Application of rnf128 gene in prognosis of gastrointestinal stromal tumor

Abstract

The present application relates to the field of biological medicine, in particular to the application of RNF128 gene in the prognosis of gastrointestinal stromal tumor (GIST). The present application provides the application of a reagent for detecting the expression amount of RNF128 gene in the preparation of a detection product for the prognosis of GIST. In the present application, the RNF128 gene is highly expressed in low-risk GIST patients and lowly expressed in high-risk GIST patients. The present application takes RNF128 gene as the core, comprehensively covers the key links of GIST treatment and transformation research, and has innovation, science and practicability. The advantages not only lie in the basic research value, but also provide an operable solution for clinical practice, which will significantly improve the diagnosis and treatment effect and the life quality of GIST patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the application of RNF128 gene in the prognosis of gastrointestinal stromal tumor. BACKGROUND

[0002] Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract, and its pathological origin is mainly Cajal interstitial cells or their precursor cells. The pathogenesis of GIST is closely related to various cell molecular abnormalities, and it is a tumor type characterized by molecular marker-driven. In some GIST patients, mutations in the KIT gene are detected. The tyrosine kinase receptor encoded by the KIT gene is in a continuous activation state after mutation, and can autonomously signal without the stimulation of external ligands, thereby driving the proliferation and survival of tumor cells. In addition, some GIST patients have platelet-derived factor receptor A (PDGFRA) gene mutations. Similar to KIT mutations, PDGFRA mutations also cause abnormal activation of tyrosine kinase 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 for localized GIST, especially in the case of no extensive metastasis, complete resection of the lesion is the key to long-term survival. However, for patients with distant metastasis or recurrence of tumors, surgery alone is difficult to achieve the goal of cure. Tyrosine kinase inhibitors (TKIs) have a milestone significance in the treatment of GIST. Imatinib, as the first generation of TKIs, is a standard treatment drug for KIT and PDGFRA mutations. It blocks the downstream signaling of mutant KIT or PDGFRA by competitively inhibiting tyrosine kinase activity, 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 kinase caused by secondary mutations, making it difficult for drugs to effectively bind to the target. At the same time, the development of drug resistance greatly limits the effect of long-term treatment. 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 development of drug resistance, and the overall efficacy is limited, especially in advanced or metastatic GIST patients, the treatment effect is not satisfactory. Metastatic GIST is currently a difficult point in clinical treatment. Therefore, the existing targeted drugs have poor mechanism for 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 an important research direction. GIST has significant heterogeneity in molecular and pathological characteristics. Different patients or different lesions of the same patient may exhibit different mutation types or molecular characteristics. For example, some patients with GIST have no KIT or PDGFRA mutations, but are accompanied by SDH deletion or BRAF mutation. This heterogeneity makes it difficult to achieve individualization of existing treatments. For patients without mutations, the effect of targeted therapy is more limited. In order to overcome the limitations of existing treatments, further research on the molecular mechanisms of GIST, discovery of new targets, and development of individualized 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 drug resistance mechanism and prevent distant metastasis is the key to improve the prognosis and quality of life of GIST patients. SUMMARY

[0005] The purpose of the present application is to provide the application of RNF128 gene in the prognosis of gastrointestinal stromal tumor, so as to solve the problems existing in the prior art. The RNF128 gene in the present application is helpful for effective diagnosis of early gastrointestinal stromal tumor patients. Therefore, the reagent for detecting the expression amount of RNF128 gene can be used for detecting the prognosis of gastrointestinal stromal tumor.

[0006] To achieve the above purpose, the present application provides the following scheme:

[0007] The present application provides the application of the reagent for detecting the expression amount of RNF128 gene in the preparation of the detection product for the prognosis of gastrointestinal stromal tumor.

[0008] In the present application, the RNF128 gene is highly expressed in low-risk patients with gastrointestinal stromal tumor and is lowly expressed in high-risk patients with gastrointestinal stromal tumor. Therefore, the RNF128 gene in the present application can be used for risk assessment of the prognosis of gastrointestinal stromal tumor.

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

[0010] The present application provides a detection product for the prognosis of gastrointestinal stromal tumor, which comprises a reagent for detecting the expression amount of RNF128 gene.

