Application of UMODL1 gene in treatment of Her-2 positive breast cancer

Abstract

The invention discloses application of a UMODL1 gene in treatment of Her-2 positive breast cancer, and belongs to the technical field of biomedicine.The high-expression gene UMODL1 in the Her-2 positive breast cancer is screened out through a bioinformatics method, the UMODL1 can enhance proliferation of the Her-2 positive breast cancer and inhibit cell apoptosis, and the UMODL1 is related to drug resistance of a breast cancer targeted drug trastuzumab. The UMODL1 can up-regulate expression of Her-2 protein and is combined with Her-2 heterodimer so as to activate downstream PI3K / AKT / mTOR and MAPK signal channels of Her-2, and the two mechanisms generate a synergistic effect so as to promote occurrence and development of Her-2 positive breast cancer and cause trastuzumab drug resistance. The research result provides a new diagnosis, treatment and prognosis prediction target for Her-2 positive breast cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of UMODL1 gene in the treatment of Her-2 positive breast cancer. Background Art

[0002] Breast cancer is one of the most common cancers in the world, and it poses a serious threat to women's health. Among breast cancers, Her-2 (Human epidermal growth factor receptor 2) positive breast cancer is a special type, characterized by the positive expression of Her-2 receptors. According to statistics, Her-2 positive breast cancer cases account for about 20%-30% of the total number of breast cancers.

[0003] Her-2 positive breast cancer grows fast, is highly invasive, metastasizes very quickly, and has a high degree of heterogeneity. For this special type of breast cancer, chemotherapy plus targeted therapy is currently the main treatment. Targeted drugs approved by the U.S. Food and Drug Administration (FDA) for the treatment of breast cancer include trastuzumab, pertuzumab, and trastuzumab emtansine (T-DM1). However, although these targeted drugs have achieved significant therapeutic effects in the treatment of Her-2 positive breast cancer, some patients still develop drug resistance during treatment. Therefore, it is particularly important to further study the pathogenesis of Her-2 positive breast cancer and the reasons why patients develop drug resistance, which is also a clinical problem that needs to be solved urgently. The UMODL1 (Uromodulin-1like) gene is located on human chromosome 21q22.3. At present, it is known that the UMODL1 gene is associated with the occurrence and development of bladder cancer, liver cancer, pancreatic cancer, ovarian cancer and colorectal adenocarcinoma, but the role of UMODL1 in Her-2 breast cancer and resistance to targeted therapy has not been reported. Therefore, whether UMODL1 can become a target for the treatment of Her-2 breast cancer and whether it is associated with resistance to targeted therapy of Her-2 positive breast cancer needs further study. Summary of the invention

[0004] In view of the above problems, the present invention screened out the highly expressed gene UMODL1 in Her-2 positive breast cancer by bioinformatics method. UMODL1 can enhance the proliferation of Her-2 positive breast cancer and inhibit apoptosis, and is related to the resistance to trastuzumab, a breast cancer targeted drug. UMODL1 can upregulate the expression of Her-2 protein, and bind to Her-2 heterodimers to activate the downstream PI3K / AKT / mTOR and MAPK signaling pathways of Her-2. The two mechanisms produce a synergistic effect, thereby promoting the occurrence and development of Her-2 positive breast cancer and leading to trastuzumab resistance. The research results provide new diagnostic, therapeutic and prognostic prediction targets for the treatment of Her-2 positive breast cancer.

[0005] One object of the present invention is to provide a reagent for detecting the expression level of UMODL1 gene and its use in preparing a Her-2 positive breast cancer diagnosis kit.

[0006] Another object of the present invention is to provide a reagent for detecting the expression level of the UMODL1 gene for use in preparing a kit for detecting the prognosis of Her-2 positive breast cancer.

[0007] Another object of the present invention is to provide the use of a UMODL1 inhibitor in the preparation of a drug for inhibiting Her-2 positive breast cancer.

[0008] Another object of the present invention is to provide a use of a UMODL1 inhibitor in the preparation of a drug for inhibiting or reversing the drug resistance of Her-2 positive breast cancer cells to trastuzumab.

[0009] Furthermore, the inhibitor is an agent for knocking down the UMODL1 gene.

[0010] Another object of the present invention is to provide a composition for inhibiting Her-2 positive breast cancer, which comprises a UMODL1 inhibitor and trastuzumab.

[0011] Furthermore, UMODL1 inhibitor and trastuzumab synergistically inhibited the phosphorylation of Her-2 and downstream PI3K / AKT / mTOR and MAPK signaling pathway proteins.

[0012] Compared with the prior art, the present invention has the following advantages:

[0013] 1. The invention provides new diagnostic, therapeutic and prognostic prediction targets for the treatment of Her-2 positive breast cancer, and provides new molecular targets for improving its trastuzumab resistance.

[0014] 2. The present invention can prepare a corresponding Her-2 positive breast cancer early diagnosis kit and a survival prognosis detection kit by detecting the UMODL1 expression level in the cancer tissue of Her-2 positive breast cancer patients, wherein the kit contains a reagent for quantitatively detecting the RNA transcription level of UMODL1, a reagent for quantitatively detecting the protein expression level of UMODL1, etc. By detecting whether the UMODL1 gene expression level in the cancer tissue of Her-2 positive breast cancer patients changes, the survival and prognosis of the patients can be predicted.

[0015] 3. Knockdown of UMODL1 can produce a synergistic effect with trastuzumab and further inhibit the growth of Her-2 positive breast tumors in nude mice.

[0016] 4. Knockdown of UMODL1 can produce a synergistic effect with trastuzumab, further reducing the phosphorylated Her-2, PI3K, AKT, MEK, and ERK. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Overexpression of UMODL1 can reverse the inhibition of trastuzumab on the proliferation of breast cancer cell lines in vitro; A: CCK-8 assay to detect the effect of overexpression of UMODL1 on the proliferation of BT-474 cells in the presence of trastuzumab; B: CCK-8 assay to detect the effect of overexpression of UMODL1 on the proliferation of SK-BR-3 cells in the presence of trastuzumab; C: Plate cloning assay to detect the effect of overexpression of UMODL1 on the clone formation of BT-474 in the presence of trastuzumab; D: Plate cloning assay to detect the effect of overexpression of UMODL1 on the clone formation of SK-BR-3 in the presence of trastuzumab. Two asterisks indicate P<0.01, and three asterisks indicate P<0.001; Ttzm: trastuzumab.

[0018] Figure 2 Overexpression of UMODL1 can reverse the promotion of apoptosis in breast cancer cell lines by trastuzumab; A: Cell apoptosis experiment detected the effect of overexpression of UMODL1 on the apoptosis level of SK-BR-3 and BT-474 cell lines in the presence of trastuzumab. Two asterisks indicate P<0.01, and three asterisks indicate P<0.001; Ttzm: trastuzumab.

[0019] Figure 3Knockdown of UMODL1 can partially restore the inhibitory effect of trastuzumab on the proliferation of trastuzumab-resistant breast cancer cell line JIMT-1 in vitro; A: CCK-8 assay to detect the effect of knockdown of UMODL1 on the proliferation ability of JIMT-1 cells in the presence of trastuzumab; B: Plate cloning assay to detect the effect of knockdown of UMODL1 on the clone formation ability of JIMT-1 in the presence of trastuzumab. Three stars indicate P<0.001, four stars indicate P<0.0001; Ttzm: trastuzumab.

