Use of TMED8 gene in preparation of drug for treating tumor and kit for diagnosing tumor

Abstract

The present application belongs to the technical field of precision medicine of tumors. Disclosed is the use of a TMED8 gene in the preparation of a drug for treating a tumor and a kit for diagnosing the tumor. The up-regulation of TMED8 gene expression can significantly inhibit the migration and proliferation abilities of pancreatic cancer cells, and can promote the apoptosis of pancreatic cancer cells; also, the TMED8 gene is related to an MAPK signaling pathway. Therefore, TMED8 can be used as an effective target for the gene diagnosis and treatment of pancreatic cancer. The expression of TMED8 in pancreatic cancer, the relationship between TMED8 and the prognosis and survival of patients, and the effect of TMED8 on the biological behavior of pancreatic cancer cells are studied, and it is explored that the action mechanism of TMED8 may be closely related to MAPK signaling pathway. TMED8 can be used as a kit for diagnosing pancreatic cancer and a drug for treating pancreatic cancer, which provides effective support for the early diagnosis and treatment of pancreatic cancer.

Description

Application of TMED8 gene in the preparation of tumor therapeutic drugs and diagnostic kits Technical Field

[0001] The present application belongs to the field of precision medicine technology for tumors, and specifically relates to the application of the TMED8 gene in the preparation of tumor treatment drugs and diagnostic kits thereof. Background Art

[0002] Pancreatic cancer (PC) is an extremely common and highly malignant tumor characterized by difficulty in early diagnosis, high metastatic potential, rapid progression, and a dismal prognosis. It is the leading cause of cancer mortality worldwide. While not directly modifiable, inherited genetic factors contribute significantly to pancreatic cancer risk. These include pathogenic variants in hereditary cancer genes, genes associated with hereditary pancreatitis, and common variants identified through genome-wide association studies. Identifying genetic alterations in cancer not only provides insights into the etiology of this cancer but also offers opportunities to guide early detection strategies. Pancreatic cancer lacks obvious symptoms in its early stages, making timely detection and diagnosis difficult. Currently, commonly used tumor markers such as CA199 and CEA have poor sensitivity and specificity for early diagnosis of pancreatic cancer. By the time a diagnosis is confirmed, most patients are already in the advanced stage. Beyond radical surgical resection, adjuvant chemotherapy and radiotherapy offer limited improvement in prognosis. Therefore, the exploration and discovery of more sensitive and specific early biomarkers and effective therapeutic targets is of great importance.

[0003] The TMED family (transmembrane emp24 domain family) is composed of type I single-pass transmembrane proteins found in all eukaryotes, and their role in human development and disease cannot be ignored. As important transport proteins in the secretory pathway, abnormalities in the protein transport pathways controlled by them can lead to a variety of diseases, including congenital malformations, diabetes, and cancer. Previous studies have shown that abnormal expression of TMED proteins is often closely associated with liver disease, pancreatic disease, and immune system disorders. They can form secretory loops during the development of diseases, maintain the normal morphology of cells and intracellular organelles, circulate a variety of important cargo components between organelles, and achieve communication between organelles, thereby continuously regulating the disease process. They play a particularly important role in the proliferation and differentiation of malignant tumor cells.

[0004] TMED8 (NCBI Gene: 283578) is a key member of this family, located between bases 77,335,021 and 77,377,094 on chromosome 14. It has a molecular weight of approximately 55 kDa and is expressed in both the nucleus and cytoplasm. Currently, there are no reports linking TMED8 to human disease or normal physiological function.

[0005] Summary of the Invention

[0006] The purpose of this application is to provide an application of the TMED8 gene in the preparation of tumor treatment drugs and diagnostic kits thereof, as well as an application of TMED8 in the preparation of tumor treatment drugs.

[0007] The present application provides an application of a reagent for detecting the expression level of TMED8 in the preparation of a product for diagnosing a disease.

[0008] Preferably, the reagent includes a reagent for detecting the expression level of TMED8 mRNA or protein.

[0009] Preferably, the method for detecting the expression level of TMED8 comprises at least one of the following: immunohistochemistry, Western Blot and real-time fluorescence quantitative PCR.

[0010] Preferably, the type of disease includes tumor.

[0011] The present application also provides a kit for diagnosing tumors, comprising at least one of the following reagents: a reagent for immunohistochemistry detection of TMED8 protein expression, a reagent for Western Blot detection of TMED8 protein expression, and a primer pair for real-time fluorescence quantitative PCR detection of TMED8 gene and / or mRNA expression.

[0012] Preferably, the tumor comprises pancreatic cancer.

[0013] The present application also provides a use of a reagent for increasing the expression level of TMED8 in the preparation of a product for treating a disease.

[0014] Preferably, the reagent comprises at least one of the following: a TMED8 agonist, a TMED8 analog, or a reagent for overexpressing TMED8.

[0015] The present application also provides a drug for treating tumors, including an agent for increasing the expression level of endogenous TMED8 or exogenous TMED8.

