A molecular marker CSAD and its application

Abstract

The present invention discloses a molecular marker, CSAD, and its application. CSAD stands for cysteine ​​sulfenic acid decarboxylase (ID: ENSG00000139631). The purpose of diagnosing type I endometrial cancer is achieved by detecting the expression level of CSAD. The expression level of CSAD in the present invention is significantly higher in endometrial cancer tissue than in benign ovarian tumor tissue, and can be used to prepare products and kits for diagnosing type I endometrial cancer. Furthermore, CSAD in the present invention can affect the proliferation, migration, and invasion capabilities of type I endometrial cancer cells. Therefore, reagents that inhibit CSAD can be used to prepare drugs for treating type I endometrial cancer.

Description

Technical Field

[0001] The present invention belongs to the field of diagnosis and treatment, and in particular relates to a molecular marker CSAD and an application thereof. Background Art

[0002] Endometrial cancer, also known as uterine corpus cancer, is an epithelial malignant tumor that originates in the endometrium. It is one of the three most common malignant tumors of the female reproductive tract and occurs primarily in perimenopausal and postmenopausal women. With increasing life expectancy and changes in lifestyle, the incidence of endometrial cancer has been rising steadily over the past two decades, with a younger age group.

[0003] Endometrial cancer is broadly divided into two subtypes: type I, which is hormone receptor-positive, low-grade, endometrioid cancer and accounts for 80% of endometrial cancers, and type II, which is hormone receptor-negative, high-grade, and non-endometrioid cancer. Most endometrial cancers (75%) are diagnosed at an early stage and have the best prognosis. The 5-year overall survival rate for patients without metastasis ranges from 74% to 91%, while that for patients with lymph node metastasis decreases to 20% to 66%. The development of type I endometrial cancer is directly related to continuous estrogen stimulation without progesterone antagonism. In the absence of progesterone antagonism, the endometrium remains in a state of chronic hyperproliferation, further progressing to endometrial cancer. A better understanding of the mechanisms of endometrial cancer development and metastasis and the identification of therapeutic targets related to metastasis will help improve the prognosis of patients with type I endometrial cancer. Cysteine ​​sulfenic acid decarboxylase (CSAD), first discovered in the liver, is the key rate-limiting enzyme for the synthesis of taurine in vivo. Taurine synthesis is regulated by CSAD by controlling the partitioning of cysteine ​​sulfenic acid between taurine synthesis and pyruvate and sulfate production. Previous studies have shown that CSAD enzyme activity and expression increase after ovariectomy and decrease after estrogen replacement. The association of CSAD with cancer has been rarely reported. Studies have found that CSAD mRNA and protein expression are elevated during hepatocarcinogenesis. Currently, the role of CSAD in the development and progression of type I endometrial tumors has not been described. Summary of the Invention

[0004] The purpose of the present invention is to provide a molecular marker CSAD and its application in response to the deficiencies of the existing technology.

[0005] The object of the present invention is achieved through the following technical solutions:

[0006] A molecular marker CSAD is a gene widely expressed in human tissues, and its sequence is shown in SEQ ID NO.1.

[0007] An application of the molecular marker CSAD, wherein CSAD is used to prepare a kit for diagnosing type I endometrial cancer.

[0008] Furthermore, the kit is used to quantitatively detect the expression level of CSAD.

[0009] Furthermore, the kit performs detection based on real-time quantitative reverse transcription PCR, in situ hybridization, chip or high-throughput sequencing.

[0010] Furthermore, the kit for diagnosing type I endometrial cancer by detecting the expression level of CSAD by real-time quantitative reverse transcription PCR includes at least a pair of specific primers, as shown in SEQ ID NO.2 and SEQ ID NO.3.

[0011] Furthermore, the kit for detecting the expression level of CSAD by in situ hybridization, chip or high-throughput sequencing platform includes a specific probe that hybridizes with the nucleic acid sequence of CSAD.

[0012] A drug for treating type I endometrial cancer, comprising an agent that inhibits CSAD.

