous cell carcinoma. Although inhibitors targeting the inhibition of cholesterol synthesis have achieved significant effects in a variety of tumor cells in clinical practice, there is still no breakthrough in the inhibitory effect on the development of esophageal squamous cell carcinoma. Summary of the invention

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

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

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

[0008] The present invention discovers that the overexpression of the TTPAL gene significantly accelerates the disease progression of esophageal squamous cell carcinoma by activating the SREBP2-mediated cholesterol synthesis pathway. The present invention uses statins, an inhibitor targeting the SREBP2 signaling pathway, to significantly inhibit the malignant phenotypes of esophageal squamous cell carcinoma with high TTPAL expression in in vitro and in vivo models. These findings not only clarify the key role of the TTPAL-SREBP2 axis in the occurrence and development of esophageal squamous cell carcinoma, but also provide a new precision treatment strategy for patients with esophageal squamous cell carcinoma with TTPAL overexpression, which can be used as a new treatment plan for tumors with TTPAL overexpression.

[0009] Furthermore, the TTPAL protein inhibitor includes small molecule compounds that inhibit the expression of the TTPAL protein or agents that knockdown the TTPAL gene.

[0010] Furthermore, the agents that knockdown the TTPAL gene include RNA interference molecules, antisense oligonucleotides, or lentiviruses.

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

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

[0013] Furthermore, when the drug uses the TTPAL protein inhibitor as one of the active ingredients, the active ingredients also include statins.

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

[0015] The present invention also provides a drug for treating esophageal squamous cell carcinoma, including the TTPAL protein inhibitor described above.

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

[0017] The present invention also provides the application of the detection reagent for the TTPAL protein in the preparation of products for the prognosis of esophageal squamous cell carcinoma.

[0018] Beneficial effects:

[0019] The present invention discovers that the 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 the judgment of poor prognosis. It also proves that TTPAL can be used as a biological target for treating esophageal squamous cell carcinoma with cholesterol synthesis inhibitor statins. Description of the drawings

[0020] Figure 1 Expression level diagrams of TTPAL in esophageal squamous cell carcinoma. Among them, A is the expression diagram of TTPAL in tumor tissues and adjacent tissues of esophageal squamous cell carcinoma patients; B is the relationship diagram between the copy number and mRNA expression of TTPAL in tumor tissues of esophageal squamous cell carcinoma patients; C is the mRNA expression diagram of TTPAL in tumor tissues and adjacent tissues of esophageal squamous cell carcinoma patients; D is the relationship diagram between the copy number variation and mRNA expression of TTPAL in tumor tissues of esophageal squamous cell carcinoma patients; E is the Western blot diagram of TTPAL protein in tumor tissues and adjacent tissues of esophageal squamous cell carcinoma patients, N is adjacent tissue, and T is tumor tissue; F is the immunohistochemical diagram of TTPAL in tumor tissues and adjacent tissues of esophageal squamous cell carcinoma patients; G is the analysis diagram of immunohistochemical results of TTPAL in tumor tissues and adjacent tissues of esophageal squamous cell carcinoma patients; H is the representative diagram of TTPAL expression in tumor tissues; I is the survival curve analysis diagram of the expression of TTPAL and esophageal squamous cell carcinoma patients.

[0021] Figure 2 Diagrams showing that TTPAL promotes the proliferation and clone formation of esophageal squamous cell carcinoma. Among them, A is the immunogram of knocking down the expression protein of TTPAL in cell lines; B is the proliferation statistical chart of KYSE180 cells after knocking down TTPAL; C is the proliferation statistical chart of ECA109 cells after knocking down TTPAL; D is the clone formation experiment diagram; E is the statistical chart of the clone formation experiment of KYSE180 cells; F is the statistical chart of the clone formation experiment of ECA109 cells.

