Application of RNF128 gene in prognosis of gastrointestinal stromal tumor

Abstract

The invention relates to the field of biological medicine, in particular to application of RNF128 gene in prognosis of gastrointestinal stromal tumor. The invention provides application of a reagent for detecting RNF128 gene expression quantity in preparation of a detection product for gastrointestinal stromal tumor prognosis. In the invention, the RNF128 gene is highly expressed in a GIST low-risk patient and is low-expressed in a GIST high-risk patient. The RNF128 gene is used as a core, key links of GIST treatment and transformation research are comprehensively covered, and innovativeness, scientificity and practicability are achieved. The method has the advantages that on the basis of basic research value, an operable solution is provided for clinical practice, and the diagnosis and treatment effect and life quality of the GIST patient can be remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the application of the RNF128 gene in the prognosis of gastrointestinal stromal tumors. Background Art

[0002] Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor in the gastrointestinal tract, and its pathological origin is mainly interstitial cells of Cajal or their precursor cells. The pathogenesis of GIST is closely related to various cell molecular abnormalities and is a type of tumor characterized by molecular marker drive. In some GIST patients, mutations in the KIT gene are detected. The tyrosine kinase receptor encoded by the KIT gene is in a constitutively active state after mutation and can transmit signals autonomously without the stimulation of external ligands, thereby driving the proliferation and survival of tumor cells. In addition, some GIST patients have mutations in the platelet-derived growth factor receptor A (PDGFRA) gene. Similar to KIT mutations, PDGFRA mutations also lead to abnormal activation of tyrosine kinases and are involved in the occurrence and progression of tumors. These mutations are highly specific in GIST and are the core basis for current diagnosis and targeted therapy.

[0003] Currently, the main treatment strategies for GIST include surgical resection and targeted drug therapy. Surgical resection is still the main treatment method for localized GIST, especially when the tumor has no extensive metastasis. Complete resection of the lesion is the key to achieving long-term survival. However, for patients with distant metastasis or recurrence of the tumor, it is difficult to achieve the purpose of cure only by surgery. Tyrosine kinase inhibitors (TKIs) have a milestone significance in the treatment of GIST. Imatinib, as the first-generation TKI, is the standard treatment drug for KIT and PDGFRA mutations. It competitively inhibits tyrosine kinase activity and blocks the downstream signal transduction of mutant KIT or PDGFRA, thereby inhibiting the proliferation and survival of tumor cells. Although targeted therapy has significantly improved the survival rate of GIST patients, especially reducing the risk of postoperative recurrence, there are still many limitations. This is mainly due to the structural changes of tyrosine kinases caused by secondary mutations, which prevent the drug from effectively binding to the target. At the same time, the development of drug resistance greatly limits the long-term treatment effect. For patients resistant to imatinib, second-generation TKIs such as sunitinib and regorafenib have been approved for treatment. However, these drugs can only delay the progression of drug resistance, and the overall efficacy is limited, especially in patients with advanced or metastatic GIST, the treatment effect is not satisfactory. Metastatic GIST is currently a difficult point in clinical treatment. It can be seen that the existing targeted drugs have poor effects on the mechanism of controlling distant metastasis, and the prognosis of patients is poor.

[0004] The mechanism of distant metastasis is complex, involving multiple signaling pathways and microenvironment regulation, which is also an important direction of current research. GIST shows significant heterogeneity in molecular and pathological characteristics. Different patients or even different lesions in the same patient may exhibit different mutation types or molecular characteristics. For example, some GIST patients do not have KIT or PDGFRA mutations, but are accompanied by SDH deficiency or BRAF mutations. This heterogeneity makes it difficult to achieve personalized treatment with existing therapies. For patients with wild-type GIST, the efficacy of targeted therapy is more limited. To overcome the limitations of existing treatments, further research on the molecular mechanism of GIST, discovery of new targets, and development of personalized treatment plans for different subtypes are important directions in the future. Especially for patients with rare mutation types or no obvious mutation characteristics, more potential markers and targets need to be explored. In addition, how to inhibit the drug resistance mechanism and prevent distant metastasis is the key to improving the prognosis and quality of life of GIST patients. Summary of the Invention

[0005] An object of the present invention is to provide the application of the RNF128 gene in the prognosis of gastrointestinal stromal tumors to solve the problems existing in the above-mentioned prior art. The RNF128 gene of the present invention helps to effectively diagnose early-stage patients with gastrointestinal stromal tumors. Therefore, reagents for detecting the expression level of the RNF128 gene can be used for detecting the prognosis of gastrointestinal stromal tumors.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides the application of a reagent for detecting the expression level of the RNF128 gene in the preparation of a detection product for the prognosis of gastrointestinal stromal tumors.

[0008] In the present invention, the RNF128 gene is highly expressed in low-risk patients with gastrointestinal stromal tumors and lowly expressed in high-risk patients with gastrointestinal stromal tumors. Thus, it can be seen that the RNF128 gene of the present invention can be used for risk assessment of the prognosis of gastrointestinal stromal tumors.