[0013] Furthermore, the reagent includes at least one of siRNA, shRNA, and ASO of CSAD.

[0014] Furthermore, the siRNA of CSAD is shown as SEQ ID NO.6 and SEQ ID NO.7, or as SEQ ID NO.8 and SEQ ID NO.9.

[0015] A use of the drug in treating type I endometrial cancer.

[0016] The beneficial effects of the present invention are as follows: the present invention provides the application of CSAD (cysteine ​​sulfenic acid decarboxylase), a molecular marker for type I endometrial cancer, in diagnosis and treatment. By performing immunochemical staining on 55 normal endometrial specimens, 26 abnormal endometrial hyperplasia specimens, and 269 type I endometrial cancer specimens, qRT-PCR verified that CSAD is indeed upregulated in type I endometrial cancer tissues. Knockdown of CSAD has a significant inhibitory effect on the proliferation of type I endometrial cancer. The above results indicate that CSAD is an important carcinogenic factor in type I endometrial cancer and can be used as a diagnostic molecular marker and therapeutic target. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 To detect the differential expression of CSAD in normal endometrium (NE), abnormal endometrial hyperplasia (AEH) and type I endometrial cancer (Type I EC) tissues using immunofluorescence;

[0018] Figure 2 The Kaplan-Meyer curve shows the correlation between abnormal expression of CSAD and the disease-free survival (DFS) and overall survival (OS) of patients;

[0019] Figure 3 is the ROC curve, showing the specificity and sensitivity of abnormal expression of CSAD in the diagnosis of type I endometrial cancer in the population;

[0020] Figure 4 Schematic diagram of the knockdown efficiency of CSAD in type Ⅰ endometrial cancer cells using two siRNAs;

[0021] Figure 5 This is a growth curve showing the effect of inhibiting CSAD expression on the proliferation of type Ⅰ endometrial cancer cells using CCK8 detection;

[0022] Figure 6 Schematic diagram showing that inhibition of CSAD expression can significantly inhibit the ability of type Ⅰ endometrial cancer cells to resist anoikis by flow cytometry;

[0023] Figure 7 Schematic diagram of western blot detection of the expression of anti-anoikis proteins BCL-2 and Bax;

[0024] In the figure, **: p < 0.01; ***: p < 0.001.

[0025] Specific implementation methods

[0026] The present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0027] The present invention provides a molecular marker for diagnosing type I endometrial cancer, CSAD (ID: ENSG00000139631), whose sequence is shown in SEQ ID NO. 1. CSAD sources include, but are not limited to, tissues and body fluid components containing nucleic acids, including blood, ascites, and the like.

[0028] The present invention also provides a use of the aforementioned molecular marker CSAD in the preparation of a kit for diagnosing type I endometrial cancer. The kit diagnoses type I endometrial cancer by quantitatively detecting the expression level of CSAD; the kit can be based on real-time quantitative reverse transcription PCR, in situ hybridization, microarrays, or high-throughput sequencing. The kit for diagnosing type I endometrial cancer by detecting the expression level of CSAD via real-time quantitative reverse transcription PCR includes at least a pair of primers that specifically amplify CSAD, with the forward primer being as shown in SEQ ID NO. 2 and the reverse primer being as shown in SEQ ID NO. 3. The kit for diagnosing type I endometrial cancer by detecting the expression level of CSAD via in situ hybridization, microarrays, or high-throughput sequencing platforms includes DNA, RNA, or other specific probes that hybridize with the nucleic acid sequence of CSAD.

[0029] The present invention also provides a drug for treating type I endometrial cancer, comprising an agent that inhibits CSAD. The agent is not limited and can inhibit either CSAD expression or CSAD functional activity; it may include siRNA, shRNA, or ASO. In the embodiments of the present invention, the siRNA sequences for CSAD are shown in SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9.

[0030] The present invention also provides a use of the above medicine in treating type I endometrial cancer.