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

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

[0013] The application provides application of an agonist of an RNF128 gene in preparation of a drug for preventing and / or treating gastrointestinal stromal tumor.

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

[0015] The application provides application of RNF128 gene as an action target in screening of a drug for treating gastrointestinal stromal tumor.

[0016] The application provides a method for screening a drug for treating gastrointestinal stromal tumor, and the method comprises the step of detecting the expression amount of RNF128 gene of a subject before and after administration.

[0017] The application discloses the following technical effects:

[0018] 1. The application discloses a key molecular mechanism of RNF128 gene, which provides a new perspective for basic research of GIST.

[0019] The application first systematically clarifies the low expression of RNF128 gene in GIST and the molecular mechanism, and discloses that the RNF128 gene affects the invasion and metastasis ability of tumor cells by regulating the ubiquitination degradation pathway. By determining the function of the RNF128 gene, the application fills the gap in the current research on the molecular mechanism of GIST, and provides a scientific basis for subsequent exploration of a new target.

[0020] 2. The application provides a potential precise prognosis method for GIST patients.

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

[0022] 3. The application provides a new treatment idea for patients with drug resistance.

[0023] The existing tyrosine kinase inhibitor (TKI) treatment has a significant drug resistance problem. The application proposes a new treatment strategy taking the RNF128 gene as a target by studying the regulation of the RNF128 gene in a signal pathway.

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

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

[0026] In summary, the present application takes RNF128 gene as the core, and comprehensively covers the key link of GIST treatment and transformation research, which has innovation, science and practicability. Its advantages not only lie in the basic research value of GIST, but also provide an operable solution for clinical practice, which will significantly improve the diagnosis and treatment effect and life quality of GIST patients. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Figure 1 is a principal component analysis result graph of GSE225819 data set; wherein, normal is a normal sample, and stromal tumor is a gastrointestinal stromal tumor patient sample with liver metastasis;

[0028] Figure 2 Figure 2 is a sample clustering tree diagram of GSE225819 data set; wherein, normal is a normal sample, and stromal tumor is a gastrointestinal stromal tumor patient sample with liver metastasis;

[0029] Figure 3 Figure 3 is a sample independence graph in determining the best soft threshold;

[0030] Figure 4 Figure 4 is a mean connectivity graph in determining the best soft threshold;

[0031] Figure 5 Figure 5 is a co-expression network graph;

[0032] Figure 6 Figure 6 is a sample trait heat map;

[0033] Figure 7 Figure 7 is a phenotype and gene correlation heat map (A, also referred to as module-trait correlation heat map) of GSE225819 data set, correlation heat map between modules (B), correlation analysis graph of blue module and gastrointestinal stromal tumor (C), and correlation analysis graph of cyan module and gastrointestinal stromal tumor (D);

[0034] Figure 8 Figure 8 is a Wayne graph of differential genes and blue module genes of GSE225819 data set;

[0035] Figure 9 Figure 9 is a LASSO regression coefficient graph;

[0036] Figure 10 LASSO regression parameter plot;

[0037] Figure 11 Differential gene volcano plot;

[0038] Figure 12 RNF128 gene box plot; wherein, normal is a normal sample, and stromal tumor is a gastrointestinal stromal tumor sample with liver metastasis;

[0039] Figure 13 RNF128 protein expression in gastrointestinal stromal tumor high-risk and low-risk patients statistical chart; wherein, A is an immunohistochemical detection of RNF128 protein expression chart; and B is an immunohistochemical score statistical chart;

[0040] Figure 14 Western blotting experiment for detecting RNF128 gene expression in gastrointestinal stromal tumor cells and normal gastric epithelial cells statistical result chart; wherein, GIST-T1 is a gastrointestinal stromal tumor cell, and GES-1 is a normal gastric epithelial cell;

[0041] Figure 15 Real-time fluorescent quantitative PCR for detecting RNF128 mRNA level statistical result chart; wherein, GIST-T1 is a gastrointestinal stromal tumor cell, and GES-1 is a normal gastric epithelial cell;