[0022] Figure 3 Diagrams showing that knocking down TTPAL inhibits the tumorigenic ability of esophageal squamous cell carcinoma in vivo. Among them: A is the diagram of tumor formation by subcutaneous inoculation in nude mice. B is the statistical chart of the tumor formation volume of the KYSE180 cell line by subcutaneous inoculation in nude mice; C is the statistical chart of the tumor formation volume of the ECA109 cell line by subcutaneous inoculation in nude mice.

[0023] Figure 4 Diagrams showing that knocking down TTPAL inhibits the cell cholesterol synthesis pathway analysis. Among them, A is the bubble chart of the enrichment analysis of differential gene signaling pathways by transcriptome sequencing; B is the Western blot diagram of SREBP2 and its downstream cholesterol synthesis pathway-related enzymes in the KYSE180 and ECA109 cell lines with knocked-down TTPAL; C is the RT-PCR result analysis diagram of SREBP2 and its downstream cholesterol synthesis pathway-related enzymes in the KYSE180 cell line with knocked-down TTPAL; D is the RT-PCR result analysis diagram of SREBP2 and its downstream cholesterol synthesis pathway-related enzymes in the ECA109 cell line with knocked-down TTPAL; E is the relationship diagram between the key transcription factor SREBP2 of cholesterol synthesis and TTPAL expression in tumor tissues of TCGA-ESCC patients; F is the relationship diagram between HMGCR and TTPAL expression in tumor tissues of TCGA-ESCC patients.

[0024] Figure 5 Analysis chart for knocking down TTPAL to inhibit cellular cholesterol content. Among them: A is the analysis chart of cholesterol content of TTPAL in KYSE180 cells; B is the analysis chart of cholesterol content of TTPAL in ECA109 cells; C is the filipin staining chart; D is the statistical chart of filipin staining in KYSE180 cells; E is the statistical chart of filipin staining in ECA109 cells; F is the analysis chart of the proliferation ability of KYSE180 cells with knocked-down TTPAL restored; G is the analysis chart of the proliferation ability of ECA109 cells with knocked-down TTPAL restored.

[0025] Figure 6 Analysis chart for in vitro experiments. Among them, A is the analysis chart of the activity of simvastatin-treated control and knocked-down TTPAL KYSE180 cells; B is the statistical chart of IC50 of simvastatin-treated control and knocked-down TTPAL KYSE180 cells; C is the experimental chart of in vitro colony formation ability; D is the statistical chart of in vitro colony formation ability experiment.

[0026] Figure 7 Experimental chart for PDX mouse model. Among them, A is the protein immunoblotting chart of TTPAL and SREBP2; B is the tumorigenesis chart of PDX mice; C is the statistical chart of PDX1 tumor volume; D is the statistical chart of PDX2 tumor volume; E is the statistical chart of PDX1 tumor weight; F is the statistical chart of PDX2 tumor weight. Detailed implementation manners

[0027] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

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

[0029] I. Immunoblotting experiment.

[0030] 1. Preparation of protein samples: After collecting cells with a cell scraper, centrifuge at 1500 rpm / min for 5 min to obtain cell pellets. Add cell lysis buffer to the cell pellets and lyse on ice for 30 min, then boil in a water bath for 10 min to obtain protein samples.

[0031] 2. Polyacrylamide gel electrophoresis: Prepare the separating gel. After the separating gel solidifies, prepare the upper stacking gel and add the protein samples, then start electrophoresis to obtain a polyacrylamide gel containing proteins.

[0032] 3. Transfer membrane and blocking: Transfer the polyacrylamide gel containing the protein to the PVDF membrane at a constant voltage of 80 V for 90 min. Then place the transferred PVDF membrane into a 5% skim milk powder solution for blocking for 60 min.

[0033] 4. Antigen-antibody immunoreaction: Prepare the antibody dilution solution, incubate at room temperature for 2 h, then wash the blocked PVDF membrane with PBST 3 times, 10 min each time. The secondary antibodies include horseradish peroxidase-labeled goat anti-rabbit IgG and horseradish peroxidase-labeled goat anti-mouse IgG. Dilute the two secondary antibodies with PBS at a volume ratio of 1:5000 to obtain the working solution of the secondary antibody. Incubate the PVDF membrane with the working solution of the secondary antibody at room temperature for 2 h, then wash the PVDF membrane with PBST 3 times and develop to obtain the results.

