Application of TMED8 gene in the preparation of pancreatic cancer drugs and diagnostic kits

Abstract

The present invention discloses the application of TMED8 gene in the preparation of pancreatic cancer drugs and diagnostic kits thereof, and belongs to the field of tumor precision medicine technology. The present invention finds that after up-regulating the expression of TMED8 gene, the migration and proliferation ability of pancreatic cancer cells are significantly inhibited, and the apoptosis of pancreatic cancer cells is promoted; the present invention also finds that TMED8 gene is related to MAPK signaling pathway. Therefore, TMED8 can be used as an effective target for genetic diagnosis and treatment of pancreatic cancer. The present invention explores for the first time the expression of TMED8 in pancreatic cancer, its relationship with patient prognosis and survival, and its effect on the biobehavior 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.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor precision medicine, and specifically relates to the application of TMED8 gene in the preparation of pancreatic cancer drugs and diagnostic kits thereof. Background Art

[0002] Pancreatic cancer (PC) is an extremely common and highly malignant tumor that is difficult to diagnose early, easily metastasizes, progresses rapidly, and has a very poor prognosis. It is the leading cause of cancer death worldwide. The regions with the highest incidence of pancreatic cancer include North America, Europe, and Australia. With the increasing aging of the world's population, smoking, obesity, diabetes, and drinking are prevalent in many parts of the world, leading to a sharp increase in the incidence of pancreatic cancer, but the relative contribution of these risk factors varies worldwide due to differences in potential prevalence and prevention strategies. Although hereditary genetic factors cannot be directly changed, they are an important component of pancreatic cancer risk, including pathogenic variants in hereditary cancer genes, genes associated with hereditary pancreatitis, and common variants identified in genome-wide association studies. Identifying genetic changes in cancer can not only provide insights into the etiology of this cancer, but also provide opportunities to guide early detection strategies. Pancreatic cancer has no obvious symptoms in the early stages, making it difficult to detect and diagnose in time. The sensitivity and specificity of tumor markers commonly used in clinical practice, such as CA199 and CEA, for the early diagnosis of pancreatic cancer are poor. Once diagnosed, most patients are already in the middle or late stages. In addition to radical surgical resection, adjuvant radiotherapy and chemotherapy have very limited effects on improving prognosis. Therefore, it is of great significance to explore and discover more sensitive and specific early markers and effective treatment targets.

[0003] The TMED family (transmembrane emp24 domain family) is composed of type I single-pass transmembrane proteins, which are found in all eukaryotes and play an important role in human development and disease. As an important transporter in the secretory pathway, once the protein transport pathway controlled by it is abnormal, it will lead to the occurrence of a variety of diseases including congenital malformations, diabetes and cancer. Previous studies have shown that abnormal expression of TMED proteins is often closely related to liver diseases, pancreatic diseases and immune system disorders. It can form a secretory loop during the development of the disease, 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, especially playing an important role in the proliferation and differentiation of malignant tumor cells.

[0004] TMED8 (NCBI Gene: 283578) is one of the important members of this family, located between bases 77,335,021-77,377,094 on chromosome 14, with a molecular weight of about 55kDa, and is expressed in both the nucleus and cytoplasm. There is no literature report on the relationship between TMED8 and human diseases and normal physiological functions. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the TMED8 gene in the preparation of a pancreatic cancer drug and a diagnostic kit thereof, specifically to provide an application of TMED8 in the preparation of a kit for disease diagnosis, and to provide an application of TMED8 in the preparation of a drug for treating tumors.

[0006] The present invention provides an application of a reagent for detecting the expression amount of TMED8 in preparing a product for diagnosing a disease.

[0007] Preferably, the reagent includes a reagent for detecting the expression level of TMED8 mRNA or protein.

[0008] 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.

[0009] Preferably, the type of disease includes tumor.

[0010] The present invention also provides a kit for diagnosing tumors, comprising at least one of the following reagents: a reagent for detecting TMED8 protein expression by immunohistochemistry, a reagent for detecting TMED8 protein expression by Western Blot, and a primer pair for detecting TMED8 gene and / or mRNA expression by real-time fluorescence quantitative PCR.