[0020] Figure 4 Knockdown of UMODL1 can partially restore the effect of trastuzumab on the apoptosis of trastuzumab-resistant breast cancer cell lines; A: Cell apoptosis experiment detected the effect of knockdown of UMODL1 on the apoptosis level of JIMT-1 cell lines in the presence of trastuzumab. Two asterisks indicate P<0.01; Ttzm: trastuzumab.

[0021] Figure 5 Overexpression of UMODL1 can promote the growth of Her-2 positive breast cancer in vivo; A: Nude mouse tumor formation experiment detected the effect of overexpression of UMODL1 on the proliferation ability of SK-BR-3 cells in vivo. Four asterisks indicate P<0.0001.

[0022] Figure 6 Knockdown of UMODL1 can inhibit the growth of Her-2 positive breast cancer in vivo; A: Nude mouse tumor formation experiment detected the effect of knockdown of UMODL1 on the proliferation ability of JIMT-1 cells in vivo. Three stars indicate P<0.001; four stars indicate P<0.0001.

[0023] Figure 7 Overexpression of UMODL1 can increase the resistance of Her-2 positive breast cancer in nude mice to trastuzumab; A: In the case of adding trastuzumab, the nude mouse tumor formation experiment detected the effect of overexpression of UMODL1 on the proliferation ability of SK-BR-3 cells in vivo. Three stars indicate P<0.001; four stars indicate P<0.0001; Ttzm: trastuzumab.

[0024] Figure 8 Knockdown of UMODL1 can reduce the resistance of Her-2 positive breast cancer to trastuzumab in nude mice; A: In the case of adding trastuzumab, the nude mouse tumor formation experiment detected the effect of knockdown of UMODL1 on the proliferation ability of trastuzumab-resistant Her-2 receptor positive breast cancer cell line JIMT-1 cells in vivo. Four asterisks indicate P<0.0001; Ttzm: trastuzumab.

[0025] Fig. 9High UMODL1 expression promotes phosphorylation of Her-2 and downstream signaling pathway proteins; A: Effect of UMODL1 overexpression on the downstream signaling pathway of Her-2 heterodimer in BT-474 cell line; B: Effect of UMODL1 overexpression on the downstream signaling pathway of Her-2 heterodimer in SK-BR-3 cell line. Two asterisks indicate P<0.01, three asterisks indicate P<0.001, and four asterisks indicate P<0.0001. Ttzm: trastuzumab.

[0026] Fig.10 Knockdown of UMODL1 inhibits the phosphorylation of Her-2 and downstream signaling pathway proteins; A: The effect of knockdown of UMODL1 on the downstream signaling pathway of Her-2 heterodimer in JIMT-1 cell line. Two asterisks indicate P < 0.01, Ttzm: trastuzumab.

[0027] Fig.11 High UMODL1 expression restored the phosphorylation inhibition of Her-2 and downstream signaling pathway proteins caused by trastuzumab; A: The effect of UMODL1 overexpression on the downstream signaling pathway of Her-2 heterodimer in BT-474 cell line added with trastuzumab; B: The effect of UMODL1 overexpression on the downstream signaling pathway of Her-2 heterodimer in SK-BR-3 cell line added with trastuzumab. Two asterisks indicate P<0.01, three asterisks indicate P<0.001, and four asterisks indicate P<0.0001. Ttzm: trastuzumab.

[0028] Fig.12 Knockdown of UMODL1 can promote the inhibitory effect of trastuzumab on the phosphorylation of Her-2 and downstream signaling pathway proteins; A: The effect of knockdown of UMODL1 on the downstream signaling pathway of Her-2 heterodimer in the JIMT-1 cell line added with trastuzumab. Two asterisks indicate P < 0.01, Ttzm: trastuzumab.

[0029] Fig.13 High UMODL1 expression restored the phosphorylation inhibition of Her-2 and downstream signaling pathway proteins caused by trastuzumab; A: Immunohistochemical analysis of the effect of UMODL1 overexpression on the downstream signaling pathway of Her-2 heterodimers with or without trastuzumab. The scale of immunofluorescence images is 20um, the scale of immunohistochemistry is 50um, and Ttzm: trastuzumab.

[0030] Fig.14Knockdown of UMODL1 can promote the inhibitory effect of trastuzumab on the phosphorylation of Her-2 and downstream signaling pathway proteins; A: Immunofluorescence and immunohistochemistry analysis of the effect of knockdown of UMODL1 on the downstream signaling pathway of Her-2 heterodimers with or without trastuzumab. The scale of immunofluorescence images is 20um; the scale of immunohistochemistry is 50um; Ttzm: trastuzumab.

[0031] Fig.15 A: A schematic diagram of the mechanism by which UMDOL1 upregulates the expression of Her-2 and activates its downstream PI3K / AKT / mTOR and MAPK signaling pathways, leading to the proliferation of Her-2-positive breast tumors and trastuzumab resistance. Ttzm: trastuzumab. DETAILED DESCRIPTION

[0032] The present invention is further described below with reference to specific examples.

[0033] Functional study of UMODL1 in Her-2 positive breast cancer

[0034] 1. Experimental Methods

[0035] 1.1 Cell culture

[0036] (1) MCF-10A complete medium is a special medium for MCF-10A cells produced by Wuhan Pronocell Life Science Co., Ltd.; MCF-7 complete medium is DMEM containing 10% fetal bovine serum; T-47D medium is RPMI-1640 containing 10% fetal bovine serum, 1‰10ug / mL insulin and 1% penicillin-streptomycin mixture (100×); BT-474 complete medium is RPMI-1640 containing 10% fetal bovine serum; SK-BR-3 complete medium is McCoy's 5A medium containing 10% fetal bovine serum; JIMT-1 (trastuzumab-resistant cell line) complete medium is DMEM containing 20% ​​fetal bovine serum; MDA-MB-231 complete medium is DMEM containing 10% fetal bovine serum.

[0037] (2) Cell recovery:

[0038] 1) Preparation: Preheat a water bath to 37°C, prepare clean disposable gloves (to prevent the cryotubes from contacting water and causing contamination) and sterile centrifuge tubes that have been preheated in a 37°C incubator and filled with 9 mL of sterile culture medium.

[0039] 2) Recovery: Take the cells that need to be recovered out of the -80℃ refrigerator, quickly immerse them in a water bath, shake the cryopreservation tube to accelerate the dissolution (it is best to dissolve all the cells within 1 minute), add the recovered cell solution to a centrifuge tube containing 9mL of culture medium in a clean bench, and centrifuge at 1200rpm / min for 5 minutes. After centrifugation, discard the supernatant and keep only the white precipitate, add 1mL of culture medium to the cryopreservation tube to resuspend the cell precipitate, then transfer the cell suspension into a culture bottle, and fill it up to 5mL of culture medium, shake it evenly, and place it in a constant temperature 37℃, 5% CO2 incubator for culture.

[0040] (3) Cell passaging: Aspirate the original culture medium, add about 2 mL of PBS, gently shake the culture bottle to rinse the cells, aspirate the PBS and discard. Then add about 1 mL of 0.25% trypsin solution (containing EDTA), gently shake the culture bottle to infiltrate all cells, and place it in an incubator for digestion. When the cells in the middle of the cell block are obviously rounded and have gaps under the microscope, the digestion can be terminated. Add 3 ml of serum-containing culture medium to terminate the digestion. Blow the cells to remove the wall, collect the cell suspension and centrifuge it at 1200 rpm / min for 3 minutes, discard the supernatant after centrifugation, add fresh culture medium, blow and mix the cells, and then inoculate the cells into a new disposable culture bottle at an appropriate density, and replenish the culture medium. After gently shaking to evenly distribute the cells, place the cells in a constant temperature of 37°C and 5% CO2 incubator for continued culture.