[0034] II. Immunohistochemistry.

[0035] 1. Sample preparation: The tissue specimens of tumors and adjacent tissues of esophageal cancer patients with direct fixation are sectioned using paraffin.

[0036] 2. Antigen retrieval: Place the sections in a citric acid antigen retrieval solution and heat for antigen retrieval in a microwave oven. After returning to room temperature, wash with PBS solution 3 times, 10 min each time.

[0037] 3. Blocking of endogenous peroxidase: Place the sections of tumors and adjacent tissues of esophageal cancer patients into a 3% H 2 O 2 solution, let stand at room temperature in the dark for 25 min, and then wash 3 times in PBS solution.

[0038] 4. Serum blocking: Drain the sections, draw a circle around the tumor tissue with an immunohistochemistry pen, and add a 3% BSA solution inside the circle to block at room temperature for 30 min.

[0039] 5. Antibody incubation: Prepare the antibody dilution solution according to the instructions and incubate overnight at 4°C. Prepare the labeled antibody dilution solution according to the instructions and incubate at room temperature for 1 h.

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

[0041] III. Experimental results.

[0042] The expression of TTPAL in tumor tissues and normal tissues and its relationship with the prognosis of esophageal squamous cell carcinoma patients were evaluated through immunoblotting experiments and immunohistochemical results of clinical samples and the TCGA database. Figure 1Results of Figures A to H showed that the increased copy number of TTAPL promoted the expression of TTPAL, and the expression of TTPAL in esophageal squamous cell carcinoma tumor tissues was higher than that in adjacent tissues. Figure 1 Figure I in [reference] showed that patients with esophageal squamous cell carcinoma with high expression of TTPAL had a worse prognosis.

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

[0044] I. Construction of an esophageal cancer cell line with knocked-down TTPAL.

[0045] 1. Lentiviral particle packaging: shTTPAL-1 and shTTPAL-2 were respectively constructed into the pLKO.1-Puromycin plasmid. After mixing with the packaging plasmids PsPAX2 and pMD2.G, transfection reagent PEI was added according to the manufacturer's instructions, and the mixture was left standing for 20 min. When the cell density reached 80%, transfection was started. After 48 h of transfection, the supernatant was collected and filtered to obtain lentivirus. The shCon group was the control group transfected with the empty plasmid. The shTTPAL-1 group was transfected with the plasmid containing shTTPAL-1, and the shTTPAL-2 group was transfected with the 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 h, 1 μg / mL of puromycin was added for screening for 1 week to obtain a stable esophageal squamous cell carcinoma cell line with knocked-down TTPAL.

[0049] II. Clone formation experiment.

[0050] Cells in the logarithmic growth phase were digested and counted. 1000 cells were seeded in a 6-well plate and cultured for another 2 weeks. The culture medium was changed every 3 days, and the growth state of the cells was observed. After fixing the cells with 1 mL of 4% paraformaldehyde for 20 min, they were washed with PBS. 1 mL of crystal violet staining solution was added, and after staining for 15 min, they were washed with PBS and photographed respectively.

[0051] III. CCK-8 experiment.

[0052] 1000 cells in the logarithmic growth phase were added to each well of a 96-well plate, and the culture plate was incubated in an incubator for 1 week. 10 μg / mL of CCK8 solution was added and incubated in a 37 °C incubator for 1 h. Detection and statistics were performed using an enzyme-linked immunosorbent assay (ELISA) reader.

[0053] IV. Experimental Results.