[0011] Preferably, the tumor comprises pancreatic cancer.

[0012] The present invention also provides an application of a reagent for increasing the expression level of TMED8 in preparing a product for treating a disease.

[0013] Preferably, the reagent comprises at least one of the following: an agonist of TMED8, an analog of TMED8, and a reagent for overexpressing TMED8.

[0014] The present invention also provides a drug for treating tumors, comprising an agent for increasing the expression level of endogenous TMED8 or exogenous TMED8.

[0015] Preferably, the treatment includes at least one of the following: inhibiting tumor cell migration, inhibiting tumor cell proliferation, promoting tumor cell apoptosis, and inhibiting activation of MAPK signaling pathway.

[0016] Beneficial effects: The present invention prepares pancreatic cancer tissue chips and uses immunohistochemistry to detect the expression of TMED8 in pancreatic cancer. The results show 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, qRT-PCR, Western Blot, cell scratch, CCK8, Transwell, flow cytometry, nude mouse subcutaneous tumor formation and other in vivo and in vitro experiments were used to explore the effect of TMED8 on the biological behavior of pancreatic cancer cells. It was found that after upregulating the expression of TMED8 gene, the migration and proliferation ability of pancreatic cancer cells were significantly inhibited, and the apoptosis of pancreatic cancer cells was promoted; the present invention 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 precision medical pancreatic cancer diagnostic kits and therapeutic drugs. The present invention explores for the first time the expression of TMED8 in pancreatic cancer and its effect on the biobehavior 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

[0017] Figure 1 For IHC detection of TMED8 expression in pancreatic cancer and adjacent tissue microarrays, representative sites were selected for display: 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;

[0018] Figure 2 Figure 2 shows the expression of TMED8 and the corresponding WB and PCR verification results;

[0019] Figure 3 This is a comparison of the expression of TMED8 in normal ductal epithelial cells and tumor cell lines of pancreatic cancer;

[0020] Figure 4 This is a diagram for validating the TMED8 overexpression efficiency in pancreatic cancer stable cell lines;

[0021] Figure 5 This is a diagram showing the effect of TMED8 overexpression on the migration ability of pancreatic cancer cells using cell scratch and Transwell experiments;

[0022] Figure 6 This is a diagram of the effect of TMED8 overexpression on the proliferation ability of pancreatic cancer cells in CCK8 and clone formation experiments;

[0023] Figure 7This is a diagram showing the effect of TMED8 overexpression on the apoptosis of pancreatic cancer cells detected by flow cytometry;

[0024] Figure 8 This is a diagram showing the effect of TMED8 overexpression on subcutaneous tumor formation of pancreatic cancer cells in nude mice;

[0025] Fig. 9 To illustrate the effect of increasing TMED8 expression on MAPK-related molecules;

[0026] Fig.10 This is a construction map of the TMED8 overexpression plasmid pLV3-CMV-TMED8(human)-3×FLAG-EF1a-CopGFP-Puro. DETAILED DESCRIPTION

[0027] The present invention provides an application of a reagent for detecting the expression amount of TMED8 in preparing a product for diagnosing a disease.

[0028] In the embodiment of the present invention, a pancreatic cancer tissue chip was prepared based on a biological sample library, and the expression of TMED8 in pancreatic cancer was detected by immunohistochemistry. The results showed that the expression level of TMED8 in pancreatic cancer tumor tissue was significantly higher than that in adjacent cancer tissue, and statistics showed that the prognosis of patients in the TMED8 high expression group was better. Therefore, the TMED8 was used as a biomarker for diagnosing pancreatic cancer.

[0029] The reagent of the present invention preferably includes a reagent 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 of the present invention preferably includes tumors, more preferably pancreatic cancer.

[0030] The present invention also provides a kit for diagnosing tumors, comprising 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 mRNA expression of TMED8 gene by real-time fluorescence quantitative PCR. The primer sequences of TMED8 and internal reference 18S are shown in Table 1.