[0031] Example 1 Differential expression of CSAD in type Ⅰ endometrial cancer

[0032] 1. Specimen source

[0033] The specimens were selected from 55 cases of normal endometrium, 26 cases of abnormal endometrial hyperplasia, and 269 cases of type I endometrial cancer surgically removed in our hospital.

[0034] 2. Case screening

[0035] All patients with type I endometrial cancer and benign ovarian tumors were newly diagnosed and had signed informed consent. None had received chemotherapy, radiotherapy, or other treatments before surgery. None had a history of other malignancies or major illnesses.

[0036] 3. Patients with type I EC were followed up postoperatively via outpatient interviews or telephone calls. Local tumor recurrence, distant metastasis, and patient survival were recorded. Disease-free survival (DFS) and overall survival (OS) were calculated from the day of surgery until recurrence or death.

[0037] 4. Reagents used

[0038] Table 1: Reagents used

[0039]

[0040]

[0041] 5. Equipment used

[0042] Table 2: Equipment used

[0043] Common instrument names Manufacturer cell culture incubator Thermo Fisher Scientific Disposable cell culture flasks / plates Thermo Fisher Scientific Low Adsorption Six-Well Plate Corning Incorporated Clean bench Thermo Fisher Scientific Ultrapure water meter Millipore Cell counting chamber Bio-rad Automatic cell counter Bio-rad pipette Eppendorf Low-temperature high-speed centrifuge Eppendorf Nuclease-free tips AXYGEN Electric hot water bath NIKON Mettler AT261 Balance Mettler Ice Maker SANYO

[0044] 6. Immunofluorescence

[0045] 1) Place wet tissue paper on the bottom of a slide box to create a humidified box. Remove slides containing sections of normal endometrium, abnormal endometrial hyperplasia, or type I endometrial cancer from the cryostat and place them in the slide box (6 slides per side) or in a humidified box (do not allow the slides to touch each other).

[0046] 2) When the slide is room-humid but not dry, spread PBS on the slices (do not overflow the slide).

[0047] 3) Diluted primary antibody (CSAD primary antibody, Invitrogen) was centrifuged at 13,500×g for 2 min at 4°C using a microcentrifuge (40–50 μl of antibody was added to each slide to cover the entire section).

[0048] 4) Use a Pasteur pipette connected to a pump to remove the PBS on the slide at one end of the slice, and add the primary antibody from the other end. Cover the humidified box and incubate at room temperature for 1 hour.

[0049] 5) Wash the slides three times with PBS (5 min / time), add new PBS buffer from one end of the slide and remove the old buffer from the other end.

[0050] 6) Dilute the secondary antibody (ready-to-use HRP-labeled anti-mouse secondary antibody, CST) and centrifuge at 1350×g for 2 min at 4°C (40-50 μl of antibody can be added to each slide).

[0051] 7) Add the secondary antibody to the sections, incubate in a humidified box at room temperature for 1 hour, and wash the slides three times with PBS (5 minutes each time).

[0052] 8) Place a coverslip on a paper towel and add one drop of Gelvatol to the center of the coverslip. Flip the slide over and place it on the coverslip (do not apply pressure). Place the slide on a work surface, cover with aluminum foil, and keep out of the light for 30 minutes to allow the Gelvatol to solidify.

[0053] 9) Observe the results under a microscope. Figure 1 As shown in the figure, the average expression level of CSAD in type I endometrial cancer tissues was significantly upregulated compared with that in normal ovarian tissues.

[0054] 7. RNA Extraction

[0055] Sample lysis:

[0056] 1) Aspirate the culture medium of the specimen tissue and wash once with an appropriate amount of PBS;

[0057] 2) Add 500 μl of Lysis Buffer and pipette vigorously ten times, transfer to an EP tube, and vortex for 10 seconds to fully lyse the cells;

[0058] Column / RNA binding:

[0059] 3) Add an equal volume of anhydrous ethanol to the lysed cells or tissue and mix thoroughly (precipitation may occur, which is normal; continue with the procedure). Shake the centrifuge tube several times or pipette vigorously 10 times to disperse any precipitate, then add the liquid to the spin column.