[0009] Preferably, the detection product includes a chip or a kit.

[0010] The present invention provides a detection product for the prognosis of gastrointestinal stromal tumors, and the detection product includes a reagent for detecting the expression level of the RNF128 gene.

[0011] Preferably, the detection product includes a chip or a kit.

[0012] The present invention provides the application of the RNF128 gene as a target in the preparation of a drug for preventing and / or treating gastrointestinal stromal tumors.

[0013] The present invention provides the use of an agonist of the RNF128 gene in the preparation of a drug for preventing and / or treating gastrointestinal stromal tumors.

[0014] The present invention provides a drug for preventing and / or treating gastrointestinal stromal tumors, and the drug comprises an agonist of the RNF128 gene.

[0015] The present invention provides the use of the RNF128 gene as a target in screening drugs for treating gastrointestinal stromal tumors.

[0016] The present invention provides a method for screening drugs for treating gastrointestinal stromal tumors, comprising the step of detecting the expression level of the RNF128 gene in a subject before and after administration.

[0017] The present invention discloses the following technical effects:

[0018] 1. The present invention reveals the key molecular mechanism of the RNF128 gene, providing a new perspective for the basic research of GIST.

[0019] The present invention for the first time systematically elucidates the low expression of the RNF128 gene in GIST and its molecular mechanism, and reveals that it affects the invasion and metastasis ability of tumor cells by regulating the ubiquitination degradation pathway. By clarifying the functional role of the RNF128 gene, the present invention fills the gap in the current research on the molecular mechanism of GIST and provides a scientific basis for subsequent exploration of new targets.

[0020] 2. The present invention provides a potential precision prognosis method for GIST patients.

[0021] The RNF128 gene can be used as a molecular marker for the prognosis of GIST. The present invention develops simple and sensitive detection methods (such as immunohistochemistry, ELISA) based on the RNF128 gene, which can be used to evaluate the disease stage and invasion and metastasis risk of patients.

[0022] 3. The present invention provides a new treatment idea for drug-resistant patients.

[0023] The existing treatment with tyrosine kinase inhibitors (TKIs) has significant drug resistance problems. The present invention proposes a new treatment strategy targeting the RNF128 gene by studying the regulatory role of the RNF128 gene in the signal pathway.

[0024] 4. The present invention proposes an innovative solution for the mechanism of distant metastasis.

[0025] Distant metastasis is the main cause of poor prognosis in GIST patients. By studying the regulatory effect of the RNF128 gene on the invasion and migration of tumor cells, the present invention finds that it can significantly inhibit the occurrence of distant metastasis. And based on molecular biology, the present invention uses gene therapy or small molecule drugs to regulate the expression of the RNF128 gene, which has lower toxic and side effects compared with traditional chemotherapy and reduces the physical burden of patients. The application in precision treatment and prognosis is expected to reduce the incidence of recurrence and metastasis, thereby reducing the consumption of medical resources and the social and economic costs.

[0026] In summary, the present invention takes the RNF128 gene as the core, comprehensively covers the key links of GIST treatment and translational research, and has innovation, scientificity and practicability. Its advantages are not only reflected in the basic research value of GIST, but also provide an operable solution for clinical practice, and will significantly improve the diagnosis and treatment effect and quality of life of GIST patients. Brief Description of the Drawings

[0027] Figure 1 It is the principal component analysis result diagram of the GSE225819 dataset; among them, normal refers to normal samples, and stromal tumor refers to samples of gastrointestinal stromal tumor patients with liver metastasis;

[0028] Figure 2 It is the sample clustering dendrogram of the GSE225819 dataset; among them, normal refers to normal samples, and stromal tumor refers to samples of gastrointestinal stromal tumor patients with liver metastasis;

[0029] Figure 3 It is the sample irrelevance diagram for determining the optimal soft threshold;

[0030] Figure 4 It is the average connectivity diagram for determining the optimal soft threshold;

[0031] Figure 5 It is the co-expression network diagram;

[0032] Figure 6 It is the sample trait heat map;

[0033] Figure 7 It is the phenotype-gene correlation heat map of the GSE225819 dataset (A, also known as the module-trait correlation heat map), the correlation heat map between modules (B), the correlation analysis diagram of the blue module and gastrointestinal stromal tumor (C), and the correlation analysis diagram of the sky blue module and gastrointestinal stromal tumor (D);

[0034] Figure 8 It is the Venn diagram of the differential genes and blue module genes of the GSE225819 dataset;

[0035] Figure 9 It is the LASSO regression coefficient diagram;

[0036] Figure 10 It is a parameter graph of LASSO regression;

[0037] Figure 11 It is a volcano plot of differential genes;

[0038] Figure 12 It is a box plot of the RNF128 gene; where, normal refers to normal samples, and stromal tumor refers to samples of patients with gastrointestinal stromal tumors accompanied by liver metastases;