[0041] (4) Cell cryopreservation: After the cells grow to 80%, discard the culture medium, wash twice with PBS, add 1 mL of trypsin, and observe under an inverted phase contrast microscope. Digest until the cell cytoplasm shrinks and the intercellular space becomes larger. Quickly add 2 mL of culture medium containing 10% FBS to terminate the digestion. The digested cells are aspirated into a 15 mL centrifuge tube and centrifuged at 1200 rpm / min for 5 min. After centrifugation, discard the supernatant, add 1 mL of FBS containing 10% DMSO, and gently blow until the cells are suspended. Then transfer the cell mixture into a cryopreservation tube. Then place the cryopreservation tube in a program cooling box at room temperature, and then store the program cooling box in a -80°C refrigerator. The next day, transfer the cryopreservation tube in the program cooling box to a liquid nitrogen tank for storage.

[0042] 1.2 shRNA transfection

[0043] (1) Cell plating: Cells were plated one day before the experiment and transfected when the cell density reached 60-80%.

[0044] (2) Preparation and transfection of complexes:

[0045] 1) Place GP-transfect-Mate transfection reagent at room temperature and mix gently before use.

[0046] 2) Add 50 μL of serum-free medium and an appropriate amount of transfection reagent to a 1.5 mL sterile centrifuge tube, mix gently with a pipette, and let stand at room temperature for 5 min; add 50 μL of serum-free medium and an appropriate amount of RNA oligo / DNA to another 1.5 mL sterile centrifuge tube, mix gently with a pipette, and let stand at room temperature for 5 min.

[0047] 3) Add the GP-transfect-Mate-medium mixture dropwise to the RNAoligo / DNA-medium mixture, mix gently with a pipette, let stand at room temperature for 15-20 minutes, and transfect immediately.

[0048] 4) While the plate is still, replace the medium in the 24-well plate with 400 μL of pre-warmed fresh culture medium in each well.

[0049] 5) Add 100 μL of the transfection mixture to each well to make the final volume reach 500 μL, and gently shake the 24-well plate to evenly distribute the complex.

[0050] 6) Cultivate the cells at 37°C and replace with complete medium after 4-6 hours. Detect mRNA expression after 24-72 hours and protein expression after 48-96 hours.

[0051] (3) The specific sequences of shRNA used in this experiment are as follows:

[0052]

[0053] 1.3 Lentiviral transfection

[0054] (1) Cell plating: The cells to be treated are plated into six-well plates one day in advance and cultured at 37°C for 16-24 hours so that the cell coverage rate is about 30% on the day of transfection.

[0055] (2) Transfection:

[0056] 1) Take out the virus from the refrigerator, slowly thaw it on ice, and dilute the virus in complete culture medium.

[0057] 2) Remove the supernatant from each well, replace the culture medium, add the virus and the corresponding infection enhancement solution, mix well, and continue to culture. Replace the medium with complete culture medium 16 hours after infection. Observe the cell morphology during the process. If there is a change, the medium can be replaced 8 hours in advance to maintain normal cell growth.

[0058] 3) 48-72 hours after lentiviral infection (reaching 70-80% confluence), the cells were cultured in a culture medium containing an appropriate concentration of puromycin. After 48 hours of screening, all the empty cells plus puromycin group died, and the remaining cells were positive cells infected with the virus.

[0059] 4) Reduce the puromycin concentration to the maintenance concentration (1 / 2 to 1 / 4 of the screening concentration), continue to screen and amplify the infected cells, collect the cells for qPCR and Western Blot, detect the expression level of the target gene, and freeze the cells with normal identification results for seed preservation.

[0060] (3) The specific sequences of the primers used in this section are as follows:

[0061]

[0062] 1.4 Total RNA extraction, reverse transcription and qRT-PCR of breast cancer cells

[0063] During the experimental operation, the operator wore gloves and masks, and the utensils, centrifuge tubes, pipette tips, etc. used in the RNA extraction process were guaranteed to be free of contamination and RNase.

[0064] (1) Obtain cells to be extracted: Aspirate the culture medium and add 1 mL of TRIzol per 10 cm2 (6-well plate or 35 mm dish) of cells to cover the cultured cells. After standing for 5 minutes, pipette 2-3 times to completely lyse the cells, and then transfer them to a 1.5 mL centrifuge tube.

[0065] (2) Separation: Add 0.2 times the volume of chloroform (i.e., add 0.2 mL of chloroform to 1 mL of TRIzol) to the centrifuge tube containing the lysate, shake vigorously to mix thoroughly, leave at room temperature for 2-3 minutes, and then centrifuge at 12000 rpm / 4°C for 10 minutes. Pipette the upper aqueous phase containing total RNA into a new centrifuge tube and record the volume of the aspirated liquid (approximately 0.5-0.6 ml can be aspirated for each milliliter of TRIzol). During the aspiration process, be careful to avoid touching the bottom and middle layers to prevent the aspiration of DNA and protein.

[0066] (3) Precipitation: Add the same volume of isopropanol to the EP tube, invert several times to mix, and precipitate the RNA. After standing at room temperature for 10 minutes, centrifuge at 12000rpm / 4℃ for 10 minutes. After centrifugation, RNA precipitate can be seen at the bottom of the tube. Discard the supernatant, add 1mL of 75% ethanol, and gently invert to mix to wash the RNA precipitate. Centrifuge at 12000rpm / 4℃ for 2 minutes and discard the supernatant. Invert and dry the RNA precipitate at room temperature for 5-10 minutes.

[0067] (4) Dissolution: Add an appropriate amount of DEPC-treated water to the centrifuge tube to dissolve the RNA precipitate. Mix well with a pipette and let stand at room temperature for 2 minutes.

[0068] (5) Determination of RNA concentration and purity by UV spectrophotometry: Dilute RNA in TE buffer at pH 8.0 and measure absorbance at 260nm and 280nm. Abs 260nm:280nm of 1.7-2.1 indicates good RNA purity. After RNA purity meets the standard, mark it and store it in a -80℃ refrigerator.

[0069] (6) mRNA reverse transcription: The conditions are 42°C for 40 min, 85°C for 5 min, and constant temperature storage at 4°C. The product is cDNA. The obtained cDNA is stored at -80°C.

[0070] (7) qRT-PCR: Quantitative real-time PCR was performed using the 7900HT fast real-time PCR system and SYBR Green PCR master mix in a total volume of 20 μL. The reaction conditions were: 50°C for 2 min, 95°C for 10 min, 95°C for 15 s, and 60°C for 60 s for 40 cycles. -ΔΔCt Methods, data were normalized according to GAPDH levels.

[0071] 1.5 Western Blot

[0072] (1) Cell protein extraction: Select cells in the logarithmic growth phase, digest and centrifuge them into EP tubes, add RIPA lysis buffer containing protease inhibitor phenylmethanesulfonyl fluoride (PMSF) according to the volume of the cell pellet, and let it stand on ice for 5 minutes. Centrifuge at 12000rpm / 4℃ for 20 minutes. The supernatant obtained after centrifugation contains the protein target product.

[0073] The rest of the Western Blot is the same as in Part 1.

[0074] 1.6 CCK-8 experiment

[0075] (1) Cell seeding

[0076] 1) The obtained stable screening cells were seeded on a 96-well plate, and the volume in each well was filled to 100 μL, and pre-cultured in an incubator at a constant temperature of 37° C. and 5% CO 2 for 24 h.