[0060] 4) Centrifuge at 4,000 × g for 1 minute (for cells larger than 1.5 × 10 6 For cell or tissue samples, 12,000 is recommended.

[0061] × g centrifugation).

[0062] Column cleaning:

[0063] 5) Add 500 μl of Wash Buffer to the RNA column and centrifuge at 12,000 × g for 1 minute. (When removing the column after centrifugation, be careful not to allow the waste liquid in the collection tube to come into contact with the RNA column to avoid contamination. Discard the waste liquid, return the RNA column to the collection tube, and centrifuge the empty tube once to completely remove any residual Wash Buffer. This method can be used to optimize the RNA extraction if the purity is insufficient according to the standard procedure.)

[0064] 6) Place the column in a clean 1.5 ml RNase-free centrifuge tube and leave it to air dry for 2 minutes with the lid open.

[0065] RNA elution:

[0066] 7) Add 20-50 μl of Elution Buffer to the center of the RNA column membrane and let it stand at room temperature for 2 minutes.

[0067] 8) Centrifuge at 12,000 × g for 1 minute (add the eluted RNA solution back to the column, let it sit for 5 minutes, and centrifuge again to improve elution efficiency and obtain more RNA). (After elution, it is recommended to keep the RNA on ice.)

[0068] 9) Determine the concentration of the eluted RNA for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments or stored at -80°C for later use.

[0069] 8. Reverse transcription and qPCR

[0070] 1) Reverse transcription

[0071] Take 1 μg RNA system as an example, as shown in Table 3. Reaction conditions: 37°C for 2 min, 55°C for 15 min, 85°C for 5 min, 4°C forever.

[0072] Table 3: RNA system

[0073] Reagents Reaction volume (μl) 5X ABScript III RT Mix 4.0 20X gDNA Remover Mix 1.0 Extracted RNA Calculate the volume by dividing 1 μg by the RNA concentration <![CDATA[RNase Free H2O]]> Fill up to 20

[0074] 2) qPCR reaction

[0075] Take a 20 μl PCR system as an example, as shown in Table 4. Reaction conditions: 95°C for 10 seconds, [95°C for 5 seconds + 60°C for 30 seconds] for 40 cycles, and 4°C forever.

[0076] Table 4: PCR system

[0077] Reagents Reaction volume (μl) 2X Universal SYBR Green Fast qPCR Mix 10 Forward primer 0.4 Reverse primer 0.4 cDNA 0.6 <![CDATA[Nuclease-free H2O]]> 8.6 Total 20

[0078] Results: After obtaining the CT value of each sample, GAPDH was used as the internal reference. -ΔΔCT Methods: Calculate the relative expression of target genes. Primer sequences are shown in Table 5.

[0079] Table 5: RT-PCR detection fragment primer sequences

[0080]

[0081]

[0082] 9. Statistical analysis

[0083] Statistical analysis was performed using Graphpad Prism 8.0.1 software. Results are expressed as mean ± SD. Normally distributed data with equal variance were compared using the Student's t-test, while normally distributed data with unequal variance were compared using the unpaired t-test with Welch's correction. Non-normally distributed experimental data were compared using the nonparametric Mann-Whitney test. P < 0.05 was considered statistically significant. Kaplan-Meier analysis was used to investigate the prognostic value of CSAD expression in type I EC. IBM SPSS Statistics 20 was used to investigate the correlation between CSAD expression and the detection of type I endometrial adenocarcinoma.