[0039] Figure 13 It is a statistical graph of the expression of RNF128 protein in high-risk and low-risk patients with gastrointestinal stromal tumors; where, A is a graph of the expression of RNF128 protein detected by immunohistochemistry; B is a statistical graph of immunohistochemical scores;

[0040] Figure 14 It is a statistical result graph of the expression of RNF128 gene detected by Western blot experiment in gastrointestinal stromal tumor cells and normal gastric epithelial cells; where, GIST-T1 is gastrointestinal stromal tumor cells, and GES-1 is normal gastric epithelial cells;

[0041] Figure 15 It is a statistical result graph of the detection of RNF128 mRNA level by real-time fluorescence quantitative PCR; where, GIST-T1 is gastrointestinal stromal tumor cells, and GES-1 is normal gastric epithelial cells;

[0042] Figure 16Statistical result graph of the RNF128 overexpression stable cell line; among them, A is the statistical graph of the RNF128 protein expression efficiency in the RNF128 overexpression stable cell line examined by Western blot, Vector is GIST-T1 cells, and RNF128-Flag is the RNF128 overexpression stable cell line; B is the statistical graph of the RNF128 gene expression efficiency in the RNF128 overexpression stable cell line examined by qRT-PCR, Vector is GIST-T1 cells, and RNF128-Flag is the RNF128 overexpression stable cell line; C is the statistical graph of the effect of overexpressing the RNF128 gene on cell proliferation measured by the CCK-8 assay, Vector is GIST-T1 cells, and RNF128-Flag is the RNF128 overexpression stable cell line; D and F are the statistical graphs of the effect of overexpressing the RNF128 gene on migration and invasion evaluated by the Transwell migration and invasion assays, Vector is GIST-T1 cells, and RNF128-Flag is the RNF128 overexpression stable cell line; E and G are the statistical graphs of the effect of increasing the expression of the RNF128 gene in gastric stromal tumors on cell invasion and migration measured by the wound healing assay, Vector is GIST-T1 cells, and RNF128-Flag is the RNF128 overexpression stable cell line; H is the graph of the wound healing condition, Vector is GIST-T1 cells, and RNF128-Flag is the RNF128 overexpression stable cell line; I is the statistical graph of the wound healing rate, Vector is GIST-T1 cells, and RNF128-Flag is the RNF128 overexpression stable cell line;

[0043] Figure 17Statistical result graph of transient transfection cell line with knocked-down RNF128 gene; among them, A is the statistical graph of the knockdown efficiency of RNF128 gene in the transient transfection cell line with knocked-down RNF128 gene examined by Western blot; B is the result graph of the knockdown efficiency of RNF128 gene in the transient transfection cell line with knocked-down RNF128 gene examined by real-time quantitative polymerase chain reaction; C is the statistical graph of the effect of knocking down RNF128 gene on cell proliferation measured by CCK-8 assay, Si-RNF128-1 is siRNF128-818, Si-RNF128-2 is siRNF128-1356; D and F are the statistical graphs of the effect of knocking down RNF128 gene on cell invasion and migration evaluated by Transwell migration and invasion assays, Si-RNF128-1 is siRNF128-818, Si-RNF128-2 is siRNF128-1356; E and G are the statistical graphs of the effect of knocking down RNF128 gene on cell invasion and migration measured by wound healing assay, Si-RNF128-1 is siRNF128-818, Si-RNF128-2 is siRNF128-1356; H is the graph of wound healing condition, Si-RNF128-1 is siRNF128-818, Si-RNF128-2 is siRNF128-1356; I is the statistical graph of wound healing rate, Si-RNF128-1 is siRNF128-818, Si-RNF128-2 is siRNF128-1356;

[0044] Figure 18 Survival analysis result graph; among them, high expression of RNF128 is the high-expression RNF128 gene group, low expression of RNF128 is the low-expression RNF128 gene group, the abscissa is survival time, the unit is days, and the ordinate is survival probability. Specific implementation mode

[0045] Example 1 Analysis of differential expression of RNF128 gene in gastrointestinal stromal tumors by GEO database

[0046] The analysis process is as follows:

[0047] The data comes from the NCBI.GEO (Gene Expression Omnibus, GEO, http: / / www.ncbi.nlm.nih.gov / geo / ) database. The GSE225819 dataset is selected, including 20 samples of patients with gastrointestinal stromal tumors (GIST) with liver metastases and 20 normal samples (NORMAL). Principal component analysis is performed to check whether the sample differences are obvious. The results are as Figure 1As shown. The results showed that the principal components of the samples of patients with gastrointestinal stromal tumors with liver metastases and normal samples were significantly different. Using the GSE225819 dataset in the GEO database, sample clustering analysis was first performed, and then the optimal soft threshold was determined to construct a co-expression network ( Figures 2 - 6 ). A heat map of the correlation between phenotypes and genes was drawn to identify the module genes most relevant to gastrointestinal stromal tumors with liver metastases ( Figure 7 ). After taking the intersection of the differentially expressed genes and module genes in the GSE225819 dataset, LASSO regression analysis was performed on the obtained genes, and finally the genes of interest were screened. A volcano plot was made for the genes of interest, and the results showed that the RNF128 gene was the most different from the phenotype of gastrointestinal stromal tumors with liver metastases. Finally, the RNF128 gene was selected as the target gene for studying the distant metastasis mechanism of gastrointestinal stromal tumors ( Figures 8 - 12 ).