[0031] Table 1 Primer sequences

[0032]

[0033] The tumor described in the present invention preferably includes pancreatic cancer.

[0034] The present invention also provides an application of a reagent for increasing the expression level of TMED8 in preparing a product for treating a disease.

[0035] In the embodiment of the present invention, by establishing a pancreatic cancer stable cell line overexpressing TMED8, the effect of TMED8 on the biological behavior of pancreatic cancer cells was explored by using in vivo and in vitro experiments such as qRT-PCR, Western Blot, cell scratch, CCK8, Transwell, flow cytometry, and nude mouse subcutaneous tumor formation; it was found that upregulating the expression of TMED8 gene significantly inhibited the migration and proliferation ability of pancreatic cancer cells and promoted the apoptosis of pancreatic cancer cells; the present invention also found that TMED8 gene is related to MAPK signaling pathway. Therefore, TMED8 can be used as an effective target for gene therapy of pancreatic cancer.

[0036] The reagent of the present invention preferably includes at least one of the following: an agonist of TMED8, an analog of TMED8, and a reagent for overexpressing TMED8.

[0037] The method for overexpressing TMED8 of the present invention preferably includes constructing a TMED8 overexpression plasmid pLV3-CMV-TMED8 (human)-3×FLAG-EF1a-CopGFP-Puro, and the plasmid sequence is shown in SEQ ID No. 5.

[0038] The present invention also provides a drug for treating tumors, comprising an agent for increasing the expression level of endogenous TMED8 or exogenous TMED8.

[0039] The reagent for increasing the expression of endogenous TMED8 of the present invention is preferably the same as the reagent for overexpressing TMED8, which is not described in detail here. The exogenous TMED8 of the present invention is preferably the same as the agonist of TMED8 and the analog of TMED8, which is not described in detail here. The treatment of the present invention preferably includes at least one of the following: inhibiting tumor cell migration, inhibiting tumor cell proliferation, promoting tumor cell apoptosis, and inhibiting activation of MAPK signaling pathway.

[0040] To further illustrate the present invention, the application of the TMED8 gene provided by the present invention in the preparation of pancreatic cancer drugs and diagnostic kits 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 invention.

[0041] Unless otherwise specified, the reagents and instruments used in the examples of the present invention are common reagent kit instruments in the art and can be purchased from reagent stores:

[0042] 1. Main reagents in the embodiments of the present invention:

[0043] IHC detection kit (Servicebio, China); 4% paraformaldehyde fixative solution (Servicebio, China); PBS buffer (Servicebio, China); bovine serum albumin (BSA) powder (Solarbio Life Sciences, China); TritonX-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 (Absin, China); HRP-labeled goat anti-rabbit secondary antibody (Abcam, USA); 95% ethanol (Sinopharm Group, China); DMEM basal medium (Cytiva, China); =Country); IMDM basal medium (Cytiva, China); RPMI basal medium (Cytiva, China); PBS basal buffer (Servicebio, China); fetal bovine serum (Gibco, USA); serum-free cell freezing solution (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, China); isopropanol (Sinopharm, China); chloroform / chloroform (Sinopharm, 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 solution pre-made powder (Servicebio, Wuhan);10× transfer buffer pre-made powder (Servicebio, Wuhan); 10× TBST pre-made powder (Servicebio, Wuhan); sodium chloride (NaCl) (Sinopharm Group, China); isopropanol (Sinopharm Group, China); ammonium persulfate (AP) (Sigma, USA); glycine (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 (New Saimei, 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 phosphorylation antibody (CST, USA); rabbit anti-human JNK antibody (CST, USA); rabbit anti-human JNK phosphorylation antibody (CST, USA); rabbit anti-human Casepase3 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-Eagle, 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); cell apoptosis detection kit: AnnexinV-PE / 7-AAD double staining (Keygen Biotechnology, China); crystal violet staining solution (Byotime, China). ;

[0044] 2. Main instruments used in the embodiments of the present invention:

[0045] Tissue chip system (UT06, Unitma, South Korea); inverted microscope (Leica, Germany); chemiluminescent gel imaging system (Bio-Rad, USA); automatic microplate reader (Bio-Rad, USA); electrophoresis system (Bio-Rad, USA); cell culture incubator (Thermo Fisher, USA).