[0077] 2) Incubate the culture plate in an incubator at 37°C and 5% CO2 for an appropriate period of time, and take samples for CCK-8 detection at 0h, 24h, 48h and 72h.

[0078] (2) CCK-8 assay: Add 10 μL of CCK-8 solution to each well and incubate in a 37°C, 5% CO2 incubator for 1-4 hours. Measure the absorbance of the sample at 450 nm using an ELISA reader and record and analyze.

[0079] 1.7 Colony formation assay

[0080] (1) Cells from each group in the logarithmic growth phase were taken, digested with 0.25% trypsin and blown into single cells, and the cells were suspended in complete culture medium (basal culture medium + 10% fetal bovine serum) for later use.

[0081] (2) The cell suspension was diluted in multiples, with 5 parallel samples in each group. Each group of cells was inoculated in a dish containing 10 ml of 37°C pre-warmed culture medium at a density of 800 cells per well of a six-well plate, and gently rotated to disperse the cells evenly. The cells were cultured in a cell culture incubator at 37°C, 5% CO2 and saturated humidity for 2 weeks, and the culture medium was changed every three days.

[0082] (3) When visible colonies appear in the culture dish, terminate the culture, discard the supernatant, and carefully rinse twice with PBS.

[0083] (4) Add 5 ml of methanol and fix the cells for 15 minutes.

[0084] (5) Discard the fixative solution and add an appropriate amount of crystal violet staining solution for 30 minutes. After staining, slowly wash away the staining solution with running water and dry.

[0085] (6) Image J software was used to calculate the clone formation rate.

[0086] Cloning rate = (number of clones / number of inoculated cells) × 100%

[0087] 1.8 Cell apoptosis assay

[0088] (1) Collect cells: Take an appropriate amount of cells in the logarithmic growth phase and inoculate them in a six-well plate. Collect the cells after the drug treatment for a corresponding period of time. Be sure to combine the supernatant and digested cells. After collection, wash the cells twice with pre-cooled PBS.

[0089] (2) Grouping: divided into unstained group, Annexin V single staining group, PI single staining group, and PI and Annexin V double staining group. The treatment groups were double stained from low to high.

[0090] (3) Staining: Dilute 4× binding buffer to 1× buffer with PBS. After aspirating the residual PBS in the centrifuge tube, add 100 μL of 1× binding buffer to each tube. Use a pipette to blow the cells to fully resuspend them. Add dye under light-proof conditions. No dye is added to the unstained group. Add 5 μL of Annexin V or PI to the single-stained group. Add 5 μL of Annexin V and PI to the double-stained group, and gently mix with a pipette.

[0091] (4) Detection: After incubation at room temperature in the dark for 15 minutes, add 300 μL of 1× binding buffer and mix well. Transfer the cell suspension to a 5 mL flow cytometer in the dark and detect on a flow cytometer within 1 hour.

[0092] 1.9 Experimental steps of subcutaneous tumor formation in nude mice

[0093] (1) 5-6 week old nude mice were selected and randomly divided into 12 groups, with 6 mice in each group.

[0094] (2) Tumor formation experiment:

[0095] 1) Pre-preparation: Melt the matrix gel at 4°C overnight, prepare an ice box, pipette and gun tip. Select the insertion site and wipe and disinfect the insertion site with an alcohol cotton ball before inoculation.

[0096] 2) Two groups of mice were subcutaneously injected with 100 μL SK-BR-3 cells, two groups of mice were subcutaneously injected with 100 μL SK-BR-3-NC cells, two groups of mice were subcutaneously injected with 100 μL UMODL1-overexpressed SK-BR-3 cells, two groups of mice were subcutaneously injected with 100 μL JIMT-1 cells, two groups of mice were subcutaneously injected with 100 μL JIMT-1-NC cells, and two groups of mice were subcutaneously injected with 100 μL UMODL1-knockdown JIMT-1 cells.

[0097] 3) When the tumor volume reached an appropriate size, mice injected with different types of tumor cells were selected to receive trastuzumab treatment (10 mg / kg).

[0098] (3) The time of tumor formation was recorded, and the maximum length and width were measured every three days after tumor formation. When the tumor volume reached an appropriate size, the nude mice were killed, the tumor tissue was weighed and photographed, and the tumor volume was measured. The tumor was routinely fixed and embedded in 10% formalin.

[0099] Calculation formula: length × width × height × 0.52

[0100] (4) This experiment was approved by the Experimental Animal Welfare Ethics Committee of China Medical University (approval number: CMU20240328).

[0101] 2 Results

[0102] 2.1 Expression of UMODL1 in mammary epithelial cells, breast cancer cell lines and trastuzumab-resistant JIMT-1 cells

[0103] The expression of UMODL1 mRNA in breast cancer cell lines MCF-10A, T-47D, MCF-7, SK-BR-3, and MDA-MB-231 was detected by qRT-PCR. The results showed that the expression of UMODL1 mRNA in breast cancer cell lines T-47D, MCF-7, SK-BR-3, and MDA-MB-231 was significantly higher than that in breast cancer cell line MCF-10A, and the most obvious was in SK-BR-3.

[0104] Western Blot was used to detect the expression of UMODL1 protein in breast cancer cell lines MCF-10A, T-47D, MCF-7, SK-BR-3, BT-474, and MDA-MB-231. The results showed that the expression of UMODL1 protein in breast cancer cell lines T-47D, MCF-7, SK-BR-3, BT-474, and MDA-MB-231 was significantly higher than that in breast cancer cell line MCF-10A ( Figure 3 B), SK-BR-3 and BT-474 are the most obvious.

[0105] Subsequently, the protein expression of UMODL1 in Her-2 receptor-positive breast cancer cell lines SK-BR-3, BT-474 and trastuzumab-resistant cell line JIMT-1 was detected by Western Blot. The results showed that at the protein level, the expression of Umodl1 in the trastuzumab-resistant cell line JIMT-1 was higher than that in SK-BR-3 and BT-474.

[0106] SK-BR-3 and BT-474 cell lines were selected to construct cell lines with stable overexpression of UMODL1, and the trastuzumab-resistant cell line JIMT-1 was selected to construct a cell line with knockdown of UMODL1 for the needs of subsequent experiments. We verified the overexpression efficiency of UMODL1 in SK-BR-3 and BT-474 cell lines at the protein level by Western Blot. Western Blot results showed that the UMODL1 protein in the overexpression UMODL1 group was significantly higher than that in the control group. Subsequently, the trastuzumab-resistant cell line JIMT-1 was transfected with three independent shRNAs, and its transfection efficiency was detected by Western Blot experiments. The experimental results showed that the expression of its UMODL1 protein was also significantly reduced.

[0107] 2.2 UMODL1 promotes the occurrence and development of Her-2 positive breast cancer

[0108] 2.2.1 Overexpression of UMODL1 promotes the proliferation of Her-2 receptor-positive breast cancer cell lines in vitro

[0109] BT-474 and SK-BR-3 cells that stably overexpressed UMODL1 were selected for CCK-8 proliferation assay. The results of CCK-8 proliferation assay showed that compared with the control group, the UMODL1 overexpression group significantly enhanced the in vitro proliferation ability of Her-2 receptor-positive cell lines SK-BR-3 and BT-474. Subsequently, a plate cloning assay was performed, and the results showed that overexpression of UMODL1 could significantly enhance the cloning ability of Her-2 receptor-positive cell lines SK-BR-3 and BT-474. The above results indicate that overexpression of UMODL1 can enhance the in vitro proliferation ability of Her-2 receptor-positive breast cancer cell lines.