[0054] The effects of knocking down TTPAL on the growth of esophageal squamous cell carcinoma were detected by colony formation and CCK-8 assays. The results are shown in Figure 2 A in the figure. The expression of TTPAL protein in esophageal squamous cell lines KYSE180 and ECA109 was knocked down using lentiviruses containing shTTPAL-1 and shTTPAL-2; as shown in Figure 2 B and C in the figure, the proliferation ability of esophageal squamous cell lines KYSE180 and ECA109 with knocked-down TTPAL decreased; as shown in Figure 2 D, E, and F in the figure, the colony formation ability of esophageal squamous cell lines KYSE180 and ECA109 with knocked-down TTPAL decreased.

[0055] Example 3: Knocking down TTPAL inhibited the tumorigenic ability of esophageal squamous cell carcinoma in vivo.

[0056] I. Tumorigenesis experiment in nude mice.

[0057] The control and TTPAL-knockdown cells were cultured to the logarithmic phase. After digesting the cells and counting, a cell and Matrigel mixture was prepared according to a volume ratio of 2:1. Subcutaneous injection was performed in 6-week-old female nude mice. The tumor growth was observed on the 21st day after inoculation, and the tumor size was measured.

[0058] II. Experimental Results.

[0059] The effects of knocking down TTPAL on the tumorigenic ability of esophageal squamous cell carcinoma in vivo were detected by subcutaneous tumorigenesis experiment in nude mice. As shown in Figure 3 the figure, knocking down TTPAL inhibited the growth of the tumor volume and weight of esophageal squamous cell lines KYSE180 and ECA109.

[0060] Example 4: Analysis of the inhibition of cell cholesterol synthesis pathway by knocking down TTPAL.

[0061] I. Transcriptome sequencing.

[0062] Total RNA was extracted from the control and TTPAL-knockdown esophageal squamous cell lines, and the obtained total RNA was converted into a cDNA library that could be sequenced. Sequencing was performed using the Illumina sequencing platform.

[0063] II. Experimental Results.

[0064] As shown in Figure 4 A in the figure, through transcriptome sequencing, it was found that the cholesterol synthesis pathway was enriched by differential gene signaling pathway enrichment analysis after knocking down TTPAL. As shown in Figure 4As shown by E and F in the TCGA database, a positive correlation was found between the expression of TTPAL and SREBP2 and their target gene HMGCR. As Figure 4 As shown by B, C, and D in , fluorescence quantitative PCR and immunoblotting experiments demonstrated that the expression of the transcription factor SREBP2 and its downstream target genes decreased after knocking down TTPAL. TTAPL 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: Analysis of the inhibition of cellular cholesterol content by knocking down TTPAL.

[0066] I. Cholesterol content determination.

[0067] Cells in the logarithmic growth phase were taken, digested and washed to obtain cell pellets, and the cells were sonicated and then reserved. According to the instructions (Total Cholesterol Detection Kit, BB-47435, Boster Biological Technology), reagents were added. After mixing, the mixture was incubated at 37 °C for 10 min. The absorbance at 510 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The shCon + cholesterol group and the shTTPAL + cholesterol group were set up. The specific operation was to add 2.5 μg / mL of exogenous cholesterol to 200 μL of the culture medium and treat until the 6th day.

[0068] II. Filipin staining experiment.

[0069] Cells were seeded in a 24-well plate with cover slips added and cultured overnight; after washing 3 times with PBS, they were fixed with paraformaldehyde for 15 min; the cells were incubated with 0.1 mg / mL of Filipin III at room temperature for 30 min; photographs were taken and counted under a fluorescence microscope.

[0070] III. Experimental results.

[0071] As Figure 5 As shown by A in , the cholesterol content determination experiment found that the cholesterol content in esophageal squamous carcinoma cells with knocked-down TTPAL decreased. As Figure 5 As shown by C, D, and E in , the Filipin staining immunofluorescence staining experiment demonstrated that the cholesterol content in esophageal squamous carcinoma cells decreased after knocking down TTPAL; Figure 5 As shown by F and G in , the CCK-8 experiment found that supplementing cholesterol could restore the proliferation ability of the esophageal squamous cell line with knocked-down TTPAL. This indicates that TTAPL inhibits the proliferation ability of esophageal squamous carcinoma cells by reducing the cholesterol content in the cells.