[0046] Example 1

[0047] Between 2020 and 2023, 79 formalin-fixed paraffin-embedded (FFPE) pancreatic cancer tissue samples, including cancer and adjacent tissues, were obtained from the Clinical Biobank of the Affiliated Hospital of Nantong University. No cancer patients received any treatment, including radiotherapy, chemotherapy, or immunotherapy, and the clinical case data were detailed and complete. The study protocol has been approved by the Human Research Ethics Committee of the Affiliated Hospital of Nantong University, China.

[0048] 1. Fabrication of tissue microarray:

[0049] (1) Based on the microscopic observation results of HE sections, the representative cancer nest areas in the wax block were marked.

[0050] (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 holes were punched using a tissue microarray instrument to make a TMA blank wax block.

[0051] (3) Select the most representative cancer nest area at the marked point on the donor wax block and collect a tissue block with a diameter of 2 mm. Take one core for each case.

[0052] (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.

[0053] (5) The tissue array block was placed in a 55°C incubator for 10 minutes for heating and fusing. Before the wax block was almost melted, it was taken out and cooled to room temperature to allow the recipient wax block to fuse with the donor tissue.

[0054] (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.

[0055] (7) Slice the tissue array block using a microtome, float the consecutive slices in cold water to allow them to unfold naturally, and then transfer the slices to 45°C warm water to unfold for about 2 minutes. After unfolding, stick them on a slide treated with an anti-slipping agent and let them dry.

[0056] (8) Bake the slices in a high-temperature oven at 60°C for 3 minutes and continue baking at 58°C for 18 hours.

[0057] (9) Store the prepared tissue microarray in a slicing box at -20°C for future use.

[0058] 2. Immunohistochemical staining:

[0059] (1) First, place the tissue chip in a 60°C constant temperature oven and bake for about 40 minutes until the paraffin melts;

[0060] (2) Immediately place the chip in xylene I, xylene II, 100% ethanol I and 100% ethanol II, soak for 5 minutes each, then place it in 95% ethanol, 90% ethanol, 80% ethanol and 70% ethanol in sequence, soak for 2 minutes each, and then rinse with clean water;

[0061] (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, 5 minutes each time.

[0062] (4) Then use an immunohistochemistry pen to draw a border line around the tissue on the chip, and then drip 3% H 2 O 2 , incubate for 10 min, and then wash three times with PBS, 5 min each time;

[0063] (5) Add primary antibody (rabbit anti-human TMED8 antibody) and incubate at 4°C overnight;

[0064] (6) Rewarm at room temperature for 1 hour, remove the primary antibody, rinse with PBS three times, 5 minutes each time, add polymer enhancer, incubate at room temperature for 20 minutes; then wash with PBS three times, 5 minutes each time;

[0065] (7) Add secondary antibody (enzyme-labeled anti-mouse / rabbit IgG polymer), incubate at room temperature for 30 min, and then wash three times with PBS, 5 min each time;

[0066] (8) Add freshly prepared DAB reagent and rinse the chip under tap water for 5 minutes to terminate the color development;

[0067] (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;

[0068] (10) Soak the chip in 80% ethanol, 90% ethanol, 95% ethanol, 100% ethanol I, 100% ethanol II, and xylene for 1 minute, and allow to air dry in a fume hood;

[0069] (11) Finally, seal the slides with neutral gum.