[0016] Preferably, the treatment includes at least one of the following: inhibiting tumor cell migration, inhibiting tumor cell proliferation, promoting tumor cell apoptosis, and inhibiting MAPK signaling pathway activation.

[0017] Beneficial effects: This application prepares pancreatic cancer tissue chips and uses immunohistochemistry to detect the expression of TMED8 in pancreatic cancer. The results suggest that the expression level of TMED8 in pancreatic cancer tumor tissue is significantly higher than that in adjacent tissues. In addition, by establishing a pancreatic cancer stable cell line overexpressing TMED8, the effects of TMED8 on the biological behavior of pancreatic cancer cells were explored using in vitro and in vivo experiments such as qRT-PCR, Western Blot, cell scratch, CCK8, Transwell, flow cytometry, and subcutaneous tumor formation in nude mice. It was found that upregulating TMED8 gene expression significantly inhibited the migration and proliferation ability of pancreatic cancer cells and promoted the apoptosis of pancreatic cancer cells. This application also found that the TMED8 gene is related to the MAPK signaling pathway. Therefore, TMED8 can be used as an effective target for genetic diagnosis and treatment of pancreatic cancer, and has important application significance in the preparation of pancreatic cancer diagnostic kits and therapeutic drugs for precision medicine. This application explores the expression of TMED8 in pancreatic cancer and its impact on the biological behavior of pancreatic cancer cells, and explores the possible mechanism of action that is closely related to the MAPK signaling pathway. TMED8 is expected to be used as a diagnostic kit and therapeutic drug for pancreatic cancer, providing effective support for the early diagnosis and treatment of pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 shows the expression of TMED8 in pancreatic cancer and adjacent tissue microarrays detected by IHC, with selected representative sites shown: ab. staining results of poorly differentiated PC cancer and adjacent tissues; cd. staining results of moderately differentiated PC cancer and adjacent tissues; ef. staining results of well-differentiated PC cancer and adjacent tissues;

[0019] Figure 2 shows the expression of TMED8 and the corresponding WB and PCR verification results;

[0020] FIG3 is a comparison of TMED8 expression in normal pancreatic ductal epithelial cells and tumor cell lines;

[0021] FIG4 is a diagram showing the validation of TMED8 overexpression efficiency in pancreatic cancer stable cell lines;

[0022] FIG5 is a diagram showing the effect of TMED8 overexpression on the migration ability of pancreatic cancer cells studied in a cell scratch assay;

[0023] FIG6 is a diagram showing the effect of TMED8 overexpression on the migration ability of pancreatic cancer cells in a Transwell experiment;

[0024] FIG7 is a diagram showing the effect of TMED8 overexpression on the proliferation of pancreatic cancer cells in a CCK8 experiment;

[0025] FIG8 is a diagram showing the effect of TMED8 overexpression on the proliferation ability of pancreatic cancer cells in a proliferative experiment;

[0026] FIG9 is a diagram showing the effect of TMED8 overexpression on the apoptosis of pancreatic cancer cells detected by flow cytometry;

[0027] FIG10 is a diagram showing the effect of TMED8 overexpression on subcutaneous tumor formation of pancreatic cancer cells in nude mice;

[0028] FIG11 is an IHC test result of TMED8 overexpression on pancreatic cancer cells after subcutaneous tumor formation in nude mice, stained with TMED8 and proliferation indicator Ki67 antibodies;

[0029] FIG12 is a heat map of transcriptome sequencing of pancreatic cancer cell lines overexpressing TMED8;

[0030] FIG13 is a volcano plot of the number of differentially expressed molecules obtained by transcriptome sequencing of pancreatic cancer cell lines overexpressing TMED8;

[0031] FIG14 is a diagram showing the KEGG enrichment analysis results of differentially expressed molecules after TMED8 overexpression;

[0032] FIG15 is a diagram showing the effect of TMED8 overexpression on MAPK-related molecules;

[0033] FIG16 is a construction map of the TMED8 overexpression plasmid pLV3-CMV-TMED8(human)-3×FLAG-EF1a-CopGFP-Puro. DETAILED DESCRIPTION

[0034] The present application provides an application of a reagent for detecting the expression level of TMED8 in the preparation of a product for diagnosing a disease.

[0035] In the examples of the present application, a pancreatic cancer tissue chip was prepared based on a biological sample library, and the expression of TMED8 in pancreatic cancer was detected using immunohistochemistry. The results showed that the expression level of TMED8 in pancreatic cancer tumor tissue was significantly higher than that in adjacent cancer tissue. Moreover, statistics showed that patients in the TMED8 high expression group had a better prognosis. Therefore, TMED8 was used as a biomarker for diagnosing pancreatic cancer.

[0036] The reagents described herein preferably include reagents for detecting the expression of TMED8 mRNA or protein, and the detection method preferably includes at least one of the following: immunohistochemistry, Western Blot, and real-time fluorescence quantitative PCR. The type of disease described herein preferably includes tumors, more preferably pancreatic cancer.