[0042] Figure 16Statistical result graph of RNF128 overexpression stable cell line; wherein, A is the statistical graph of RNF128 protein expression efficiency in RNF128 overexpression stable cell line checked by Western blotting experiment, Vector is GIST-T1 cell, RNF128-Flag is RNF128 overexpression stable cell line; B is the statistical graph of RNF128 gene expression efficiency in RNF128 overexpression stable cell line checked by qRT-PCR, Vector is GIST-T1 cell, RNF128-Flag is RNF128 overexpression stable cell line; C is the statistical graph of the influence of overexpression of RNF128 gene on cell proliferation determined by CCK-8 experiment, Vector is GIST-T1 cell, RNF128-Flag is RNF128 overexpression stable cell line; D and F are the statistical graphs of the influence of overexpression of RNF128 gene on migration and invasion evaluated by Transwell migration and invasion experiment, Vector is GIST-T1 cell, RNF128-Flag is RNF128 overexpression stable cell line; E and G are the statistical graphs of the influence of increasing RNF128 gene expression of gastric stromal tumor on cell invasion and migration determined by wound healing experiment, Vector is GIST-T1 cell, RNF128-Flag is RNF128 overexpression stable cell line; H is the wound healing condition graph, Vector is GIST-T1 cell, RNF128-Flag is RNF128 overexpression stable cell line; I is the statistical graph of wound healing rate, Vector is GIST-T1 cell, RNF128-Flag is RNF128 overexpression stable cell line;

[0043] Figure 17Statistical results of the knockdown of the RNF128 gene in the transient cell line; wherein, A is a statistical graph of the knockdown efficiency of the RNF128 gene in the transient cell line in which the RNF128 gene is knocked down by Western blotting; B is a result graph of the knockdown efficiency of the RNF128 gene in the transient cell line in which the RNF128 gene is knocked down by real-time quantitative polymerase chain reaction; C is a statistical graph of the effect of the knockdown of the RNF128 gene on cell proliferation determined by CCK-8 experiment, Si-RNF128-1 is siRNF128-818, and Si-RNF128-2 is siRNF128-1356; D and F are statistical graphs of the effect of the knockdown of the RNF128 gene on cell invasion and migration determined by Transwell migration and invasion experiment, Si-RNF128-1 is siRNF128-818, and Si-RNF128-2 is siRNF128-1356; E and G are statistical graphs of the effect of the knockdown of the RNF128 gene on cell invasion and migration determined by wound healing experiment, Si-RNF128-1 is siRNF128-818, and Si-RNF128-2 is siRNF128-1356; H is a wound healing condition graph, Si-RNF128-1 is siRNF128-818, and Si-RNF128-2 is siRNF128-1356; I is a statistical graph of the wound healing rate, Si-RNF128-1 is siRNF128-818, and Si-RNF128-2 is siRNF128-1356;

[0044] Figure 18 Survival analysis results; wherein, high expression of RNF128 is a high expression RNF128 gene group, low expression of RNF128 is a low expression RNF128 gene group, the abscissa is survival time, in days, and the ordinate is survival probability. DETAILED DESCRIPTION

[0045] Example 1 GEO database analysis of differential expression of RNF128 gene in gastrointestinal stromal tumor

[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, and the GSE225819 data set is selected, including 20 samples of gastrointestinal stromal tumor (GIST) patients with liver metastasis and 20 normal samples (NORMAL), principal component analysis is performed to check whether the sample difference is obvious, and the result is as follows: Figure 1The results show that the principal components of gastrointestinal stromal tumor samples with liver metastasis and normal samples are significantly different. Using the GSE225819 dataset in the GEO database, first perform sample clustering analysis, then determine the optimal soft threshold, and construct the co-expression network ( Figures 2-6 ). Draw a phenotype and gene correlation heatmap to find the most relevant module genes to gastrointestinal stromal tumor with liver metastasis ( Figure 7 ). After taking the intersection of the differential genes and the module genes of the GSE225819 dataset, perform LASSO regression analysis on the obtained genes, and finally screen the interested genes. The result of the volcano plot shows that the RNF128 gene is most different from the gastrointestinal stromal tumor with liver metastasis phenotype, and the RNF128 gene is finally selected as the target gene for studying the distant metastasis mechanism of gastrointestinal stromal tumor ( Figures 8-12 ).

[0048] Example 2: Detection of RNF128 gene expression in gastrointestinal stromal tumor (stromal tumor)

[0049] (I) Immunohistochemical detection of RNF128 protein expression in gastrointestinal stromal tumor patient lesion tissue

[0050] 1. Collect patient clinical data

[0051] The surgical specimens of 30 gastrointestinal stromal tumor patients were collected, and the clinical pathological data (including tumor size, nuclear division number, risk classification, metastasis status, etc.) of the patients were obtained. The clinical pathological data is shown in Table 1.