[0070] 3. Result judgment:

[0071] According to the degree of staining of cells in the tissue, the positive intensity can be divided into 3 levels, no positive staining is level 0 (negative), light yellow is level 1 (weak positive), brown is level 2 (moderate positive), and brown is level 3 (strong positive); cell positive ratio = number of positive cells / total number of cells, which can be divided into 4 levels, and the ratios of levels 0 to 4 are 0% to 5%, 6% to 25%, 26% to 50%, 51% to 75% and >75% respectively. The chip results were scored by 3 pathology experts based on independent reading, and then divided into four groups of "-, +, ++, +++" according to the quartiles of the scores for statistics and analysis.

[0072] The results of immunohistochemical staining of 79 pancreatic cancer tumor and adjacent tissue specimens were as follows: Figure 1 As shown in the figure, TMED8 protein is mainly expressed in the cytoplasm of tumor cells and the stroma of cancer tissues, and is brown-yellow, while most adjacent tissues have low or no expression. The statistical results show that TMED8 is indeed highly expressed in pancreatic cancer tissue microarrays *P<0.05.

[0073] Eight pairs of pancreatic cancer and adjacent tissues were selected for WB validation. Figure 2 As shown, it is consistent with the above.

[0074] Example 2

[0075] 1. Cell transfection:

[0076] (1) Transformation and extraction of plasmid: 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, turn on the electric constant temperature water bath and raise it to 42°C, and put the DH5α competent state stored in a -80°C ultra-low temperature refrigerator on ice to melt. When the competent state melts, the transformation experiment is carried out immediately;

[0077] The TMED8 overexpression plasmid ( Fig.10) and the melted competent cells in a volume ratio of 1:3, flick the tube wall and blow 2-3 times to mix, let it stand on ice, and immediately put it in a 42°C water bath for 45 seconds for incubation after 25 minutes, and then immediately transfer it to ice bath for 3 minutes. Add the transformed plasmid to a shaking tube containing 40 μL of antibiotic-free LB liquid culture, use a constant temperature shaker, pre-shake at 200 rpm for 1 hour at 37°C, add 4 mL of liquid culture containing 50 μg / mL ampicillin after the pre-shaking, shake at 250 rpm at 37°C overnight, and control the specific time within 14-16 hours to avoid the growth of miscellaneous bacteria; use Axygen plasmid extraction kit to extract the bacterial solution according to the standard process, measure and record the obtained plasmid concentration, and mix the remaining bacterial solution with glycerol for seed preservation and store at -80°C.

[0078] (2) Establishment of stable transfected cell lines: An appropriate amount of wild-type cells to be transfected (PANC-1 and BXPC-3 pancreatic cancer cell lines) were seeded in a six-well plate, and the density was controlled to be 60% to 70% after 6 hours of attachment. 80 μL of 1640 basal medium without serum and double antibody was fused with 3 μg of plasmid. The tube wall was gently tapped to mix the reagents. Do not use a pipette to avoid DNA breakage. After standing at room temperature for 5 minutes, 3 μL of Lipofectamine was added. 2000 transfection reagent, stand at room temperature for 20-30 minutes, add to the complete culture medium of the 6-well plate, mix gently with a pipette, and culture in a cell culture incubator at 37°C and 5% carbon dioxide for 24-48 hours. Observe once in the first 24 hours, and then observe the cell state every 12 hours. If the cell state 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, puromycin is used to screen and kill the wild-type cells that have not been successfully transfected. The cells that have been successfully transfected are screened out, and the stable transfection cell lines are established. After subculture and seed preservation, the total protein or total RNA of the cells is collected for WB or qRT-PCR experiments to verify the overexpression efficiency of the cells. The results are as follows Figure 4 shown.

[0079] 2. CCK-8 cell proliferation activity detection

[0080] Use the medium dish to pre-culture the overexpression stable cells and wild-type control cell lines to be tested, and digest and resuspend when the cell confluence in the dish reaches 90%. Note that the amount of cells 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, according to the cell growth characteristics, plant them in a 96-well plate at a number of 5,000 to 10,000 per well. Each group has 5 to 6 replicate wells to reduce the experimental error caused by the operation technique; note that the number of control cells and treatment group cells must be equal to achieve the purpose of controlling variables. Start the detection of cell proliferation activity 6 to 12 hours after the cells adhere to the wall. Because the cell proliferation activity needs to be continuously detected for 5 days according to the cell doubling cycle, the experimental operation and result detection should be performed at the same time point every day.