[0084] 10. Results

[0085] Figure 2The survival curves in Figure 2 show the correlation between CSAD expression and disease-free survival (DFS) and overall survival (OS) in 269 patients with type I EC. Univariate and multivariate Cox regression models were used to test significance. Kaplan-Meier analysis showed that (using immunohistochemistry (IHC) scores ranging from 0 to 9, with a total score <4 considered as the CSAD expression decreased group (Low) and a total score ≥4 considered as the CSAD expression increased group (High)) that increased CSAD expression was significantly associated with shorter DFS (P = 0.044) and OS (P = 0.045). In addition, Cox univariate proportional hazards analysis showed that menopausal status, International Federation of Gynecology and Obstetrics (FIGO) stage, histological differentiation, myometrial invasion, LVSI, LNM, and CSAD expression significantly shortened DFS and OS. In addition, peritoneal lavage cytology was also associated with worse OS. Figure 3 The receiver operating characteristic (ROC) regression curve showed the correlation between CSAD expression and type I endometrial adenocarcinoma. Immunohistochemistry (IHC) scoring was used, ranging from 0 to 9, with a total score <4 considered decreased CSAD expression and a total score ≥4 considered increased CSAD expression. The ROC curve was used to test the model's fit. The analysis showed a correlation between CSAD expression and the detection of type I endometrial adenocarcinoma, with an area under the ROC curve (AUC) of 0.724, sensitivity of 84.76, and specificity of 56.10.

[0086] Example 2 Effect of CSAD expression on the proliferation of type Ⅰ endometrial cancer cells

[0087] 1. Cell Recovery

[0088] Human type I endometrial cancer cell lines HEC1A and IK cells stored in a -80°C freezer or liquid nitrogen were removed and placed in a 37°C electric hot water bath to shake for rapid thawing. The cell suspension was then pipetted into a 15ml centrifuge tube and centrifuged at 1000rpm for 5 minutes. The supernatant was discarded and 1ml of fresh complete culture medium was added. The tube was gently pipetted and mixed thoroughly, and then pipetted into a culture flask. A sufficient amount of culture medium was then added according to the size of the culture flask. After mixing thoroughly in the flask, the tube was placed flat in a cell culture incubator at 37°C and 5vol% CO2. The medium was changed the next day.

[0089] 2. Conventional Cell Culture

[0090] Human type Ⅰ endometrial cancer cell lines HEC1A and IK were cultured in RPMI-1640 medium containing 10% FBS and placed in a 5 vol% CO2, 37°C cell culture incubator for routine culture. The medium was changed every 2-3 days. Cells were passaged whenever the cell confluence reached 90%, and cells in the logarithmic growth phase were used for subsequent experiments.

[0091] 3. siRNA Synthesis

[0092] We selected the target site in the CSAD region and designed it according to the principle of sequence determination. We commissioned Shanghai Jima Pharmaceutical Technology Co., Ltd. to synthesize two siRNAs targeting CSAD (si-CSAD):

[0093]

[0094] This sequence was determined for use in this example (in the sense and antisense duplexes of the synthesized siRNA oligo, the two bases at the 3'-end were replaced by T instead of U, which did not affect the inhibitory effect, reduced costs, and enhanced resistance to enzyme degradation). A universal random negative sequence (si-NC) was also synthesized for calculating the transfection rate. 48 hours after transfection, qRT-PCR was used to detect the siRNA transfection efficiency. The results are shown in Figure 2. Figure 4 The knockdown efficiency was greater than 50%.

[0095] 4. Cell Transfection with DharmaFECT

[0096] 1) HEC1A and IK cells were seeded at a density of 100,000 cells / well in six-well plates. When the cells reached 50%-60% confluency and adherent growth, transfection was performed. Three groups were used: si-NC, si-CSAD#1, and si-CSAD#2.

[0097] 2) Working fluid configuration:

[0098] Solution A: 2 μl of siRNA mixed with 150 μl of OPTI-MEM;

[0099] Solution B: 4 μl of DharmaFECT mixed with 150 μl of OPTI-MEM;

[0100] After standing at room temperature for 5 minutes, mix solution A and solution B evenly. After standing at room temperature for 20 minutes, prepare the working solution. Do not mix vigorously after that.

[0101] 3) Add 1.7 ml of culture medium to each well of a 6-well cell plate, then slowly add 300 μl of the above working solution dropwise. Add 100 μl of the above working solution to each well of a 96-well cell plate, and gently shake the cell plate back and forth to mix the mixture with the culture medium in the wells.

[0102] 4) Culture in a CO2 incubator at 37°C for 48 hours.