[0037] This application also provides a tumor diagnostic kit comprising at least one of the following reagents: a reagent for immunohistochemistry to detect TMED8 protein expression, a reagent for Western blot detection of TMED8 protein expression, and a primer pair for real-time fluorescence quantitative PCR to detect TMED8 mRNA expression. The primer sequences for TMED8 and the internal reference 18S are shown in Table 1.

[0038] Table 1 Primer sequences

[0039] The tumor described in the present application preferably includes pancreatic cancer.

[0040] The present application also provides a use of a reagent for increasing the expression level of TMED8 in the preparation of a product for treating a disease.

[0041] In the examples of this application, a stable pancreatic cancer cell line overexpressing TMED8 was established. The effects of TMED8 on the biological behavior of pancreatic cancer cells were investigated using in vitro and in vivo experiments, including qRT-PCR, Western Blot, cell scratch assays, CCK8, Transwell assays, flow cytometry, and subcutaneous tumor formation in nude mice. Upregulation of TMED8 gene expression significantly inhibited the migration and proliferation of pancreatic cancer cells and promoted apoptosis. The application also found that TMED8 is associated with the MAPK signaling pathway. Therefore, TMED8 may be an effective target for gene therapy of pancreatic cancer.

[0042] The reagents described in the present application preferably include at least one of the following: a TMED8 agonist, a TMED8 analog, and a reagent for overexpressing TMED8.

[0043] The method for overexpressing TMED8 described in the present application preferably includes constructing a TMED8 overexpression plasmid pLV3-CMV-TMED8 (human)-3×FLAG-EF1a-CopGFP-Puro, the plasmid sequence of which is shown in SEQ ID No. 5.

[0044] The present application also provides a drug for treating tumors, including an agent for increasing the expression level of endogenous TMED8 or exogenous TMED8.

[0045] The agents described herein for increasing endogenous TMED8 expression are preferably the same as those described above for overexpressing TMED8 and are not further described herein. The exogenous TMED8 described herein is preferably the same as the TMED8 agonists and TMED8 analogs described above and are not further described herein. The treatment described herein preferably includes at least one of the following: inhibiting tumor cell migration, inhibiting tumor cell proliferation, promoting tumor cell apoptosis, and inhibiting activation of the MAPK signaling pathway.

[0046] To further illustrate the present application, the application of the TMED8 gene provided by the present application in the preparation of pancreatic cancer drugs and diagnostic kits thereof is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present application.

[0047] Unless otherwise specified, the reagents and instruments used in the examples of this application are common reagent kits and instruments in the field and can be purchased from reagent stores:

[0048] 1. Main reagents in the examples of this application:

[0049] IHC detection kit (Servicebio, China); 4% paraformaldehyde fixative solution (Servicebio, China); PBS buffer (Servicebio, China); bovine serum albumin (BSA) powder (Solarbio Life Sciences, China); Triton X-100 (Solarbio Life Sciences, China); xylene (Sinopharm Group, China); hematoxylin staining solution (Byotime, China); hematoxylin differentiation solution (Byotime, China); anhydrous ethanol (Sinopharm Group, China); sodium citrate antigen retrieval solution (Beyotime, China); rabbit anti-TMED8 antibody (Invitrogen, USA); rabbit anti-Ki67 antibody (Absin, China); HRP-conjugated goat anti-rabbit secondary antibody (Abcam, USA); 95% ethanol (Sinopharm Group, China); DMEM basal medium (Cytiva, China) China); IMDM basal medium (Cytiva, China); RPMI basal medium (Cytiva, China); PBS basal buffer (Servicebio, China); fetal bovine serum (Gibco, USA); serum-free cell freezing medium (New Saimei, China); EDTA-containing trypsin digestion solution (New Saimei, China); penicillin-streptomycin solution (New Saimei, China); tetracycline hydrochloride solution (Shanghai Biotechnology, China); puromycin (Sangon, USA); cell culture dishes (Nest, China); Lipofectamine 2000 (Invitrogen, USA); 6-well plates, 96-well plates, and 24-well plates (Nest, China); 8 μm transparent PET film for cell culture chambers (BD Falcon, USA); 1.5 mL, 4 mL, and 10 mL centrifuge tubes and cell cryopreservation tubes (Biosharp, USA); Trizol (Novizan, China); cell scraper (Nest, China); DEPC water (Shanghai Biotech, China); LC eight-tube strips (Roche, Switzerland); enzyme-free pipette tips and centrifuge tubes (Biosharp, China); anhydrous ethanol (Sinopharm Group, China); isopropanol (Sinopharm Group, China); chloroform / trichloromethane (Sinopharm Group, China); (1) RIPA strong lysis buffer (Byotime, China); protease inhibitor CM (Amresco, USA); phosphatase inhibitor PI (Amresco, USA); BCA protein assay kit (Byotime, China); protein loading buffer 4× Loading Buffer (Novizan, China); Marker standard protein (Novizan, China); PVDF membrane (Sigma, USA); TEMED tetramethylethylenediamine (Sigma, USA); 0× electrophoresis buffer pre-made powder (Servicebio, Wuhan);10× transfer buffer powder (Servicebio, Wuhan); 10× TBST powder (Servicebio, Wuhan); sodium chloride (NaCl) (Sinopharm Group, China); isopropyl alcohol (Sinopharm Group, China); ammonium persulfate (AP) (Sigma, USA); glycine (Sinopharm Group, China); sodium dodecyl sulfate (SDS) (Sinopharm Group, China); tris (hydroxymethylaminomethane) (Tris) (Sinopharm Group, China); methanol (Sinopharm Group, China); skim milk powder (Yili Dairy, China); ultrasensitive ECL chemiluminescence kit (Xinsaime, China); 30% polyacrylamide solution (Servicebio, China); Tween (Sinopharm Group, China); mouse anti-human GAPDH antibody (Proteintech, China); rabbit anti-human TMED8 antibody (Invitrogen, USA); rabbit anti-human ERK1 / 2 antibody (CST, USA); rabbit anti-human ERK phosphorylation antibody (CST , USA); rabbit anti-human P38 antibody (CST, USA); rabbit anti-human P38 phospho-antibody (CST, USA); rabbit anti-human JNK antibody (CST, USA); rabbit anti-human JNK phospho-antibody (CST, USA); rabbit anti-human Casepase 3 antibody (Santa, Japan); rabbit anti-human CD63 antibody (Abcam, USA); rabbit anti-human CD81 antibody (Abcam, USA); rabbit anti-human CD9 antibody (Abcam, USA); mouse anti-human Ki-67 antibody (Wuhan Tri-Tek Co., Ltd., China); HRP-labeled goat anti-rabbit IgG antibody (Invitrogen, USA); HRP-labeled goat anti-mouse IgG antibody (Invitrogen, USA); CCK-8 kit (Byotime, China); cell cycle analysis kit (Formex Biotechnology, China); apoptosis detection kit: Annexin V-PE / 7-AAD double staining (Keygen Biotechnology, China); crystal violet staining solution (Byotime, China).

[0050] 2. Main instruments used in the examples of this application:

[0051] Tissue chip system (UT06, Unitma, South Korea); inverted microscope (Leica, Germany); chemiluminescence gel imaging system (Bio-Rad, USA); automatic microplate reader (Bio-Rad, USA); electrophoresis system (Bio-Rad, USA); cell culture incubator (Thermo Fisher Scientific, USA).

[0052] Example 1

[0053] Between 2020 and 2023, 79 formalin-fixed, paraffin-embedded (FFPE) pancreatic cancer tissue samples, including both cancerous and adjacent tissues, were obtained from the Clinical Biobank of the Affiliated Hospital of Nantong University. None of the cancer patients received any treatment, including radiotherapy, chemotherapy, or immunotherapy, and their clinical records were complete. The study protocol was approved by the Human Research Ethics Committee of the Affiliated Hospital of Nantong University, China.

[0054] 1. Fabrication of tissue microarrays:

[0055] (1) Based on the microscopic observation results of HE sections, the representative cancer nest areas in the paraffin block were marked.

[0056] (2) Paraffin wax and beeswax were mixed at a ratio of 1:1 to make a TMA blank receptor wax block. A 10×7 point tissue array was designed on the wax block, and then a tissue microarray instrument was used to punch holes to make a TMA blank wax block.

[0057] (3) The most representative cancer nest area was selected at the marked point on the donor wax block, and a tissue block with a diameter of 2 mm was collected. One core was taken for each case.

[0058] (4) The obtained tissue cores are transferred to the holes of the recipient wax block, with a certain distance between each tissue core, and the corresponding adjacent cancer tissues are taken as controls.

[0059] (5) The tissue array block was placed in a 55°C incubator for 10 minutes to heat and fuse. Before the wax block melted, it was taken out and cooled to room temperature to allow the recipient wax block to fuse with the donor tissue.

[0060] (6) Before slicing, precool the tissue chip at 4°C for about 4 hours, and then use a fully automatic tissue slicer to trim the tissue array block at a speed of 20 mm / rev until all tissues are intact.

[0061] (7) Slice the tissue array block using a microtome. Float the serial slices in cold water to allow them to unfold naturally. Then transfer the slices to 45°C warm water and unfold them for about 2 minutes. After unfolding, stick them on a slide treated with anti-slipping and let them dry.

[0062] (8) Place the slices in a high-temperature oven at 60°C for 3 minutes and continue baking at 58°C for 18 hours.

[0063] (9) Store the prepared tissue microarray in a slicing box and store at -20°C until ready for use.