[0110] 2.2.2 Overexpression of UMODL1 inhibits cell apoptosis

[0111] Cell apoptosis was detected by flow cytometry in the control Her-2 receptor-positive cell line and the Her-2 receptor-positive cell line overexpressing UMODL1. The results showed that the apoptosis rate of SK-BR-3 and BT-474 cells overexpressing UMODL1 was significantly reduced. The above results indicate that overexpression of UMODL1 can significantly inhibit the apoptosis of SK-BR-3 and BT-474.

[0112] 2.2.3 Knockdown of UMODL1 inhibits the proliferation of Her-2 positive breast cancer cell lines in vitro

[0113] Untreated trastuzumab-resistant JIMT-1 cell lines and JIMT-1 cell lines with UMODL1 knockdown were selected to explore the effect of knockdown of UMODL1 on the proliferation ability of Her-2 positive breast cancer cell lines. Through CCK-8 proliferation experiment, we compared the proliferation ability of JIMT-1 cells in the control group and the sh-UMODL1 group. The CCK-8 results showed that the proliferation ability of the sh-UMODL1 group was significantly weakened compared with the control group. This result shows that knockdown of UMODL1 can inhibit the proliferation ability of JIMT-1 cells. Subsequently, a plate cloning experiment was performed, and the results showed that knockdown of UMODL1 can significantly weaken the cloning ability of JIMT-1 cell lines. The above results show that knockdown of UMODL1 can significantly inhibit the in vitro proliferation ability of Her-2 positive breast cancer cell lines.

[0114] 2.2.4 Knockdown of UMODL1 promotes cell apoptosis

[0115] Cell apoptosis was detected by flow cytometry in the control group trastuzumab-resistant cell line JIMT-1 cells and the JIMT-1 cell line with UMODL1 knockdown. The results showed that the apoptosis rate of JIMT-1 cells with UMODL1 knockdown was significantly increased. The above results indicate that knocking down UMODL1 can significantly promote the apoptosis of JIMT-1 cells, a trastuzumab-resistant breast cancer cell line.

[0116] 2.3 UMODL1 can enhance the resistance of Her-2 positive breast cancer to trastuzumab

[0117] 2.3.1 Overexpression of UMODL1 can reverse the inhibition of trastuzumab on the proliferation of breast cancer cell lines in vitro

[0118] Through CCK-8 proliferation assay, SK-BR-3 and BT-474 cell lines that overexpressed UMODL1 and added trastuzumab were compared with SK-BR-3 and BT-474 cell lines that did not overexpress UMODL1 but added trastuzumab. The results showed that compared with the cell lines that did not overexpress UMODL1, the proliferation ability of the cell lines successfully transfected with UMODL1 was significantly enhanced, and the inhibitory effect of trastuzumab on them was significantly weakened ( Figure 1 A, B). Subsequently, a plate cloning experiment was performed, and the results showed that the clone formation ability of SK-BR-3 and BT-474 cell lines overexpressing UMODL1 was significantly enhanced, and the effect of trastuzumab on inhibiting cancer cell cloning was significantly weakened ( Figure 1 C, D). The above results indicate that overexpression of UMODL1 can reverse the inhibition of trastuzumab on the proliferation of SK-BR-3 and BT-474 breast cancer cell lines in vitro.

[0119] 2.3.2 Overexpression of UMODL1 can partially reverse the effect of trastuzumab on apoptosis in breast cancer cell lines

[0120] Flow cytometry was used to explore the changes in the response of SK-BR-3 and BT-474 cell lines overexpressing UMODL1 to trastuzumab compared with those without upregulation of UMODL1. The results showed that the apoptosis rate of SK-BR-3 and BT-474 cell lines overexpressing UMODL1 and adding trastuzumab was significantly reduced ( Figure 2 A). The above results indicate that high expression of UMODL1 can partially reverse the promoting effect of trastuzumab on apoptosis of SK-BR-3 and BT-474 cells.

[0121] 2.3.3 Knockdown of UMODL1 can partially restore the inhibitory effect of trastuzumab on the proliferation of trastuzumab-resistant breast cancer cell lines in vitro

[0122] The Her-2 positive breast cancer cell line JIMT-1 is a trastuzumab-resistant cell line, and the inhibitory effect of trastuzumab on it is significantly weakened. The CCK-8 proliferation assay was used to examine the effect of knocking down UMODL1 on the drug resistance of JIMT-1. The experimental results showed that compared with the control group JIMT-1, the cell viability of the JIMT-1 cell line with knocking down UMODL1 and adding trastuzumab was significantly reduced ( Figure 3A). Then, a plate cloning experiment was performed to compare the proliferation ability of the two groups of cells. The results showed that the cloning ability of JIMT-1 cells after knocking down UMODL1 and then adding trastuzumab was significantly weakened compared with the control group ( Figure 3 B). The above results indicate that knocking down UMODL1 can partially restore the inhibitory effect of trastuzumab on the proliferation of JIMT-1 cell lines in vitro.

[0123] 2.3.4 Knockdown of UMODL1 partially restores the effect of trastuzumab on promoting apoptosis in trastuzumab-resistant breast cancer cell lines

[0124] Flow cytometry was used to detect the changes in the control group trastuzumab-resistant Her-2 positive breast cancer cell line JIMT-1 and the UMODL1 knockdown group JIMT-1 after the addition of trastuzumab. The results showed that compared with the control group JIMT-1, the apoptosis rate of the cells knocked down UMODL1 was significantly increased after the addition of trastuzumab ( Figure 4 A). The above experimental results show that knocking down UMODL1 can partially restore the apoptosis-promoting effect of trastuzumab on JIMT-1 cell line.

[0125] 2.4 UMODL1 promotes the tumorigenicity of Her-2 positive breast cancer in nude mice

[0126] 2.4.1 Overexpression of UMODL1 promotes the growth of Her-2-positive breast cancer in vivo

[0127] CCK-8 and plate cloning experiments proved that UMODL1 has the effect of promoting the occurrence and development of Her-2 positive breast cancer in vitro. Then we subcutaneously injected normal SK-BR-3 cell lines and SK-BR-3 cell lines overexpressing UMODL1 into nude mice to establish a transplanted tumor model. The results showed that compared with the control group SK-BR-3, the volume and weight of the transplanted tumors of SK-BR-3 cells overexpressing UMODL1 were significantly increased ( Figure 5 A). The above experimental results prove that overexpression of UMODL1 can promote the growth of Her-2 positive breast cancer in vivo.

[0128] 2.4.2 Knockdown of UMODL1 inhibits the growth of Her-2-positive breast cancer in vivo

[0129] Normal JIMT-1 cell lines and JIMT-1 cell lines with UMODL1 knockdown were subcutaneously injected into nude mice to establish a transplanted tumor model. The results showed that compared with the control group JIMT-1, the volume and weight of the JIMT-1 cell transplanted tumor in the UMODL1 knockdown group were significantly reduced ( Figure 6 A). The above experimental results prove that knocking down UMODL1 can inhibit the growth of Her-2 positive breast cancer in vivo.

[0130] 2.5UMODL1 can enhance the resistance of Her-2 positive breast cancer to trastuzumab in nude mice

[0131] 2.5.1 Overexpression of UMODL1 can enhance the resistance of Her-2 positive breast cancer to trastuzumab in nude mice

[0132] On the basis of the above experiments, the SK-BR-3 cell line overexpressing UMODL1 was subcutaneously injected into nude mice to establish a transplanted tumor model, and trastuzumab was added to explore the effect of UMODL1 on trastuzumab resistance in vivo. The results showed that compared with the control group without overexpressing UMODL1, the volume and weight of the transplanted tumor in the experimental group injected with trastuzumab and overexpressing UMODL1 increased significantly ( Figure 7 A). The above experimental results show that overexpression of UMODL1 can increase the resistance of Her-2 positive breast cancer to trastuzumab in nude mice.