[0048] Example 2 Detection of the expression of the RNF128 gene in gastrointestinal stromal tumors (stromal tumors)

[0049] (I) Immunohistochemical detection of the expression of RNF128 protein in the diseased tissues of patients with gastrointestinal stromal tumors

[0050] 1. Collection of patient clinical data

[0051] Surgical specimens of 30 patients with gastrointestinal stromal tumors were collected, and the clinicopathological data of the patients (including tumor size, mitotic count, risk grade, metastasis status, etc.) were obtained. The clinicopathological data are shown in Table 1.

[0052] Table 1 Clinicopathological data

[0053]

[0054] 2. Immunohistochemical method

[0055] Immunohistochemistry (IHC): Immunostaining of RNF128 protein was performed on tumor tissue sections to observe its expression and distribution in tumor tissues. The experimental steps are as follows:

[0056] The selected tissue sections were placed in an incubator (70 °C) and baked for 1 - 2 h.

[0057] The tissue sections were successively immersed in xylene × 2, 100% alcohol, 95% alcohol, and 85% alcohol for 10 min, 10 min, 5 min, 5 min, and 5 min respectively.

[0058] The tissue sections were taken out and rinsed with running water for 15 min, taking care not to wash off the tissues.

[0059] Wash the tissue sections 3 times with phosphate buffered saline (PBS buffer), 5 minutes each time.

[0060] Immerse the tissue sections in citrate solution, place them in a microwave oven, and microwave at medium-high power for 15 minutes. After antigen retrieval, let them cool naturally at room temperature.

[0061] Wash the tissue sections with phosphate buffered saline 3 times, 5 minutes each time.

[0062] Prepare the primary antibody against RNF128 (the primary antibody against RNF128 is diluted at a volume ratio of 1:300, purchased from Proteintech, catalog number 26015-1-AP), and dilute it with phosphate buffered saline or antibody diluent.

[0063] Drop the prepared primary antibody against RNF128 onto the tissue sections, draw a circle around it with a grease pen to prevent the antibody from overflowing, then place it in a wet box and incubate overnight at 4°C.

[0064] Take out the wet box the next day, open the lid, and rewarm at room temperature for 30 minutes.

[0065] Then recover the primary antibody on the tissue sections and wash 3 times with phosphate buffered saline, 5 minutes each time.

[0066] Wipe the washed tissue sections with paper, drop the corresponding rabbit / mouse secondary antibody against the primary antibody of RNF128, and incubate at room temperature for 1 hour.

[0067] Wash the tissue sections with phosphate buffered saline, 3 times, 5 minutes each time.

[0068] Prepare diaminobenzidine chromogenic solution (DAB chromogenic solution), and the preparation method is: 850 μL of double-distilled water (ddH2O) + 1 drop of diaminobenzidine reagent.

[0069] Drop the prepared diaminobenzidine chromogenic solution onto the tissue, observe under a microscope to avoid over-staining.

[0070] Rinse the tissue sections with running water for 20 minutes, taking care not to wash away the tissue.

[0071] Stain with hematoxylin for 8 seconds.

[0072] Rinse with running water for 20 minutes, taking care not to wash away the tissue.

[0073] Air dry naturally and mount with neutral resin.

[0074] 3. Immunohistochemical scoring method

[0075] Select 10 fields of view for each tissue section to observe, and the observation content includes the depth of staining and the proportion of stained cells to all cells.

[0076] The scoring process was completed by two independent pathologists, and the relevant scoring methods are as follows: The stained cells were scored from 0 to 4 according to the occupancy ratio; among them, the occupancy ratio was 0%, scored 0 points; the occupancy ratio was between 1% and 25%, scored 1 point; the occupancy ratio was between 26% and 50%, scored 2 points; the occupancy ratio was between 51% and 75%, scored 3 points; the occupancy ratio was between 75% and 100%, scored 4 points.

[0077] The staining was scored from 0 to 3 according to the intensity; among them, no staining was scored 0 points; the staining was light yellow, scored 1 point, the staining was brownish yellow, scored 2 points, and the staining was brownish brown, scored 3 points.

[0078] The total score = the score of the occupancy ratio multiplied by the score of the staining intensity. Finally, the total scores were arranged from high to low, and taking the median as the boundary, the total scores were divided into a low-risk group (low risk) and a high-risk group (high risk).

[0079] 4. Result analysis

[0080] The results are as Figure 13 shown. The RNF128 protein was highly expressed in the low-risk group (low-risk patients with gastrointestinal stromal tumors) and lowly expressed in the high-risk group (high-risk patients with gastrointestinal stromal tumors).