[0081] 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. After standing in a 37°C incubator for 1 to 2 hours, the absorbance was measured at 460 nm using an enzyme reader, and the data was recorded. After all the tests were completed, the data were processed and compared and analyzed.

[0082] The results are as follows Figure 6 As shown, overexpression of TMED8 inhibited the proliferation ability of pancreatic cancer cells.

[0083] 3. Cell scratch healing assay

[0084] Place a brand new sterile packaged six-well plate upside down on the clean bench, and use a ruler and a marker to draw nine-square grid coordinate lines on the bottom of the plate to facilitate positioning when taking photos under a microscope in the subsequent scratch experiment. Appropriate amounts of wild-type and overexpression cell lines were seeded in six-well plates, with three replicate wells for the control group and the overexpression group. According to their growth cycle, fresh complete nutrient medium was used to culture the cells to a density of 90%, and then a ruler disinfected with alcohol and a 1 mL sterile blue gun 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 drawn in each well to ensure the repeatability of the experimental results. After the scratching was completed, 1× PBS buffer was added to clean the floating cells and cell fragments scratched out. 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 0h at the beginning of the experiment, and photos were taken. Every 24h, the healing of the cell scratches was observed under a microscope and photos were taken. After 72h, the final results of the experiment were integrated and analyzed.

[0085] The results are as follows Figure 5As shown, overexpression of TMED8 inhibited the migration ability of pancreatic cancer cells.

[0086] 4. Cell colony formation assay

[0087] 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 cells of the control group and the overexpression group in 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 and 5% carbon dioxide for 5 to 7 days. Due to the small initial number of cells, no liquid change is required during colony formation. After 5 to 7 days, the culture plates are taken out and the colony formation state is observed under a microscope. After washing and discarding with 1×PBS, 1 mL of 4% paraformaldehyde fixative is added to each well for 2 hours, rinsed with PBS 3 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.

[0088] The results are as follows Figure 6 As shown, overexpression of TMED8 inhibited the formation of pancreatic cancer cell colonies, that is, inhibited the proliferation ability of pancreatic cancer cells.

[0089] 5. Transwell cell migration assay

[0090] 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 small chamber with 8μm transparent PET film of the matching model, place the small chamber in the culture well of the 24-well plate, add 600μL of complete nutrient medium containing 5% fetal bovine serum outside the small chamber groove, that is, in the culture well; count and aspirate 30,000 cells, fully resuspend with 200μL of basic nutrient medium without fetal bovine serum, transfer to the inner groove of the small chamber with a pipette, and culture for 24 to 72 hours according to the cell growth cycle. The cells fully pass through the biofilm and observe the migration ability of the cells; take out the chamber, discard the culture medium in the inner tank, put it in a clean 24-well plate, soak it in 4% paraformaldehyde fixative for 2 hours, then rinse it gently with double distilled water, add crystal violet staining solution and stain it for 1 hour, then rinse it with double distilled water after staining. 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 part of the membrane; after the staining is completed, turn the chamber upside down, dry it at room temperature, observe it with an ordinary optical microscope, and select a suitable field of view to take pictures.

[0091] The results are as follows Figure 5 As shown, overexpression of TMED8 inhibited the migration ability of pancreatic cancer cells.

[0092] 6. Flow cytometry

[0093] Flow cytometry of cell apoptosis: PE fluorescein-labeled Annexin V dye and 7-AAD dye were used to double-stain the cells to be tested to detect cell apoptosis; first, the cells were digested and collected using trypsin without EDTA, and then washed 2 to 3 times with 1×PBS cell washing solution. The cells were counted. The number of cells in each sample was generally controlled at 10,000 to 50,000 cells. 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 dye and 5 μL of 7-AAD dye were added to each 100 μL suspension. 400 μL of 7-AAD dye was added, mixed gently, and then stained in a dark and room temperature state. After reacting for 5 to 10 minutes, 400 μL of buffer was added and mixed, and the mixture was transferred to a flow cytometry test tube. The detection was completed within 1 hour. PE is an 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 a 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.