[0133] 2.5.2 Knockdown of UMODL1 can reduce the resistance of Her-2 positive breast cancer to trastuzumab in nude mice

[0134] The trastuzumab-resistant cell line JIMT-1 with UMODL1 knocked down was subcutaneously injected into nude mice to establish a transplanted tumor model, and trastuzumab was added. The results showed that compared with the control group, the transplanted tumor volume and weight of the experimental group injected with trastuzumab-added JIMT-1 cells with UMODL1 knockdown were significantly reduced ( Figure 8 A). The above experiments have shown that knocking down UMODL1 can reduce the resistance of Her-2 positive breast cancer to trastuzumab in nude mice.

[0135] in conclusion

[0136] 1. The expression level of UMODL1 in breast cancer tissue is significantly higher than that in adjacent tissues. High expression of UMODL1 is associated with poor overall survival and recurrence-free survival. The expression of UMODL1 is significantly correlated with Her-2 positive breast cancer in breast cancer pathological classification. In Her-2 positive breast cancer, high UMODL1 expression indicates a shorter disease-free survival. High expression of UMODL1 is also found to be associated with trastuzumab resistance.

[0137] 2. Among breast cancer cell lines of different types, the expression of UMODL1 was highest in Her-2 receptor-positive breast cancer cell lines, and the expression of UMODL1 was even higher in the trastuzumab-resistant cell line JIMT-1.

[0138] 3. Successfully overexpressed UMODL1 in BT-474 and SK-BR-3, knocked down UMODL1 in JIMT-1, and completed subsequent functional experiments. Proliferation and apoptosis-related cell phenotype experiments proved that upregulating UMODL1 can enhance the proliferation ability and clone formation ability of Her-2 positive breast cancer cell lines and inhibit cell apoptosis; knocking down UMODL1 can weaken the proliferation ability and clone formation ability of Her-2 positive breast cancer cell lines and promote cell apoptosis. In the case of adding trastuzumab, upregulating UMODL1 can partially restore the inhibitory effect of trastuzumab on the proliferation ability and clone formation ability of Her-2 positive breast cancer cell lines, and inhibit cell apoptosis; knocking down UMODL1 can produce a synergistic effect with trastuzumab, further weakening the proliferation ability and clone formation ability of Her-2 positive breast cancer cell lines and promoting cell apoptosis.

[0139] 4. Upregulation of UMODL1 can enhance the proliferation of breast tumors in nude mice; knockdown of UMODL1 can weaken the proliferation of breast tumors in nude mice. When trastuzumab is used in nude mice, upregulation of UMODL1 can further enhance the resistance of Her-2 positive breast tumors to trastuzumab; knockdown of UMODL1 can produce a synergistic effect with trastuzumab and further inhibit the growth of Her-2 positive breast tumors in nude mice.

[0140] 5. The expression of UMODL1 is positively correlated with the expression of Her-2 receptor. UMODL1 can upregulate the expression of Her-2: upregulating UMODL1 can increase the expression of Her-2 protein; knocking down UMODL1 can reduce the expression of Her-2 protein.

[0141] Study on the mechanism of UMODL1 leading to trastuzumab resistance in breast cancer

[0142] 1. Experimental Methods

[0143] 1.1 Cell culture

[0144] The cell culture method was the same as in the previous part.

[0145] 1.2 Total RNA extraction, reverse transcription and qRT-PCR of breast cancer cells

[0146] Total RNA extraction, reverse transcription and qRT-PCR of breast cancer cells were the same as the previous part.

[0147] 1.3 Western Blot Experimental Procedure

[0148] The Western Blot part is the same as the previous part.

[0149] 1.4 Immunohistochemistry (conventional method, no further details here)

[0150] 1.5 Dual luciferase reporter assay

[0151] (1) Cell culture: Add an appropriate volume of 0.25% trypsin to the cultured cells to digest the cells. After the cells become round, add complete medium to terminate the reaction. Collect the cells and centrifuge for 3 minutes. Discard the supernatant, count the cells, and inoculate the cells in a 12-well plate at a density of 50%. Place the culture plate in an incubator at 37°C and 5% CO2 and culture overnight. Transfect the cells 24 hours later.

[0152] (2) Cell transfection:

[0153] 1) Preparation of transfection reagent: Solution 1 is 150 μL optimized solution + 9 μL liposome 3000; Solution 2 is 150 μL optimized solution + 3 μg plasmid. Mix solutions 1 and 2 and place at room temperature for 15 minutes.

[0154] 2) Transfection: Add the mixture dropwise into the wells, mix well, and culture at 37°C and 5% CO2 for 48 h.

[0155] (3) Luciferase activity detection:

[0156] 1) Lyse cells: discard the old cell culture medium and rinse with PBS, add 100 μL of cell lysis solution to each well, centrifuge at 1000 r / min for 15 min. After cell lysis, transfer cells from each well to a sterilized centrifuge tube, centrifuge at 10000 r / min for 5 min at 4°C, and take the supernatant.

[0157] 2) Equilibrate the samples, firefly luciferase detection reagent, and Renilla luciferase detection reagent to room temperature.

[0158] 3) Add 100 μL of firefly luciferase detection reagent to each well, mix well, and immediately place the centrifuge tube into the fluorescence detector to calculate the firefly luciferase activity value F.

[0159] 4) Add 100uL of Renilla luciferase detection reagent to each well, mix well, and immediately place the centrifuge tube into the fluorescence detector to calculate the Renilla luciferase activity value R.

[0160] 5) Calculate the relative luciferase activity based on the detected F value and R value.

[0161] 1.6 Co-IP Experiment

[0162] (1) Collect cells expressing both UMODL1 and Her-2 proteins. Aspirate the cell culture medium, rinse once with PBS, add an appropriate amount of RIPA cell lysis buffer (containing protease inhibitors) and lyse on ice for 30 min. Transfer the cell lysate to a 1.5 ml EP tube, centrifuge at maximum speed for 30 min at 4°C, and collect the supernatant.

[0163] (2) Take a small amount of lysate as the input group for subsequent Western blot analysis; add 1 μg of UMODL1 / Her-2 antibody to the lysate of the experimental group and incubate overnight with slow shaking at 4°C; add IgG antibody to the lysate of the control group and incubate overnight.

[0164] (3) Add 10 μL of pretreated protein A agarose beads to the cell lysate incubated with the antibody overnight, and incubate at 4°C with slow shaking for 2-4 h to allow the antibody to couple to the protein A agarose beads.

[0165] (4) Centrifuge at 3000 rpm / min for 3 min at 4°C to remove the agarose beads to the bottom of the tube. Aspirate the supernatant and add 1 ml of lysis buffer to the remaining agarose beads in the tube to wash three times. Then add 15 μl of 2× SDS loading buffer and boil in boiling water for 5 min.

[0166] (5) After centrifugation, Western blot analysis was performed and the binding proteins were determined by co-immunoprecipitation.

[0167] 1.7 ChIP experiments

[0168] (1) Cell lysis and fixation: Add an appropriate amount of RIPA cell lysis buffer to the cultured cells to lyse the cells. Add 1% formaldehyde to fix the cells for 10 min, and then add glycine to react for 5 min to terminate cross-linking.