[0081] Example 3 Survival analysis

[0082] After immunohistochemical detection, taking the median as the boundary, 30 patients with gastrointestinal stromal tumors were divided into a high-expression RNF128 gene group (15 cases) and a low-expression RNF128 gene group (15 cases) according to the expression level of the RNF128 gene. Among them, the high-expression RNF128 gene group was consistent with the patients with gastrointestinal stromal tumors with low and very low risk grades in Table 1, and the low-expression RNF128 gene group was consistent with the patients with gastrointestinal stromal tumors with high risk grades in Table 1; then, survival analysis was performed, and the results are as Figure 18 shown. The results showed that the prognosis of the low-expression RNF128 gene group was worse and the survival time was shorter compared with the high-expression RNF128 gene group.

[0083] Example 4 Detection of the expression of the RNF128 gene in gastrointestinal stromal tumor cells (GIST-T1 cells) and normal gastric epithelial cells (GES-1 cells) by Western blot

[0084] 1. Cell culture;

[0085] 2. Steps for cell protein extraction;

[0086] 3. Steps for Western blot:

[0087] Western blot (WB): Extract cellular or tissue proteins, separate them by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and detect their protein expression using an anti-RNF128 antibody.

[0088] The specific experimental steps are as follows:

[0089] Remove GIST-T1 cells and GES-1 cells from the 6-well plate in the cell incubator.

[0090] Use a Pasteur pipette to aspirate the supernatant in the wells, add phosphate-buffered saline for rinsing. If the cells do not adhere firmly, this process should be carried out carefully or skipped directly. Rinse 2-3 times, add 1-2 mL of cell protein lysate to each well, scrape the cells in the 6-well plate with a cell scraper, and transfer the cell suspension to a plastic centrifuge tube (EP tube). This process should be carried out on ice.

[0091] Place the plastic centrifuge tube containing the cell suspension in a 4°C refrigerator for lysis for 30 min. After lysis, place the plastic centrifuge tube in a pre-cooled 4°C ultracentrifuge and centrifuge at 12,000 rpm for 10 min. Then transfer the supernatant to a new plastic centrifuge tube, add protein loading buffer according to the ratio of supernatant:protein loading buffer = 5:1 (volume ratio), place the plastic centrifuge tube in a 100°C metal bath, take out the plastic centrifuge tube after 5-7 min and store it at -20°C.

[0092] Determine the concentration of the gel according to the molecular weight of the target protein (a high-concentration gel should be prepared for small-molecular-weight proteins, and a low-concentration gel should be prepared for large-molecular-weight proteins). According to the molecular weight of the target protein such as RNF128 protein (40-70 kDa), prepare 20 mL of 10% separating gel. The preparation scheme is 5.3 mL of double-distilled water + 6.7 mL of 30% acrylamide + 200 μL of 10% sodium dodecyl sulfate (SDS) + 200 μL of 10% ammonium persulfate (AP) + 7.6 mL of tris(hydroxymethyl)aminomethane hydrochloride buffer (Tris-HCl buffer, pH 8.8). Mix the prepared separating gel with a 5 mL pipette and inject it into a special glass plate. The glass splint should be pre-tested for leakage. Then quickly and gently inject anhydrous ethanol into the glass plate with a pipette for liquid sealing. This process can make the gel surface more flat. Let it stand at room temperature for 30 min. A clear dividing line can be seen under the anhydrous ethanol, indicating that the separating gel has completely solidified. Pour out the upper anhydrous ethanol, and let it stand at room temperature for 3-5 min. Wait for the anhydrous ethanol to volatilize completely before proceeding to the next step.

[0093] After the separating gel is prepared, the preparation of the 5% stacking gel can be started. The preparation formula is 4.1 mL of double-distilled water + 1 mL of 30% acrylamide + 60 μL of 10% sodium dodecyl sulfate + 60 μL of 10% ammonium sulfate + 750 μL of tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 6.8). After preparation, use a 5 mL pipette to mix the liquid evenly, inject it into the glass plate, and then quickly insert a special 10-well or 15-well comb into the stacking gel. Bubbles should be avoided during this process. Then let it stand at room temperature for 15 min and observe whether the gel has completely solidified.

[0094] Prepare the protein samples, shake and mix them in advance. Add the freshly prepared electrophoresis buffer to the gel, add 10 μL of protein sample to each well (equal volume loading), and 1 - 2 μL of protein marker. Electrophorese at 80 V for 30 min, and then adjust to 120 V and electrophorese for 1 h.

[0095] After electrophoresis, use a nitrocellulose membrane (NC membrane) for electrotransfer at a current of 300 mA for 100 min.

[0096] Prepare the skim milk blocking solution in advance. Rinse the electrotransferred nitrocellulose membrane one or two times in TBST buffer, then soak it in the skim milk blocking solution, and gently shake it at room temperature for 90 min. Then wash it twice with TBST buffer under fast shaking conditions, 5 min for each time.

[0097] Cut the membrane according to the molecular weight of the target protein required, soak it in the prepared primary antibody (the RNF128 primary antibody is diluted at a volume ratio of 1:1000, and the GAPDH primary antibody is diluted at a volume ratio of 1:3000), and gently shake it overnight at 4 °C.