[0064] 2. Immunohistochemical staining:

[0065] (1) First, bake the tissue chip in a 60°C constant temperature oven for about 40 minutes until the paraffin melts;

[0066] (2) Immediately place the chip in xylene I, xylene II, 100% ethanol I, and 100% ethanol II for 5 minutes each, then place it in 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol in sequence for 2 minutes each, and then rinse with clean water;

[0067] (3) Place antigen retrieval solution in the immunohistochemistry box in advance and preheat it to 95°C in a water bath. Then place the chip in the container and treat it at 95°C for 20 minutes for antigen retrieval. Then cool it naturally to room temperature and rinse it with PBS three times for 5 minutes each time.

[0068] (4) Then, draw a border line around the tissue on the chip with an immunohistochemistry pen, then drip 3% H2O2 inside the circle, incubate for 10 minutes, and then wash with PBS three times, 5 minutes each time;

[0069] (5) Add primary antibody (rabbit anti-human TMED8 antibody) and incubate overnight at 4°C;

[0070] (6) Rewarm at room temperature for 1 hour, remove the primary antibody, rinse with PBS three times for 5 minutes each, add polymer enhancer, and incubate at room temperature for 20 minutes; then wash with PBS three times for 5 minutes each;

[0071] (7) Add secondary antibody (enzyme-labeled anti-mouse / rabbit IgG polymer) and incubate at room temperature for 30 min, then wash three times with PBS for 5 min each;

[0072] (8) Add freshly prepared DAB reagent and rinse the chip under tap water for 5 minutes to terminate the color development.

[0073] (9) Place the chip in hematoxylin stain for 2 minutes, and immediately rinse it slowly with running water. Then, place it in 0.5% HCl ethanol solution, soak it for 2 minutes, and finally rinse it with running water for 15 minutes.

[0074] (10) Soak the chip in 80% ethanol, 90% ethanol, 95% ethanol, 100% ethanol I, 100% ethanol II, and xylene for 1 minute in sequence and allow to air dry in a fume hood.

[0075] (11) Finally, seal the slides with neutral gum.

[0076] 3. Result judgment:

[0077] Based on the degree of cell staining within the tissue, the positive intensity can be divided into three levels: no positive staining is level 0 (negative), light yellow is level 1 (weakly positive), brown is level 2 (moderately positive), and tan is level 3 (strongly positive). The cell positive ratio is calculated as the number of positive cells divided by the total number of cells and can be divided into four levels. The ratios of levels 0 to 4 are 0% to 5%, 6% to 25%, 26% to 50%, 51% to 75%, and >75%, respectively. The microarray results were scored by three pathology experts based on independent review and were divided into four groups (-, +, ++, and +++) according to the quartiles for statistical analysis.

[0078] Figure 1 shows immunohistochemical staining results for 79 pancreatic cancer and adjacent tissue specimens. TMED8 protein expression is primarily in the cytoplasm of tumor cells and in the stroma of cancerous tissues, appearing brownish-yellow. However, expression is low or absent in most adjacent tissues. Statistical results demonstrate that TMED8 is highly expressed in pancreatic cancer tissue microarrays. *P < 0.05.

[0079] Eight pairs of pancreatic cancer and adjacent tissues were selected for WB verification. The results are shown in Figure 2 and are consistent with the above.

[0080] Example 2

[0081] 1. Cell transfection:

[0082] (1) Transformation and extraction of plasmids: Take out the plasmid powder of TMED8 overexpression plasmid pLV3-CMV-TMED8(human)-3×FLAG-EF1a-CopGFP-Puro stored in a -20°C refrigerator, add 40 μL of DEPC water to dissolve it, and at the same time, turn on the electric constant temperature water bath and raise it to 42°C. Place the DH5α competent vector stored in a -80°C ultra-low temperature refrigerator on ice to melt. When the competent vector melts, the transformation experiment is carried out immediately;

[0083] The TMED8 overexpression plasmid (Figure 16) was mixed with the thawed competent medium in a volume ratio of 1:3. The tube was gently tapped and blown 2-3 times to mix. The tube was allowed to stand on ice. After 25 minutes, the tube was immediately placed in a 42°C water bath for incubation with a precise time of 45 seconds. Then, the tube was immediately transferred to an ice bath for 3 minutes. The transformed plasmid was added to a shake tube containing 40 μL of antibiotic-free LB liquid culture. The tube was pre-shaken at 200 rpm at 37°C for 1 hour. After the pre-shaking, 4 mL of liquid culture containing 50 μg / mL ampicillin was added. The tube was shaken at 250 rpm at 37°C overnight. The specific time was controlled within 14-16 hours to avoid the growth of contaminants. The bacterial solution was extracted using the Axygen plasmid extraction kit according to the standard procedure. The obtained plasmid concentration was measured and recorded. The remaining bacterial solution can be mixed with glycerol for seed preservation and stored at -80°C.