[0169] (2) Ultrasonic disruption of chromatin: Wash with pre-cooled PBS to obtain a mixed solution, add an appropriate amount of protease inhibitor, centrifuge and discard the supernatant, and collect the cells. Ultrasonic disruption of the collected cells, centrifugation to remove impurities, retain 300 μL for subsequent experiments, and store the rest at -80°C.

[0170] (3) Antibody incubation

[0171] 1) Divide the sample into three 100 μL fractions, add 900 μL buffer and 60 μL protein A to each fraction, and flip and mix at 4°C for 1-2 hours. Let the mixture settle at 4°C for 10 minutes, and centrifuge at 3000 rpm / min for 1 minute.

[0172] 2) 20 μL of supernatant was extracted from each group of samples as input. 1-10 μg UMODL1 antibody was added to the anti-UMODL1 group, 1 μg RNA polymerase II was added to the input group, and 1 μg human serum IgG was added to the IgG group, and then cultured overnight at 4°C.

[0173] (4) Immune complex precipitation

[0174] 1) Add 60 μL of protein A to each sample and mix by inversion at 4°C for 1-2 hours. Let the solution stand for 10 minutes and centrifuge to remove the supernatant.

[0175] 2) Washing: Add 1 ml of pre-cooled solution, invert at 4°C for 10 minutes, let it stand for 10 minutes and centrifuge to remove the supernatant.

[0176] 3) Elution: Add 250 μL elution buffer to each tube, invert for 15 minutes at room temperature, centrifuge and collect the supernatant. Repeat the washing once. The final elution buffer is 500 μL per tube.

[0177] 4) Mix well and decrosslink at 65°C overnight.

[0178] (5) Recovery of DNA samples: After the cross-linking is resolved, add 1 μL RNase A to each tube and incubate at 37°C for 1 h. Recover DNA fragments and dissolve them in 100 μL ddH20.

[0179] (6) The DNA fragments were analyzed by agarose gel electrophoresis.

[0180] 1.8 Molecular docking of UMODL1 and Her-2

[0181] (1) The UniProt online database (https: / / www.uniprot.org / ) was used to obtain the structures of known ligands of UMODL1 protein, Her-2, and Her-2 heterodimer, and the structures were analyzed.

[0182] (2) The structure of UMODL1 was prepared using MOE QuikPrep, and the MOE molecular docking software was applied to simulate the intramolecular interaction between UMODL1 and Her-2.

[0183] (3) According to the guidelines provided in the MOE protein-protein docking tutorial, the interaction energy model including Evdw, Eele and Egb / vi was used to select the docking solution with the lowest energy and the strongest binding affinity as the molecular docking result.

[0184] 2. Conclusion

[0185] 2.1 UMODL1 can upregulate the transcription of Her-2

[0186] 2.1.1 Her-2 is also highly expressed in human breast cancer pathological specimens with high UMODL1 expression

[0187] 60 Luminal A type, 68 Luminal B type (Her-2 receptor positive) or Her-2 positive type, and 63 triple negative breast cancer pathological specimens were collected for UMODL1 immunohistochemical staining. The results showed that among the 68 Her-2 positive pathological specimens, 27 pathological specimens had high expression of UMODL1 and 41 had low expression of UMODL1; among the 123 Her-2 negative pathological specimens, 3 pathological specimens had high expression of UMODL1 and 120 pathological specimens had low expression of UMODL1. The above results show that the expression of UMODL1 protein is positively correlated with the expression of Her-2 protein.

[0188] 2.1.2 Her-2 is also highly expressed in breast cancer cell lines with high UMODL1 expression

[0189] To further explore the relationship between UMODL1 and Her-2, SK-BR-3 and BT-474 cell lines with stable overexpression of UMODL1 were constructed, and the relationship between UMODL1 and Her-2 was verified by qRT-PCR and Western Blot experiments. The results showed that the protein level of Her-2 in SK-BR-3 and BT-474 in the UMODL1 overexpression group was higher than that in the control group.

[0190] 2.1.3 Her-2 is also lowly expressed in breast cancer cell lines with UMODL1 knockdown

[0191] To further explore the relationship between UMODL1 and Her-2, we knocked down UMODL1 in the JIMT-1 cell line, and used qRT-PCR and Western Blot experiments to explore the changes in Her-2 protein. The results showed that the Her-2 protein level in the JIMT-1 in the UMODL1 knockdown group was lower than that in the control group.

[0192] 2.1.4 UMODL1 can upregulate the transcription of Her-2

[0193] Through qRT-PCR experiments, it was found that UMODL1 regulates Her-2 at the pre-transcriptional level. Therefore, it is hypothesized that UMODL1 can upregulate the transcription of Her-2. In order to further explore the relationship between UMODL1 and Her-2, dual luciferase reporter experiments of UMODL1 and Her-2 and chromatin immunoprecipitation (ChIP) experiments were performed. The results showed that UMODL1 enhanced the transcriptional activity of Her-2.

[0194] 2.2UMODL1 can specifically bind to Her-2 heterodimers and activate the downstream signaling pathway of Her-2

[0195] 2.2.1 The structure of UMODL1 is similar to the ligand structure of Her-2 heterodimer

[0196] To further explore the reason why UMODL1 causes trastuzumab resistance, we downloaded the protein structures of UMODL1 and Her-2 from the Uniprot website, and analyzed the protein structures of UMODL1, the protein structures of known ligands of Her-2 heterodimers, and the protein structures of Her-2. The results showed that UMODL1 has three EGF domains, which is similar to the structures of Her-2 heterodimer ligands such as EGF, HB-EGF, TGF-α, BTC, NRG1, and NRG2 (Table 1). We then used MOE software to simulate the interaction between UMODL1 and Her-2 proteins, and the results showed that there was a significant binding affinity between the two proteins.

[0197] Table 1 Domain structure of UMODL1 and HER-2 heterodimer ligand

[0198]

[0199] 2.2.2 UMODL1 and Her-2 heterodimers specifically bind

[0200] To further explore the interaction between UMODL1 and Her-2 heterodimers, SK-BR-3 cells were stably transfected with Flag-UMODL1 lentiviral vector, and protein immunoprecipitation experiments were performed to verify the mass spectrometry results. Flag tag antibodies were used to obtain proteins that interact with UMODL1, and the results showed that UMODL1 can bind to Her-2 heterodimers. Subsequently, Her-2 antibodies were used to obtain proteins that can interact with Her-2 heterodimers, and the results showed that Her-2 heterodimers can bind to UMODL1.

[0201] 2.2.3 High UMODL1 expression can specifically bind to Her-2 heterodimers and promote the phosphorylation of Her-2 and downstream signaling pathway proteins

[0202] After confirming that the UMODL1 protein can specifically bind to the Her-2 heterodimer, we further explored the functional changes caused by the binding of the two. The phosphorylation levels of Her-2 and the iconic proteins PI3K, AKT, MEK, and ERK in the downstream signaling pathway of Her-2 were detected in normal BT-474 and SK-BR-3 and BT-474 and SK-BR-3 stably transfected with UMODL1 lentiviral vectors. The results showed that the phosphorylation levels of Her-2 and PI3K, AKT, MEK, and ERK in the cell lines overexpressing UMODL1 were significantly increased ( Fig. 9 A, B). The above experimental results indicate that high UMODL1 expression can bind more to Her-2 heterodimers and activate its downstream signaling pathway.