[0098] The next day, take out the strips, wash them 3 times with TBST buffer, 5 min for each time. Then place the strips in the corresponding rabbit / mouse secondary antibody (the rabbit / mouse secondary antibody is diluted at a volume ratio of 1:3000), incubate at room temperature for 1 h, and then wash the membrane 3 times with TBST buffer, 5 min for each time.

[0099] Finally, use a chemiluminescence instrument to expose the strips.

[0100] The results are as Figure 14 shown. The results show that the expression level of RNF128 protein is lower in gastrointestinal stromal tumor cells than in normal gastric epithelial cells.

[0101] Example 5 Detection of RNF128 Gene Expression by Real-Time Fluorescent Quantitative PCR (Q-PCR)

[0102] Real-time fluorescence quantitative PCR: Using specific primers, the mRNA expression level of RNF128, which is lower in gastrointestinal stromal tumor cells (GIST-T1) than in normal gastric epithelial cells (GES-1), was detected by real-time fluorescence quantitative PCR.

[0103] The experimental steps are as follows:

[0104] 1. Extract total RNA from cells and colon tissues

[0105] (1) Preparation before the experiment: Prepare cell / tissue RNA extraction kits (Yeasen), 1.5 mL RNase-free centrifuge tubes, absolute ethanol, high-pressure gun heads, etc. It should be noted that when using this kit for the first time, 24 mL and 52 mL of absolute ethanol should be added to the binding solution BD and the washing solution W respectively according to the instructions, mixed well before use, and marked.

[0106] (2) Pretreatment of samples (adherent cells): Aspirate the supernatants of GIST-T1 cells and GES-1 cells in the six-well plate, wash them 3 times with phosphate buffer solution, and then add 350 µL of lysis solution LB, and pipette and mix repeatedly until no cell clumps can be seen.

[0107] (3) RNA extraction:

[0108] 1) First, place the DNA removal / RNA adsorption universal column in a 2 mL collection tube, then add the processed tissue or cell homogenate to the column, and after centrifuging the column in a centrifuge, collect the filtrate containing RNA.

[0109] 2) Add an equal volume of binding solution BD to the RNA filtrate and immediately gently pipette and mix until fully mixed.

[0110] 3) Take a new DNA removal / RNA adsorption universal column, add all the above mixture to the new DNA removal / RNA adsorption universal column, centrifuge for 30 s (rotation speed 13000 rpm), and discard the filtrate.

[0111] 4) Add 700 µL of protein removal solution to the DNA removal / RNA adsorption universal column, let it stand at room temperature for 30 s, centrifuge for 30 s (rotation speed 13000 rpm), discard the filtrate, and place the DNA removal / RNA adsorption universal column back into the 2 mL collection tube.

[0112] 5) Add 500 L of washing solution W to the DNA removal / RNA adsorption universal column, centrifuge for 30 s (rotation speed 13000 rpm), and discard the filtrate. Repeat this step once, and place the DNA removal / RNA adsorption universal column back into the 2 mL collection tube.

[0113] 6) Centrifuge the empty column for 2 min (at a speed of 13,000 rpm) to remove the residual wash buffer W.

[0114] 7) Prepare new 1.5 mL RNase-free centrifuge tubes in advance and number them. Place the DNA removal / RNA adsorption universal column into the new centrifuge tube, add enzyme-free and sterile water to the center of the column (about 30 µL for cells and about 50 µL for tissues), let it stand at room temperature for 1 min, and then centrifuge for 1 min (at a speed of 13,000 rpm). The filtrate collected is the RNA solution.

[0115] 8) Use a nucleic acid concentration detector to measure the RNA concentration in each sample. If the concentration is too high, it can be diluted with enzyme-free and sterile water and stored at -80 °C for a long time.

[0116] 2. Reverse transcription of RNA to synthesize cDNA

[0117] (1) Remove residual genomic DNA: First, prepare the mixture shown in Table 2 in an RNase-free centrifuge tube, pipette and mix well, and incubate at 42 °C for 2 min.

[0118] Table 2 Reaction system for removing residual genomic DNA

[0119]

[0120] (2) Prepare the reverse transcription reaction system: As shown in Table 3, directly add 5 µL of 4×Hifair® III SuperMix plus to the reaction tube in step (1) to prepare a 20 µL system, and pipette and mix well.

[0121] Table 3 Reverse transcription reaction system

[0122]

[0123] (3) Set the reverse transcription program: Set the reverse transcription program according to Table 4.

[0124] Table 4 Reverse transcription program

[0125]

[0126] (4) The reverse transcription product cDNA can be immediately used for real-time fluorescence quantitative PCR reaction, or stored at -20 °C for a short time. If long-term storage is required, it is recommended to aliquot and store in a -80 °C refrigerator to avoid repeated freezing and thawing.

[0127] 3. cDNA amplification

[0128] (1) Dilute the cDNA stock solution: Add 80 µL of enzyme-free water to 20 µL of the cDNA stock solution to dilute it 5-fold.