[0084] (2) Establishment of stable transfected cell lines: seed an appropriate amount of wild-type cells to be transfected (PANC-1 and BXPC-3 pancreatic cancer cell lines) in a six-well plate, and control the density to reach 60% to 70% after 6 hours of attachment. Fuse 80 μL of 1640 basal medium without serum and double antibodies with 3 μg of plasmid, gently tap the tube wall to mix the reagents, do not use a pipette to avoid DNA breakage, let it stand at room temperature for 5 minutes, and then add 3 μL of Lipofectamine 2000 transfection reagent, stand at room temperature for 20 to 30 minutes, add to the complete culture medium of 6-well plate, mix gently with a pipette, and culture in a cell culture incubator at 37°C and 5% carbon dioxide for 24 to 48 hours. Observe once in the first 24 hours, and observe the cell status every 12 hours thereafter. If the cell status deteriorates due to the toxicity of the transfection reagent, change the medium immediately; after the transfection is stable, observe the cell transfection efficiency under a fluorescence microscope using the fluorescent label of the plasmid. After successful transfection, use the corresponding resistance, generally use puromycin to kill the wild-type cells that have not been successfully transfected, screen out the successfully transfected cells, establish a stable transfected cell line, and collect the total protein or total RNA of the cells after subculture for WB or qRT-PCR experiments to verify the overexpression efficiency of the cells. The results are shown in Figure 4.

[0085] 2. CCK-8 cell proliferation activity assay

[0086] Use medium dishes to pre-culture the overexpressing stable cells and wild-type control cell lines to be tested. When the cell confluence in the dish reaches 90%, digest and resuspend them. Note that the cell weight in the dish must be properly controlled. Cells growing in a crowded state may trigger their own apoptosis, affecting the accuracy of subsequent results. Count the cells and, based on cell growth characteristics, seed them in a 96-well plate at a rate of 5,000 to 10,000 cells per well. Set up 5 to 6 replicate wells for each group to reduce experimental errors caused by manipulation. Note that the number of cells in the control and treatment groups must be equal to achieve the purpose of controlling variables. Cell proliferation activity testing begins 6 to 12 hours after the cells have attached. Because cell proliferation activity needs to be continuously tested for 5 days based on the cell doubling cycle, experimental operations and results testing should be performed at the same time each day.

[0087] CCK8 reagent and the basal culture medium of the corresponding cells were mixed at a volume ratio of 1:9 to obtain a fresh working solution. The culture medium in the original wells was discarded, and after washing with 1× PBS buffer, 100 μL of 10% CCK8 working solution was added to each well. The cells were allowed to react in a 37°C incubator for 1 to 2 hours. The absorbance was measured at 460 nm using a microplate reader, and the data was recorded. After all the tests were completed, the data were processed and compared and analyzed.

[0088] The results are shown in FIG6 , and overexpression of TMED8 inhibited the proliferation ability of pancreatic cancer cells.

[0089] 3. Cell scratch healing assay

[0090] Place a brand new sterile packaged six-well plate upside down on the clean bench, and use a ruler and marker to draw nine-square coordinate lines on the bottom of the plate to facilitate positioning when taking pictures under a microscope during the subsequent scratch experiment. Appropriate amounts of wild-type and overexpression cell lines were seeded in six-well plates, with three replicate wells for each control and overexpression group. According to their growth cycle, fresh complete nutrient medium was used to culture the cells to a density of 90%. Then, an alcohol-sterilized ruler and a 1 mL sterile blue pipette tip were used to scratch the cells. During the process, attention was paid to verticality and uniform force. The size of the scratches in each replicate well should be controlled to be basically equal to reduce the result error caused by the operation and better control the experimental variables. Three scratch lines were also made in each well to ensure the reproducibility of the experimental results. After the scratching was completed, 1× PBS buffer was added to clean the floating cells and cell debris. The force should be gentle to prevent the cells at the scratch boundary from being washed away. The complete medium with low serum concentration was replaced to maintain cell growth while controlling the growth rate for subsequent observation. The time point was recorded as 0 h at the start of the experiment and photos were taken. The healing of the cell scratches was observed under a microscope and photos were taken every 24 h. The final experimental results were integrated and analyzed after 72 h.

[0091] The results are shown in Figures 5 and 6 , showing that overexpression of TMED8 inhibited the migration ability of pancreatic cancer cells.

[0092] 4. Cell colony formation assay

[0093] Use trypsin containing EDTA to digest the cells to be tested into a single state, collect the cell suspension, count the cells, and seed the control group and overexpression group cells into 6-well plates at a number of 500 per well. Each group has 3 replicate wells, and 2 mL of fresh culture medium is added to each well. The plates are placed in a cell culture incubator and cultured at 37°C under 5% carbon dioxide for 5 to 7 days. Due to the small initial number of cells, no medium change is required during colony formation. After 5 to 7 days, the culture plates are removed and the colony formation state is observed under a microscope. After washing with 1× PBS and discarding the solution, 1 mL of 4% paraformaldehyde fixative is added to each well for 2 hours, rinsed with PBS three times, and then stained with crystal violet stain for 1 hour. The working solution is discarded, rinsed with double distilled water, and then photographed after drying to observe the difference in colony formation between the two groups of cells.