[0203] 2.2.4 Knockdown of UMODL1 can reduce the phosphorylation of Her-2 and downstream signaling pathway proteins

[0204] To further confirm the above experimental results, sh-UMODL1 was transfected into JIMT-1 to knock down UMODL1, and then the phosphorylation levels of Her-2 and the marker proteins PI3K, AKT, MEK, and ERK in the downstream signaling pathway of Her-2 were detected in normal JIMT-1 and JIMT-1 with UMODL1 knockdown. The results showed that the phosphorylation levels of Her-2 and PI3K, AKT, MEK, and ERK in the cell lines with UMODL1 knockdown were significantly reduced ( Fig.10 A). The above experimental results show that knocking down UMODL1 can reduce the phosphorylation of Her-2 and downstream signaling pathway proteins PI3K, AKT, MEK, and ERK.

[0205] 2.3 Mechanism of UMODL1 antagonism to trastuzumab

[0206] 2.3.1 UMODL1 overexpression can partially restore the phosphorylation inhibition of Her-2 and downstream signaling pathway proteins caused by trastuzumab

[0207] In order to explore the drug resistance mechanism caused by UMODL1, trastuzumab was added to SK-BR-3 and BT-474 cells overexpressing UMODL1, and then the phosphorylation levels of Her-2 and the marker proteins PI3K, AKT, MEK, and ERK in the downstream signaling pathway of Her-2 were detected. The results showed that compared with BT-474 and SK-BR-3 cells only added with trastuzumab, the phosphorylation levels of Her-2 and PI3K, AKT, MEK, and ERK were higher in SK-BR-3 and BT-474 cell lines with trastuzumab added and UMODL1 overexpression ( Fig.11A, B). The above experimental results indicate that UMODL1 overexpression can partially restore the phosphorylation inhibition of Her-2 and downstream signaling pathway proteins caused by trastuzumab.

[0208] 2.3.2 Knockdown of UMODL1 can synergize with trastuzumab to inhibit the phosphorylation of Her-2 and downstream signaling pathway proteins

[0209] To further confirm the above experimental results, we knocked down UMODL1 in the trastuzumab-resistant cell line JIMT-1 and added trastuzumab. We then detected changes in the phosphorylation levels of Her-2 and the marker proteins PI3K, AKT, MEK, and ERK in the downstream signaling pathway of Her-2. The results showed that after adding trastuzumab, the phosphorylation levels of Her-2 and PI3K, AKT, MEK, and ERK in JIMT-1 with UMODL1 knocked down were lower ( Fig.12 A). The above experimental results show that knocking down UMODL1 can produce a synergistic effect with trastuzumab, thereby jointly inhibiting the phosphorylation of Her-2 and PI3K, AKT, MEK, and ERK.

[0210] 2.4 In vivo mechanism of UMODL1 antagonism to trastuzumab

[0211] 2.4.1 UMODL1 overexpression can partially restore the phosphorylation inhibition of Her-2 and downstream signaling pathway proteins caused by trastuzumab in vivo

[0212] In order to further verify the above experimental results in vivo, the SK-BR-3 cell line overexpressing UMODL1 was subcutaneously injected into nude mice to establish a transplanted tumor model, and trastuzumab was injected into the nude mice. Subsequently, immunofluorescence technology was used to verify whether the in vivo mechanism of UMODL1 resistance to trastuzumab was the same as in vitro. The changes in the phosphorylation levels of Her-2 and the marker proteins PI3K, AKT, MEK, and ERK of the Her-2 downstream signaling pathway in the tumors of the control group injected with trastuzumab and the UMODL1-overexpressing group injected with trastuzumab were detected. The results were consistent with the in vitro experiments: that is, the phosphorylation levels of Her-2 and PI3K, AKT, MEK, and ERK in the experimental group were higher than those in the control group. Immunohistochemistry was also used to verify the effect of upregulating UMODL1 on Ki-67 ( Fig.13 A).

[0213] 2.4.2 Knockdown of UMODL1 can synergize with trastuzumab in vivo to inhibit the phosphorylation of Her-2 and downstream signaling pathway proteins

[0214] We then subcutaneously injected the JIMT-1 cell line with UMODL1 knockdown into nude mice to establish a transplanted tumor model, and injected trastuzumab into the nude mice. Immunofluorescence technology was then used to detect changes in the phosphorylation levels of Her-2 and the marker proteins PI3K, AKT, MEK, and ERK in the Her-2 downstream signaling pathway in the control group injected with trastuzumab and the UMODL1 knockdown group injected with trastuzumab. As expected, compared with the control group, the phosphorylation levels of Her-2 and PI3K, AKT, MEK, and ERK in the experimental group were lower ( Fig.14 A). The above experimental results show that knocking down UMODL1 in vivo can produce a synergistic effect with trastuzumab, thereby partially restoring the drug sensitivity of drug-resistant tumors to trastuzumab.

[0215] 2.5 Scientific hypothesis that UMODL1 promotes the proliferation of Her-2 positive breast tumors and causes trastuzumab resistance by regulating Her-2

[0216] This study found that UDMOL1 can upregulate the transcription of Her-2 and activate its downstream PI3K / AKT / mTOR and MAPK signaling pathways, thereby leading to the proliferation of Her-2 positive breast tumors and resistance to trastuzumab targeted therapy (such as Fig.15 A).

[0217] in conclusion:

[0218] 1. The oncogene UMODL1 was screened through multiple databases and found to be most highly expressed in Her-2 positive breast cancer. UMODL1 can enhance the proliferation of Her-2 positive breast cancer and inhibit apoptosis, and is associated with resistance to the breast cancer targeted drug trastuzumab. UMODL1 can upregulate the expression of Her-2 protein and bind to Her-2 heterodimers to activate the downstream PI3K / AKT / mTOR and MAPK signaling pathways of Her-2. The two mechanisms produce a synergistic effect, thereby promoting the occurrence and development of Her-2 positive breast cancer and leading to trastuzumab resistance.

[0219] 2. UMODL1 can bind to Her-2 heterodimers and activate its downstream PI3K / AKT / mTOR and MAPK signaling pathways. When UMODL1 is upregulated, phosphorylated Her-2, PI3K, AKT, MEK, and ERK increase; when UMODL1 is knocked down, phosphorylated Her-2, PI3K, AKT, MEK, and ERK decrease. In the case of trastuzumab, upregulating UMODL1 can partially restore the reduction of phosphorylated Her-2, PI3K, AKT, MEK, and ERK caused by trastuzumab; knocking down UMODL1 can produce a synergistic effect with trastuzumab, further reducing phosphorylated Her-2, PI3K, AKT, MEK, and ERK.

[0220] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a reagent for detecting the expression level of the UMODL1 gene in the preparation of a Her-2 positive breast cancer diagnostic kit.

2. Use of a reagent for detecting the expression level of the UMODL1 gene in the preparation of a kit for prognosis detection of Her-2 positive breast cancer.

3. Application of UMODL1 inhibitors in the preparation of drugs for inhibiting Her-2 positive breast cancer.

4. Application of UMODL1 inhibitors in the preparation of drugs for inhibiting or reversing the resistance of Her-2 positive breast cancer cells to trastuzumab.

5. The use according to claim 4, characterized in that: The inhibitor is an agent for knocking down the UMODL1 gene.

6. A composition for inhibiting Her-2 positive breast cancer, characterized in that: The composition includes a UMODL1 inhibitor and trastuzumab.

7. The composition according to claim 6, characterized in that UMODL1 inhibitor and trastuzumab synergistically inhibit the phosphorylation of Her-2 and downstream PI3K / AKT / mTOR and MAPK signaling pathway proteins.

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