[0129] (2) Design primers: All primer sequences are shown in Table 5. Before use, the primers need to be diluted to 10 μmol / µL with enzyme-free water.

[0130] Table 5 Primer Sequences

[0131]

[0132] (3) Prepare the reaction system: Prepare the real-time fluorescence quantitative PCR reaction system on an ice plate according to Table 6, add it to an 8-strip tube, and centrifuge and mix well to remove air bubbles.

[0133] Table 6 Real-time Fluorescence Quantitative PCR Reaction System

[0134]

[0135] (4) Run on the machine: Place the loaded 8-strip tube into the real-time fluorescence quantitative PCR instrument. The program is set as 95°C for 30 s (pre-denaturation), 95°C for 3 s (denaturation), 60°C for 20 s (annealing / extension). Among them, the denaturation and annealing / extension stages are repeated 40 times in total, and the melting curve is set according to the default settings of the instrument.

[0136] (5) Result analysis: After the experiment, according to the cycle threshold (CT value), use the 2 -△△Ct method to calculate the relative expression level of each gene.

[0137] The results are as Figure 15 shown. The results show that the expression level of RNF128 mRNA is lower in gastrointestinal stromal tumor cells than in normal gastric epithelial cells.

[0138] Example 6 Effects of RNF128 Gene on the Biological Behavior of Gastrointestinal Stromal Tumor Cells

[0139] 1. Select the GIST-T1 cell line as the research object, and construct a stable RNF128 overexpression cell line (HBLV-h-RNF128-3flag-Zsgreen-PURO, RNF128-Flag) and a transient RNF128 gene knockdown cell line (si-RNF128);

[0140] The steps to construct the stable RNF128 overexpression cell line are as follows:

[0141] (1) Cell preparation: Inoculate well-conditioned GIST-T1 cells into a 24-well plate so that the cell concentration is 3×10 5 / mL, 500 μL / well, and the number of cells in each well is about 1.5×10 5cells to ensure that the cell confluence rate ranges from 30% to 50% when infected with the virus the next day. Place them in an incubator at 37°C and 5% CO2 for overnight culture. (2) Perform lentivirus packaging to obtain recombinant virus. The steps for lentivirus packaging are as follows: 1) Select HEK-293T / HEK-293FT as the tool cells, PEI as the transfection reagent, and psPAX2 and pMD2.G as the helper plasmids. 2) Prepare the cell state: When the HEK-293T cells in a 10-cm culture dish reach 70%-80% confluence, start packaging the virus. Transfect with serum-containing medium. The system used for one 10-cm dish is: the mass ratio of the target plasmid (HBLV-3xflag-ZsGreen-PURO containing the RNF128 gene), psPAX2, and pMD2.G is = 9:6:3, and the amount of PEI used is 2.5 times the mass of the plasmid. 3) Replace the cell medium with fresh medium before transfection, add 15 mL of medium, and start timing from after transfection. Change the medium 12 h later. 4) Collect the virus solution: Collect the virus solution at two time periods, 36 h and 72 h. 5) Filter the virus solution: Filter the virus solution using a sterile syringe and a 0.22-μm filter head. 6) Concentrate the virus: Concentrate with polyethylene glycol 8000 (PEG8000) + 4 M sodium chloride (NaCl). For 20 mL of the original virus solution, add 5.5 mL of polyethylene glycol 8000 and 2 mL of 4 M sodium chloride, and shake on a rolling shaker at 4°C for 12 h (PEG8000 formulation: Weigh 44 g of polyethylene glycol 8000 and make up the volume to 100 mL with double-distilled water). 7) Centrifuge the virus: Place the concentrated virus solution in a 4°C centrifuge and centrifuge at 4500 rpm for 30 min, and the virus precipitate can be seen. 8) Resuspend the virus and store: Discard the supernatant and only keep the precipitate. Resuspend with 1 mL of medium, aliquot the resuspended solution into 1.5-mL plastic centrifuge tubes, and store at -80°C. Before infecting with the recombinant virus, aspirate the original cell medium, add 1 / 2 volume of fresh medium, and add an appropriate volume of the recombinant virus for infection (virus amount per well (μL) = MOI × number of cells / virus titer (TU / mL) × 1000). (3) On the second day after infection (about 24 h), aspirate the culture solution containing the recombinant virus, replace it with fresh complete culture solution, and continue culturing at 37°C. (4) 48 h after infection, for the recombinant virus carrying the GFP reporter gene, the GFP expression efficiency can be preliminarily observed through a fluorescence microscope. For the recombinant virus carrying the puromycin (Puromycin) resistance gene, replace it with fresh complete culture solution containing an appropriate concentration of puromycin to screen for stable transduced cell lines. (5) Perform Western blot experiments and real-time quantitative polymerase chain reaction (qRT-PCR) on the stable transduced cell lines obtained by screening to detect the overexpression efficiency of the RNF128 gene in these cells. At the same time, use GIST-T1 cells as a control (Vector), and the results are as Figure 16as shown in A and B in []. The results showed that: this step successfully constructed a stable cell line with overexpressed RNF128.