[0094] The results are shown in Figures 7 and 8 , indicating that overexpression of TMED8 inhibited the formation of pancreatic cancer cell colonies, ie, inhibited the proliferation ability of pancreatic cancer cells.

[0095] 5. Transwell cell migration assay

[0096] Digest, resuspend and count the cells to be tested, and set aside for later use; take out the 24-well cell culture plate and the matching chamber with 8μm transparent PET film, place the chamber in the culture well of the 24-well plate, add 600μL of complete nutrient medium containing 5% fetal bovine serum outside the chamber groove, that is, into the culture well; count and aspirate 30,000 cells, fully resuspend them with 200μL of basic nutrient medium without fetal bovine serum, transfer them to the inner groove of the chamber with a pipette, and culture for 24 to 72 hours according to the cell growth cycle. The cells fully pass through the biomembrane and the migration ability of the cells is observed; the chamber is removed, the culture medium in the inner tank is discarded, and it is placed in a clean 24-well plate. It is fixed with 4% paraformaldehyde fixative for 2 hours, then gently rinsed with double distilled water, and then crystal violet staining solution is added for 1 hour. After staining, it is washed with double distilled water. If the staining is too dark and affects the observation results, methanol can be used for decolorization and rinsing to elute the crystal violet in the blank area of ​​the membrane; after the staining is completed, the chamber is turned upside down and dried at room temperature. It is observed with an ordinary optical microscope and a suitable field of view is selected for taking pictures.

[0097] The results are shown in Figures 5 and 6 , showing that overexpression of TMED8 inhibited the migration ability of pancreatic cancer cells.

[0098] 6. Flow cytometry

[0099] Flow cytometry detection of cell apoptosis: PE-fluorescein-labeled Annexin V dye and 7-AAD dye were used to double-stain the cells to detect cell apoptosis; first, the cells were digested and collected using EDTA-free trypsin, washed 2-3 times with 1×PBS cell washing solution, and the cells were counted. The number of cells in each sample was generally controlled at 10,000-50,000. No fixation was required to prevent subsequent fluorescence quenching; the collected clean cell pellet was resuspended in 100 μL buffer, and 1 μL of Annexin V was added to each 100 μL suspension. V dye and 5μL 7-AAD dye, mix gently and stain in the dark at room temperature. After 5 to 10 minutes of reaction, add 400μL of buffer and mix well. Transfer to a professional flow cytometer tube and complete the detection within 1 hour. PE is orange-red fluorescence with an excitation wavelength of Ex=488nm and an emission wavelength of Em=578nm. It is recommended to use the FL2 channel. 7-AAD dye is red fluorescence with an excitation wavelength of Ex=546nm and an emission wavelength of Em=647nm. It is recommended to use the FL3 channel for detection. Fluorescence compensation adjustment is required during subsequent data analysis.

[0100] The results are shown in FIG9 , and overexpression of TMED8 promoted the apoptosis of pancreatic cancer cells.

[0101] 7. Subcutaneous tumor formation in nude mice

[0102] Four-week-old male BALB / c immunodeficient nude mice were selected for subcutaneous tumor formation experiments. The weight of each nude mouse was weighed and recorded before the experiment. PANC-1 control cells and overexpressing cells were digested with trypsin and then counted. Each nude mouse was injected with approximately 2×10 cells of each type. 7 The cells were resuspended in sterile 1× PBS solution. The cell suspension must be kept on ice before animal experiments to prevent cell damage and death. Equal numbers of PANC-1 cells overexpressing TMED8 and PANC-1 control cells were injected into the left and right axillae of the same nude mouse. The mice were observed daily thereafter. After 4 to 6 weeks of tumor formation, the mice were sacrificed and dissected. The tumors were removed, weighed, and measured in volume before being fixed for subsequent immunohistochemistry experiments. Control experiments performed on the same nude mouse facilitated control of interfering factors caused by mouse heterogeneity, allowing for a more intuitive assessment of the effect of TMED8 on tumor growth in vivo.

[0103] The results are shown in Figures 10 and 11 , indicating that TMED8 inhibited the tumor-forming ability of pancreatic cancer cells under the skin of nude mice, that is, inhibited the proliferation ability of pancreatic cancer cells in animals.

[0104] 8. Western Blot

[0105] (1) Extraction of total protein from cells and tissues: During the entire process, the cell samples from which total protein is to be obtained should be placed on ice. The culture medium in the dish should be discarded and washed with 1× PBS solution. 0.2 to 0.4 mL of RIPA strong lysis buffer should be added. After lysis for about half an hour, the samples should be scraped with a cell scraper and transferred to a 1.5 mL centrifuge tube. The tube should be placed on an electric rotary table at 4°C and rotated overnight to allow the protein to be fully lysed. The tube should be centrifuged at 7500 rpm for 15 minutes. The supernatant should be aspirated and the cell debris pellet should be discarded. 1 / 4 of the total volume of the supernatant should be added with sample buffer (Loading Buffer) and boiled for at least 30 minutes to ensure that the protein is fully heated and denatured. After cooling to room temperature, the tube should be aliquoted and stored in a -20°C or -80°C refrigerator to prevent the protein from being degraded by repeated freezing and thawing.