[0103] 5. Cell proliferation assay: Repeat the following experimental steps three times:

[0104] 1) With a cell density of 3*10 3 / well, inoculated into 96-well plates, 100 μl per well, and 3 replicates per group.

[0105] 2) After the cells are completely attached, transfection is performed according to the above steps, and CCK8 assay is performed at 0 h, 24 h, 48 h, 72 h, and 96 h after transfection.

[0106] 3) Add 10 μl of CCK8 solution to each well, taking care not to create bubbles in the wells to avoid affecting the OD reading.

[0107] 4) Incubate at 37°C for 2 h in the dark. Measure the absorbance at 450 nm using a microplate reader. Plot a cell growth curve with time as the horizontal axis and absorbance as the vertical axis.

[0108] 6. Statistical Analysis

[0109] Statistical analysis was performed using Graphpad Prism 8.0.1 software and IBM SPSS Statistics 20. Results are expressed as mean ± SD. Normally distributed data with equal variances were compared using the Student's t-test, while normally distributed data with unequal variances were compared using the unpaired t-test with Welch's correction. Nonnormally distributed data were analyzed using the nonparametric Mann-Whitney test. P < 0.05 was considered statistically significant.

[0110] 7. Results

[0111] See the results Figure 4-5 , indicating that inhibiting CSAD expression can significantly inhibit the proliferation of type Ⅰ endometrial cancer cells.

[0112] Example 3 Effect of CSAD expression on the anoikis resistance of type Ⅰ endometrial cancer cells

[0113] 1. Establishment of a Cell Anoikis Model: Type I endometrial cancer cells were digested with 0.25% trypsin and plated into six-well plates (made from Corning Inc.) using ultra-low-adhesion polystyrene, a neutral, hydrophilic hydrogel surface. Approximately 250,000 cells were added to each well. Cells were cultured in suspension under these conditions, and their morphology and growth were observed using an inverted microscope. The medium was changed every 2-3 days depending on the cell status (centrifugation at 800 rpm for 3 minutes, supernatant discarded, and resuspended in fresh complete medium before addition to the plate). Adherent cells were also cultured as a control.

[0114] 2. Apoptosis Assay: Cell inoculation and transfection were performed as in Example 2. 72 hours after transfection, floating cells were collected from the supernatant. Adherent cells were digested with EDTA-free trypsin, and floating and adherent cells from the same well were collected in a 15 ml centrifuge tube. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant discarded, and the pellet collected. The pellet was then washed twice with PBS. The pellet was suspended in 500 μl of 1× binding buffer, and 5 μl of FITC-Annexin V-labeled annexin and 10 μl of PI were added. The cells were incubated at room temperature for 15 minutes in the dark. Cells were collected on a flow cytometer, and the apoptosis rate between the experimental and control groups was calculated and compared.

[0115] 3. Detection of anti-anoikis proteins BCL-2 and Bax:

[0116] 1) Total cell protein extraction: Wash cells twice with pre-chilled PBS and add 100 μl of RIPA (High-Performance Protein Lysis Buffer) containing 1% PMSF to each well. After thorough lysis on ice for half an hour, scrape the protein into a 1.5 ml EP tube and centrifuge at 14,000 rpm at 4°C for 30 min. Collect the supernatant, discard the pellet, and store at -80°C.

[0117] 2) Semi-quantitative protein concentration by Western blot:

[0118] ① Protein denaturation: Mix the protein sample with 5× Loading buffer at a ratio of 4:1, and then boil at 100°C for 10 minutes to denature.

[0119] ②SDS-PAGE electrophoresis: Mount a 12% precast gel on the electrophoresis stand, add 1x electrophoresis buffer, remove the comb, and add an appropriate amount of protein sample to each well. Maintain a constant voltage of 120V. Stop electrophoresis when the indicator reaches 1 cm from the edge of the glass.