[0052] Table 1. Clinical pathological data

[0053]

[0054] 2. Immunohistochemical method

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

[0056] Place the selected tissue sections in an incubator (70°C) and bake for 1-2 hours.

[0057] Place the tissue sections in xylene x 2, 100% alcohol, 95% alcohol, 85% alcohol in sequence, and soak for 10 min, 10 min, 5 min, 5 min, and 5 min, respectively.

[0058] Take out the tissue sections and rinse with running water for 15 min. Be careful not to wash the tissue down.

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

[0060] Submerge the tissue sections in the citrate solution and put them in the microwave oven, microwave for 15 min at high-medium fire. After the antigen retrieval is completed, let them cool naturally at room temperature.

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

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

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

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

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

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

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

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

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

[0070] Rinse the tissue sections with running water for 20 min, without washing the tissue.

[0071] Stain with hematoxylin for 8 s.

[0072] Rinse with running water for 20 min, without washing the tissue.

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

[0074] 3. Immunohistochemical scoring method

[0075] Observe 10 fields of view on each tissue section, 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 method was as follows: stained cells were scored from 0 to 4 points based on the proportion of cells; among them, a proportion of 0% was scored as 0 points; a proportion of 1%-25% was scored as 1 point; a proportion of 26%-50% was scored as 2 points; a proportion of 51%-75% was scored as 3 points; and a proportion of 75%-100% was scored as 4 points.

[0077] Staining was scored on a scale of 0 to 3 depending on the intensity; no staining was scored as 0, pale yellow staining was scored as 1, brownish yellow staining was scored as 2, and tan staining was scored as 3.

[0078] The total score = the proportion score multiplied by the staining intensity score. Finally, the total scores are arranged from high to low, and the median is used as the boundary to divide the total scores into low-risk group (low risk) and high-risk group (high risk).

[0079] 4. Results Analysis

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

[0081] Example 3 Survival Analysis

[0082] After immunohistochemistry testing, 30 patients with gastrointestinal stromal tumors were divided into high-expression RNF128 gene groups (15 cases) and low-expression RNF128 gene groups (15 cases) based on the expression level of the RNF128 gene, using the median as the boundary. Among them, the high-expression RNF128 gene group was consistent with the gastrointestinal stromal tumor patients with low-risk and very low-risk risk in Table 1, and the low-expression RNF128 gene group was consistent with the gastrointestinal stromal tumor patients with high-risk risk in Table 1. Subsequently, survival analysis was performed, and the results were as follows: Figure 18 The results showed that the low-expression RNF128 gene group had a worse prognosis and shorter survival time than the high-expression RNF128 gene group.

[0083] Example 4 Western blotting assay to detect RNF128 gene expression in gastrointestinal stromal tumor cells (GIST-T1 cells) and normal gastric epithelial cells (GES-1 cells)

[0084] 1. Cell culture;

[0085] 2. Cell protein extraction step;

[0086] 3. Western blotting experimental steps:

[0087] Western blot: The protein of cells or tissues was extracted, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then detected by anti-RNF128 antibody.

[0088] The specific experimental steps are as follows:

[0089] GIST-T1 cells and GES-1 cells were taken out of the cell incubator using a 6-well plate.

[0090] The supernatant in the hole was sucked out with a Pasteur tube, and phosphate buffered saline solution was added for rinsing. If the cell adhesion is not firm, this process should be careful or directly skipped. Rinse 2-3 times, add 1-2 mL of cell protein lysate to each hole, scrape the cells in the six-well plate with a cell scraper, and transfer the cell suspension to a plastic centrifuge tube (EP tube). This process should be operated on ice.

[0091] Place the plastic centrifuge tube containing the cell suspension in the 4°C refrigerator for lysis for 30 min. After lysis, place the plastic centrifuge tube in the pre-cooled 4°C ultracentrifuge, centrifuge at 12000 rpm for 10 min, then transfer the supernatant to a new plastic centrifuge tube, and 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, and after 5-7 min, take it out and store it in -20°C.