[0072] Example 6: In vitro experimental analysis.

[0073] I. Experimental method.

[0074] The control group and the shTTPAL-1 group of esophageal squamous cell carcinoma cell lines with knocked-down TTPAL were treated with 50 μM simvastatin, and the CCK-8 and colony formation assay methods in Example 1 were used to detect the differences in the sensitivity of the control and knocked-down TTPAL esophageal squamous cell carcinoma cell lines to simvastatin.

[0075] II. Experimental results.

[0076] The detection results are shown in Figure 6 A and B below. Compared with the control group, the IC50 of the esophageal squamous cell carcinoma cell line with knocked-down TTPAL for simvastatin decreased significantly; Figure 6 C and D below show that the esophageal squamous cell carcinoma cells with knocked-down TTPAL were more sensitive than the control, and the colony formation ability was significantly lower than that of the control group.

[0077] Example 7: PDX mouse model experiment.

[0078] I. Establishment of PDX model.

[0079] Fresh tumor tissue specimens from esophageal squamous cell carcinoma patients were obtained and divided into three groups, namely PDX1 group, PDX2 group and PDX3 group, and western blot detection was performed. Then the fresh tumor tissues of the PDX1 group and the PDX2 group were cut into small tissue blocks about 10 mm 3 in size under sterile conditions and implanted into immunodeficient mice. When the tumor volume reached about 500 mm 3 in size, in vivo passage was carried out. The control group was intraperitoneally injected with dimethyl sulfoxide at a concentration of 3 mg / kg (mouse weight), and the simvastatin group was intraperitoneally injected with simvastatin at a concentration of 3 mg / kg (mouse weight). The two groups were injected every two days for 5 weeks.

[0080] II. Experimental results.

[0081] The experimental results are shown in Figure 7 A below. Western blot of the three tumor tissues showed that the expression of SREBP2 in the tumor tissues with low expression of TTPAL was also reduced. Figure 7 As shown in B, C, D, E and F below, simvastatin could significantly inhibit the tumors with high expression of TTPAL, but had no obvious therapeutic effect on the tumors with low expression of TTPAL. The cholesterol synthesis inhibitor statin drugs could effectively inhibit the tumorigenesis of esophageal squamous cell carcinoma cells with high expression of TTPAL.

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

[0083] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made by those skilled in the art once they learn of the basic inventive concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

[0084] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Application of TTPAL protein inhibitors in the preparation of drugs for the treatment of esophageal squamous cell carcinoma.

2. The use according to claim 1, characterized in that: The TTPAL protein inhibitor includes a small molecule compound that inhibits the expression of the TTPAL protein or an agent that knocks down the TTPAL gene.

3. The use according to claim 2, characterized in that: The agent for knocking down the TTPAL gene includes RNA interference molecules, antisense oligonucleotides or lentiviruses.

4. The use according to claim 3, characterized in that: The coding sequence of the RNA interference molecule is CGAGCCATATACTTGACCTTA.

5. The use according to claim 1, characterized in that: The drug has the TTPAL protein inhibitor as the only active ingredient or one of the active ingredients.

6. The use according to claim 5, characterized in that: When the drug has the TTPAL protein inhibitor as one of the active ingredients, the active ingredients also include statins.

7. The use according to claim 6, characterized in that: The statins include simvastatin, atorvastatin, rosuvastatin and pitavastatin.

8. A drug for treating esophageal squamous cell carcinoma, characterized in that: The invention comprises the TTPAL protein inhibitor as claimed in claim 1.

9. The drug for treating esophageal squamous cell carcinoma according to claim 8, characterized in that: The drugs also include statins, which include simvastatin, atorvastatin, rosuvastatin and pitavastatin.

10. Use of the detection reagent for TTPAL protein according to claim 1 in preparing a prognosis product for esophageal squamous cell carcinoma.

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