[0106] (2) Preparation of SDS-PAGE gel: Wash and dry the gel plate and clamp it on the gel rack. Add double-distilled water to check for leaks. After the leak test is passed, hang it vertically to dry. Prepare the separation gel according to the proportion of the components of different concentrations (see Table 2). Inject the prepared separation gel gently and evenly into the pre-mounted gel plate. Be careful to avoid the generation of bubbles during the process. Let the isopropanol gel stand for about 40 minutes. Shake the gel plate gently. After confirming that the gel has solidified, remove the isopropanol and gently rinse with double-distilled water to dry. Then add the prepared upper concentrated gel and quickly insert the comb vertically (see Table 3). In this step, it is especially important to avoid the generation of bubbles, otherwise the sample wells are prone to deformation and sample leakage.

[0107] Table 2 Preparation of 10 mL SDS-PAGE separation gel fractions of different concentrations (mL)

[0108] Table 3: Ratios for preparing 8 mL of upper concentrated gel

[0109] 9. TMED8 may regulate the biological functions of PAAD tumor cells through the MAPK pathway, thereby affecting the occurrence and development of pancreatic cancer

[0110] PANC-1 control and overexpression cell lines (each group had 3 replicates) were selected for transcriptome sequencing. A total of 248 differentially expressed genes were obtained through data analysis, of which 103 were upregulated and 145 were downregulated. They were visualized using heat maps and volcano maps, respectively. Functional annotation and KEGG enrichment analysis were performed on them. The MAPK signaling pathway with significant correlation was selected for further study, and the expression levels of key molecules ERK, JNK, P38 and their phosphorylation were verified by Western blotting.

[0111] The results are shown in Figures 10 to 15 , indicating that TMED8 inhibited the activation of the MAPK pathway.

[0112] Although the above embodiment provides a detailed description of the present application, it is only a part of the embodiments of the present application, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present application.

Claims

1. Application of a reagent for detecting the expression level of TMED8 in the preparation of products for diagnosing tumors.

2. The application according to claim 1, characterized in that: The detecting of the expression level of TMED8 includes detecting the expression level of TMED8 mRNA or protein.

3. The use according to claim 1 or 2, characterized in that: The reagent for detecting the expression level of TMED8 includes at least one of the following: an immunohistochemistry reagent, a Western Blot reagent, and a real-time fluorescence quantitative PCR reagent.

4. The use according to claim 1, wherein the tumor comprises pancreatic cancer.

5. A kit for diagnosing tumors, characterized in that: The method comprises at least one of the following reagents: a reagent for detecting the expression of TMED8 protein by immunohistochemistry, a reagent for detecting the expression of TMED8 protein by Western Blot, and a primer pair for detecting the expression of TMED8 gene and / or mRNA by real-time fluorescence quantitative PCR.

6. The kit according to claim 5, characterized in that The nucleotide sequences of the primer pairs for detecting the mRNA expression of TMED8 by real-time fluorescence quantitative PCR are shown in SEQ ID No.1 to SEQ ID No.

2.

7. The kit according to claim 5 or 6, characterized in that When the mRNA expression level of TMED8 is detected by real-time fluorescence quantitative PCR, primers for detecting the internal reference gene 18S are also included, and the primer sequences are shown in SEQ ID No.3 to SEQ ID No.

4.

8. Use of a reagent for increasing the expression level of TMED8 in the preparation of a product for treating tumors.

9. The use according to claim 8, characterized in that: The reagent for increasing the expression level of TMED8 includes at least one of the following: an agonist of TMED8, an analog of TMED8, and a reagent for overexpressing TMED8.

10. The use according to claim 9, characterized in that: The reagent for overexpressing TMED8 includes the TMED8 overexpression plasmid pLV3-CMV-TMED8 (human)-3×FLAG-EF1a-CopGFP-Puro, and the nucleotide sequence of the plasmid is shown in SEQ ID No.

5.

11. A drug for treating tumors, characterized in that: It includes reagents that increase the expression level of endogenous TMED8 or exogenous TMED8.

12. A method for treating tumors, characterized in that: The following steps are involved: Including increasing the expression level of endogenous TMED8.

13. The method according to claim 12, characterized in that: The tumor includes pancreatic cancer.

14. The method according to claim 12 or 13, characterized in that: The treatment includes at least one of the following: inhibiting tumor cell migration, inhibiting tumor cell proliferation, promoting tumor cell apoptosis, and inhibiting MAPK signaling pathway activation.