[0092] Determine the concentration of the gel according to the molecular weight of the target protein (high concentration gel for small molecular weight proteins, and low concentration gel for large molecular weight proteins). According to the molecular weight of the target protein such as RNF128 protein (40-70 KDa), configure 20 mL of 10% separation gel, and the configuration 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 sulfate (AP) + 7.6 mL of Tris-HCl buffer (pH 8.8). Mix the prepared separation gel with a 5 mL pipette, pour it into a special glass plate, and check whether the glass clamp plate leaks in advance. Then gently pour anhydrous ethanol into the glass plate with a pipette to seal the liquid. This process can make the gel plane more flat. Place at room temperature for 30 min, and a clear dividing line can be seen under the anhydrous ethanol, indicating that the separation gel has completely solidified. Pour out the upper anhydrous ethanol, and let it stand at room temperature for 3-5 min. After the anhydrous ethanol is completely volatilized, proceed to the next step.

[0093] After the separation gel is prepared, you can begin preparing a 5% stacking gel. The preparation is as follows: 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, and 750 µL of Tris-HCl buffer (pH 6.8). After preparation, use a 5 mL pipette to mix the liquid thoroughly and pour it into the glass plate. Then, quickly insert a dedicated 10- or 15-well comb into the stacking gel, avoiding the formation of bubbles. Let it stand at room temperature for 15 minutes and observe whether the gel is completely solidified.

[0094] Prepare the protein sample and vortex to mix in advance. Add the prepared electrophoresis buffer to the gel. Add 10µL of protein sample to each well (equal volume addition) and 1-2µL of protein marker. Run the gel at 80V for 30min, then adjust to 120V for 1h.

[0095] After electrophoresis, the cells were electrotransferred to a nitrocellulose membrane (NC membrane) at a current of 300 mA for 100 min.

[0096] Prepare skim milk blocking solution in advance, rinse the electro-transferred nitrocellulose membrane one or two times in TBST buffer, then soak it in skim milk blocking solution, shake it slowly at room temperature for 90 minutes, and then wash it twice with TBST buffer under fast shaking conditions, each time for 5 minutes.

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

[0098] The next day, the strips were removed and washed three times with TBST buffer for 5 minutes each time. The strips were then placed in the corresponding rabbit / mouse secondary antibody (rabbit / mouse secondary antibody was diluted at a volume ratio of 1:3000), incubated at room temperature for 1 hour, and then washed three times with TBST buffer for 5 minutes each time.

[0099] Finally, the strips were exposed using a chemiluminescence instrument.

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

[0101] Example 5 Detection of RNF128 gene expression by real-time fluorescence quantitative PCR (Q-PCR)

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

[0103] The experimental steps are as follows:

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

[0105] (1) Preparation before experiment: Prepare cell / tissue RNA extraction kit (Yixing), 1.5 mL RNase-free centrifuge tube, anhydrous ethanol, high-pressure gun head and other items. It is worth noting that before using the kit for the first time, 24 mL and 52 mL of anhydrous ethanol should be added to the binding liquid BD and the rinsing liquid W respectively according to the instructions, mix well after use, and make a mark.

[0106] (2) Sample (adherent cell) pretreatment: Remove the supernatant of GIST-T1 cells and GES-1 cells in the six-well plate, and wash with phosphate buffered saline for 3 times, then add 350 µL of lysis buffer LB, and mix well with a pipette until no cell clumps are visible.

[0107] (3) RNA extraction:

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

[0109] 2) Add an equal volume of binding liquid BD to the RNA filtrate, and immediately mix well by blowing gently.

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

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

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

[0113] 6) Centrifuge at 13000 rpm for 2 min to remove residual rinse W.

[0114] 7) Prepare new 1.5 mL RNase-free centrifuge tubes in advance and number them. Place the DNA clean-up / RNA adsorption universal column into the new centrifuge tube, add 30 μL of enzyme-free sterile water (about 30 μL for cells and about 50 μL for tissues) into the center of the column, stand at room temperature for 1 min, then centrifuge at 13000 rpm for 1 min, collect the obtained filtrate as the RNA solution.

[0115] 8) Measure the RNA concentration in each sample using a nucleic acid concentration detector. If the concentration is too high, dilute it with enzyme-free sterile water, and store it at -80°C for long-term preservation.