[0120] ③ Transfer: Cut a 0.22 μm PVDF membrane to the desired gel size, leaving a notch in the upper left corner as a distinguishing mark. Activate with methanol for 15 seconds, then wash once in membrane equilibration solution with shaking for 1 minute. Arrange the membranes in the order of sponge, PVDF membrane, gel, and sponge, and secure them with the electroporation clamp. Be careful to avoid air bubbles. Carefully place the clamp in the electroporation tank and transfer the membrane using the standard transfer procedure.

[0121] ④Immunotoxicity: Blocking: After electroporation, immerse the membrane in 5 vol% skim milk, place it on a shaker and shake slowly, and block it at room temperature for 1 hour. Primary antibody incubation: Remove the blocking solution and wash off the excess milk with 1×TBST solution. Locate the target protein according to the marker band on the membrane. Cut the membrane and immerse it in the primary antibody dilution solution. Incubate it at 4°C overnight. Primary antibody washing: Recover the primary antibody, add 1×TBST solution, and wash it 3 times at room temperature with rapid shaking for 10 minutes each time. Change 1×TBST each time. Secondary antibody incubation: Immerse the membrane in the corresponding secondary antibody (1:5000) and incubate it on a shaker at room temperature for 1 hour with slow shaking. Secondary antibody washing: Recover the secondary antibody, add 1×TBST solution, and wash it 2 times with 10 minutes each time. Finally, wash the membrane once with 1×TBS solution for 10 minutes.

[0122] ⑤ Development: Aspirate 1 ml each of ECL Solution A and Solution B in the dark, mix thoroughly, and immerse the membrane in the mixture for 1 min. Use a GE ImageQuant LAS 4000mini instrument for automatic exposure and image storage. Analyze band grayscale values ​​using Image J software. The relative expression level of the target protein = grayscale value of the target protein / grayscale value of Gapdh.

[0123] 5. Statistical Analysis

[0124] Statistical analysis was performed using Graphpad Prism 8.01 software. Results are expressed as mean ± SD. Normally distributed data with equal variance were compared using the Student's t-test, while normally distributed data with unequal variance were compared using the unpaired t-test with Welch's correction. Non-normally distributed data were analyzed using the nonparametric Mann-Whitney test. P < 0.05 was considered statistically significant.

[0125] 6. The results are as follows Figure 6 、 Figure 7 As shown, Figure 7 It showed that inhibition of CSAD significantly suppressed the expression of the anti-anoikis gene BCL2 while up-regulating the expression of the Bax gene; Figure 6 This indicates that inhibiting CSAD expression can significantly inhibit the ability of type I endometrial cancer cells to resist anoikis, and increase cell apoptosis; it further explains that inhibiting CSAD expression is by inhibiting the expression of the anti-anoikis gene BCL2, thereby significantly inhibiting the ability of type I endometrial cancer cells to resist anoikis.

[0126] The above examples are only for understanding the method and concept of the present invention. Those skilled in the art may make several improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of a reagent for quantitatively detecting the expression level of CSAD in the preparation of a kit for diagnosing type I endometrial cancer, characterized in that: The sequence of the molecular marker CSAD is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that The kit performs detection based on real-time quantitative reverse transcription PCR, in situ hybridization, chip or high-throughput sequencing.

3. The application according to claim 2, characterized in that: The kit for diagnosing type I endometrial cancer by detecting the expression level of CSAD by real-time quantitative reverse transcription PCR comprises at least a pair of specific primers, as shown in SEQ ID NO.2 and SEQ ID NO.

3.

4. The application according to claim 2, characterized in that: The kit for detecting the expression level of CSAD by in situ hybridization, chip or high-throughput sequencing platform includes a specific probe that hybridizes with the nucleic acid sequence of CSAD.

5. Use of an agent for inhibiting CSAD in the preparation of a drug for treating type I endometrial cancer, characterized in that: The CSAD sequence is shown in SEQ ID NO.1, wherein the reagent for inhibiting CSAD is siRNA of CSAD, and the CSAD siRNA is shown in SEQ ID NO.6 and SEQ ID NO.7, or in SEQ ID NO.8 and SEQ ID NO.9.

Images