[0142] The steps for constructing a transient cell line with knocked-down RNF128 gene are as follows:

[0143] (1) Culture GIST-T1 cells in a T25 cell culture flask until they reach 90% density per microscope field, then start the plating work. After plating, it is expected that the cell density in the six-well plate is 40%-60%, and the number of cells is about 10 5 / well. (2) Observe the cell status the next day. If the cells are in good condition and firmly adherent, transfection can be carried out. (3) In this invention, siRNA transfection reagent (siRNA-mateplus reagent) is used for transfection. Prepare a sterile plastic centrifuge tube in advance. First, configure the siRNA system. Add 100 µL of optim and 5 µL of siRNA to each well (siRNF128-635 is shown in SEQ ID NO.5, specifically: 5’-CUGGAGCCGUCAUCUUUAATT-3; siRNF128-818 is shown in SEQ ID NO.6, specifically: 5’-GGGUGAAUCACUAUUCAAUTT-3’; siRNF128-1356 is shown in SEQ ID NO.7, specifically: 5’-GCAGUCAACAAAUGAAAGUTT-3’; siRNF128-1473 is shown in SEQ ID NO.8, specifically: 5’-GACUGCUGUUCGAGAAAUUTT-3’; si-NC is shown in SEQ ID NO.9, specifically: 5’-UUCUCCGAACGUGUCACGUTT-3’), and mix well with a pipette. Then configure the transfection reagent system. Add 100 µL of optim and 5 µL of siRNA transfection reagent to each well, and mix well. (4) Add the transfection reagent system to the siRNA system, mix well, and do not let it stand. (5) Take out the six-well plate in the incubator, aspirate the medium in it, and replace it with serum-free DMEM medium, 1 mL per well. (6) Add the prepared transfection reagent to the wells, and then gently shake the six-well plate horizontally and vertically to mix it well. This process should be gentle. (7) Place the six-well plate in the incubator. After 6-8 h, change the medium, and add 2 mL of serum-containing DMEM medium to each well. (8) After 60 h, perform protein extraction and Western blot experiment. The results are as Figure 17 shown in A and B in []. The results showed that: this step successfully constructed a transient cell line with knocked-down RNF128 gene, namely si-RNF128-653, si-RNF128-818, si-RNF128-1356, si-RNF128-1473, and si-NC.

[0144] 2. Cellular biological behaviors

[0145] Proliferation assay: The CCK-8 and colony formation assays were used to detect the proliferation ability of cells in different treatment groups; invasion and migration assays: The Transwell chamber assay (Transwell cell migration and invasion assay) and wound healing assay (The wound healing assay is a simple method to detect cell motility and can be used to detect the invasion and metastasis ability of adherent-growing tumor cells. The faster the wound heals, the faster the cells crawl, indicating a higher degree of cell malignancy) were used to detect the invasion and migration ability of tumor cells. The results are shown in Figure 16 C-I in Figure 17 and Figure 16 C-I in Figure 17 It can be seen from C-I in Figure 16 and C-I in

[0146] that knockdown of the RNF128 gene promoted the growth, migration, and invasion of GIST-T1 cells and increased the wound healing rate; overexpression of the RNF128 gene decreased the proliferation ability of GIST-T1 cells and reduced the invasion and migration rates of GIST-T1 cells. In other words, exogenous increase in RNF128 gene expression could inhibit the growth, migration, and invasion of gastrointestinal stromal tumor cells. The expression level of the RNF128 gene was negatively correlated with the malignancy of gastrointestinal stromal tumors. Increasing the expression level of the RNF128 gene would slow down the healing and migration and reduce the malignancy of gastrointestinal stromal tumors ( Figure 16 H and I in

[0146] The above-described embodiments are only descriptions of the preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a reagent for detecting the expression level of the RNF128 gene in the preparation of a detection product for the prognosis of gastrointestinal stromal tumors.

2. The application according to claim 1, wherein The detection product includes a chip or a kit.

3. A detection product for the prognosis of gastrointestinal stromal tumors, characterized in that, The detection product includes a reagent for detecting the expression level of the RNF128 gene.

4. The detection product according to claim 3, wherein The detection product includes a chip or a kit.

5. Use of the RNF128 gene as a target in the preparation of a drug for preventing and / or treating gastrointestinal stromal tumors.

6. Use of an agonist of the RNF128 gene in the preparation of a drug for preventing and / or treating gastrointestinal stromal tumors.

7. A drug for preventing and / or treating gastrointestinal stromal tumors, characterized in that, The drug includes an agonist of the RNF128 gene.

8. Use of the RNF128 gene as a target in screening for drugs for treating gastrointestinal stromal tumors.

9. A method for screening drugs for treating gastrointestinal stromal tumors, characterized in that, It includes the step of detecting the expression level of the RNF128 gene in a subject before and after drug administration.

Images