[0116] 2. Synthesis of cDNA by RNA reverse transcription

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

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

[0119]

[0120] (2) Reverse transcription reaction system preparation: As shown in Table 3, directly add 5 μL of 4×Hifair® Ⅲ SuperMix plus into the reaction tube of step (1), prepare a 20 μL system, and mix by blowing.

[0121] Table 3 Reverse transcription reaction system

[0122]

[0123] (3) Reverse transcription program setting: The reverse transcription program is set according to Table 4.

[0124] Table 4 Reverse transcription program

[0125]

[0126] (4) The product cDNA of reverse transcription can be used immediately for real-time fluorescent quantitative PCR reaction, or can be stored at -20°C for short-term preservation. If long-term preservation is required, it is recommended to store it in a -80°C refrigerator after aliquoting 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 cDNA stock solution to dilute it 5 times.

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

[0130] Table 5 Primer sequences

[0131]

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

[0133] Table 6 Real-time fluorescent quantitative PCR reaction system

[0134]

[0135] (4) Machine: place the 8-tube tube with the added sample into the real-time fluorescent quantitative PCR instrument, and set the program to 95°C for 30s (pre-denaturation), 95°C for 3s (denaturation), and 60°C for 20s (annealing / extension). Among them, the denaturation and annealing / extension stages are repeated for 40 cycles, and the melting curve is set according to the instrument default.

[0136] (5) Result analysis: after the experiment, according to the cycle threshold (CT value), the relative expression amount of each gene was calculated by using 2 -△△Ct method.

[0137] The results are shown in Table 6. The results show that the expression of RNF128 mRNA in gastrointestinal stromal tumor cells is lower than that in normal gastric epithelial cells. Figure 15

[0138] Example 6 Influence of RNF128 gene on biological behavior of gastrointestinal stromal tumor cells

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

[0140] The steps for constructing the RNF128 overexpression stable cell line are as follows:

[0141] (1) Cell preparation: inoculate GIST-T1 cells in good condition into a 24-well plate to make the cell concentration 3×10 5 / mL, 500 μL / well, and the number of cells in each well is about 1.5×10 5 ​One, to ensure that the cell confluence rate is between 30-50% when infected with virus the next day. Place in a 37°C, 5% CO2 incubator overnight. (2) Perform lentivirus packaging to obtain recombinant virus. The steps for lentivirus packaging are as follows: 1) Select tool cells: HEK-293T / HEK-293FT, and the transfection reagent is PEI, and the helper plasmid is psPAX2 and pMD2.G. 2) Cell state preparation: when the HEK-293T cells in a 10 cm culture dish reach 70-80% confluence, begin packaging the virus. Transfect with serum-containing medium, and 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) Before transfection, replace the medium with fresh medium, add 15 mL of medium, and start timing from after transfection. Replace the medium after 12 h. 4) Virus liquid collection: collect virus liquid at two time periods of 36 h and 72 h. 5) Filter the virus liquid: filter the virus liquid using a sterile syringe and a 0.22 μm filter head. 6) Virus concentration: PEG8000 + 4M NaCl concentration, 20 mL of virus stock solution + 5.5 mL of PEG8000 and 2 mL of 4M NaCl, roll on a shaker at 4°C for 12 h (PEG8000 formula: weigh 44 g of PEG8000, and make up to 100 mL with double distilled water). 7) Virus centrifugation: centrifuge the concentrated virus liquid at 4500 rpm for 30 min at 4°C, and the virus precipitate can be seen. 8) Resuspend the virus and store it: discard the supernatant and only keep the precipitate. Resuspend with 1 mL of medium, and divide the resuspension into 1.5 mL plastic centrifuge tubes, and store at -80°C. Before infection with the recombinant virus, remove the original medium from the cells, add 1 / 2 volume of fresh medium, and add the appropriate volume of recombinant virus for infection (add virus volume (μL) per well = MOI x cell number / virus titer (TU / mL) x 1000). (3) The next day after infection (about 24 h), remove the culture medium containing the recombinant virus, replace it with fresh complete culture medium, and continue to culture at 37°C. (4) 48 h after infection, for recombinant viruses carrying the GFP reporter gene, the GFP expression efficiency can be preliminarily observed by fluorescence microscopy. For recombinant viruses carrying the puromycin resistance gene, replace it with fresh complete culture medium containing an appropriate concentration of puromycin, and screen for stably transduced cell lines. (5) Perform Western blotting and real-time quantitative polymerase chain reaction (qRT-PCR) detection on the stably transduced cell lines screened to detect the overexpression efficiency of the RNF128 gene in the cells, and use GIST-T1 cells as a control (Vector). The results are as follows Figure 16A and B in FIG. 1. The results show that the RNF128 overexpression stable cell line is successfully constructed in this step.