[0094] The results are as follows Figure 7 As shown, overexpression of TMED8 promoted the apoptosis of pancreatic cancer cells.

[0095] 7. Subcutaneous tumor formation in nude mice

[0096] Four-week-old male BALB / c immunodeficient nude mice were selected for subcutaneous tumor formation experiments. Each nude mouse was weighed and recorded before the experiment. PANC-1 control cells and overexpressed cells were digested with trypsin and then counted. Each nude mouse was injected with approximately 2×10 cells of each type. 7 It is appropriate to resuspend the cells in a sterile 1× PBS solution. Note that the cell suspension must be placed on ice before animal experiments to avoid cell damage and death; equal amounts of PANC-1 cells overexpressing TMED8 and PANC-1 control cells were injected into the left and right armpits of the same nude mouse; the mouse status needs to be observed every day thereafter, and the nude mouse is killed and dissected after 4 to 6 weeks of tumor formation. The tumor is removed for weighing and volume measurement and then fixed for subsequent immunohistochemical experiments; it is important to perform control experiments on the same nude mouse in order to control the interference factors caused by mouse heterogeneity and more intuitively determine the effect of TMED8 on tumor growth in vivo.

[0097] The results are as follows Figure 8 As shown, TMED8 inhibited the tumor-forming ability of pancreatic cancer cells under the skin of nude mice, that is, it inhibited the proliferation ability of pancreatic cancer cells in animals.

[0098] 8. Western Blot

[0099] (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 the sample should be 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 sample should be scraped with a cell scraper to assist in lysis. The sample should be transferred to a 1.5 mL centrifuge tube and placed on an electric rotary machine at 4°C for overnight rotation. After the protein is fully lysed, it should be centrifuged at 7500 rpm for 15 minutes. The supernatant should be aspirated and the cell debris precipitate should be discarded. Sample buffer (Loading Buffer) with a volume of 1 / 4 of the total volume of the supernatant should be added and boiled for at least 30 minutes to ensure that the protein is fully heated and denatured. After cooling to room temperature, the sample should be aliquoted and stored in a -20°C or -80°C refrigerator to prevent protein degradation due to repeated freezing and thawing.

[0100] (2) Preparation of SDS-PAGE gel: Wash and dry the gel plate and clamp it on the gel preparation rack. Add deionized water to check for leaks. After the leak test is passed, let it dry vertically. Prepare the separation gel according to the component ratio 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 colloid has solidified, remove the isopropanol and gently rinse it with deionized 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.

[0101] Table 2 Preparation of 10mL SDS-PAGE separation gel components of different concentrations (mL)

[0102]

[0103] Table 3 Proportion scheme for preparing 8 mL upper concentrated gel

[0104]

[0105] 9. TMED8 may regulate the biological functions of PAAD tumor cells through the MAPK pathway, thereby affecting the occurrence and development of pancreatic cancer

[0106] PANC-1 control and overexpression cell lines (each group had 3 replicate samples) were selected for transcriptome sequencing. A total of 248 differentially expressed genes were obtained through data analysis, including 103 upregulated genes and 145 downregulated genes, which were visualized using heat maps and volcano maps, respectively. Functional annotation and KEGG enrichment analysis were performed on them, and the MAPK signaling pathway with significant correlation was selected for further study. The expression levels of key molecules ERK, JNK, P38 and their phosphorylation were verified by WB.

[0107] The results are as follows Fig. 9As shown, TMED8 inhibited the activation of the MAPK pathway.

[0108] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a reagent for increasing the expression of TMED8 in the preparation of a product for treating pancreatic cancer, characterized in that: The reagent is a TMED8 overexpression plasmid, and the nucleotide sequence of the TMED8 overexpression plasmid is shown in SEQ ID No.5.

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