[0142] The steps for constructing the transient cell line for knocking down the RNF128 gene are as follows:

[0143] (1) The GIST-T1 cells in the T25 cell culture bottle are cultured to a density of 90% per field, and then the plating work is started. It is expected that the cell density in the six-well plate is 40%-60% after plating, and the cell number is about 10 5 / well. (2) The next day, the cell state is observed. If the cell state is good and the adhesion is firm, the transfection can be carried out. (3) The siRNA transfection reagent (siRNA-mate plus reagent) is used for transfection in the present application. The sterile plastic centrifuge tube is prepared in advance. First, the siRNA system is configured. 100 µL of optim and 5 µL of siRNA (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') are added to each well, and mixed uniformly with a pipette gun. Then, the transfection reagent system is configured. 100 µL of optim and 5 µL of siRNA transfection reagent are added to each well, and mixed uniformly. (4) The transfection reagent system is added to the siRNA system, and mixed uniformly without resting. (5) The six-well plate in the incubator is taken out, and the culture medium therein is aspirated and replaced with 1 mL of serum-free DMEM medium per well. (6) The prepared transfection reagent is added to the well, and then the six-well plate is shaken horizontally left and right, up and down to mix it thoroughly. This process should be gentle. (7) The six-well plate is placed in the incubator, and 2 mL of serum-containing DMEM medium is added to each well after 6-8 h. (8) The protein extraction and Western blotting experiment are carried out after 60 h. The results are shown in FIG. 1A and FIG. 1B. Figure 17 A and B in FIG. 1. The results show that the RNF128 overexpression stable cell line is successfully constructed in this step.

[0144] 2. Cell biological behavior

[0145] Proliferation experiment: CCK-8 and colony formation experiment were used to detect the proliferation ability of cells in different treatment groups; invasion and migration experiment: Transwell chamber experiment (Transwell cell migration and invasion experiment) and wound healing experiment (wound healing experiment is a simple method for detecting cell movement, which can be used to detect the invasion and metastasis ability of adherent tumor cells, the faster the healing, the faster the cell crawling, representing the higher degree of cell malignancy) were used to detect the invasion and migration ability of tumor cells, and the results are shown in C-I of Figure 16 and C-I of Figure 17 . As shown in C-I of Figure 16 and C-I of Figure 17 , RNF128 gene knockdown promotes the growth, migration and invasion of GIST-T1 cells, and increases the wound healing rate; overexpression of RNF128 gene can reduce the proliferation ability of GIST-T1 cells, and reduce the invasion and migration rate of GIST-T1 cells. In other words, exogenous increase of RNF128 gene expression can inhibit the growth, migration and invasion of gastrointestinal stromal tumor cells, the expression amount of RNF128 gene and the malignant degree of gastrointestinal stromal tumor are negatively correlated, and increasing the expression amount of RNF128 gene will make the healing migration slower, and reduce the malignant degree of gastrointestinal stromal tumor (H and I of Figure 16 ).

[0146] The above-described embodiments are only to describe the preferred modes of the present application, and do not limit the scope of the present application, and various modifications and improvements to the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope determined by the claims of the present application.

Claims

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

2. The use according to claim 1, characterized in that The detection product includes a chip or a kit.

3. Application of RNF128 gene in the preparation of drugs for preventing and / or treating gastrointestinal stromal tumors.

4. The use of RNF128 gene as a target in screening drugs for treating gastrointestinal stromal tumors, characterized in that: The drug for treating gastrointestinal stromal tumors can increase the expression level of the RNF128 gene.

5. A method for screening drugs for treating gastrointestinal stromal tumors, characterized in that: The method comprises the steps of detecting the expression level of the RNF128 gene of the subject before and after administration; the drug for treating gastrointestinal stromal tumors can increase the expression level of the RNF128 gene.

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