SiRNA of targeted AK4 gene and application of siRNA

Abstract

The invention discloses siRNA of a targeted AK4 gene and application of the siRNA, and belongs to the technical field of molecular biotechnology and genetic engineering. The invention discloses siRNA of a targeted AK4 gene. The sequences of the siRNA are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and / or SEQ ID NO.3, SEQ ID NO.4 and / or SEQ ID NO.5 and SEQ ID NO.6. The siRNA of the targeted AK4 gene provided by the invention can effectively inhibit cell proliferation and cloning, inhibit cell migration, promote cell apoptosis and promote improvement of cell sensitivity. The result shows that siRNA of the targeted AK4 gene can be used for preparing a cancer cell metastasis inhibitor and / or a medicine for treating cancers and a medicine for promoting improvement of cell sensitivity, promoting cell apoptosis, inhibiting cell migration and inhibiting cell proliferation and cloning. The invention provides a new technical means for developing new targets for tumor treatment, and has very important application value and prospect in clinical treatment of tumors and development of targeted drugs.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biotechnology and genetic engineering technology. More specifically, it relates to an siRNA targeting the AK4 gene (RefSeq ID: NM_013410) and its application. Background Art

[0002] Gastric cancer (GC) is one of the most common malignant tumors worldwide. Currently, chemotherapy remains the main treatment method for gastric cancer. The first-line drugs for gastric cancer chemotherapy are mainly platinum combined with paclitaxel and 5-fluorouracil. However, most patients have poor or no response to chemotherapy. The reason for the failure of more than 90% of advanced gastric cancer treatments is chemotherapy resistance. For preoperative chemotherapy, palliative treatment, advanced patients, and metastatic gastric cancer patients, cisplatin is the preferred drug for systemic treatment. However, with the long-term application of cisplatin, its inhibitory ability against tumors gradually weakens. Among them, hypoxia plays an important role in tumor drug resistance. Hypoxia can lead to tumor cell drug resistance by changing drug metabolism, promoting the formation of tumor stem cells, regulating signal pathways, and affecting the immune environment, but its specific role in gastric cancer is still unclear.

[0003] AK4 is a member of the adenylate kinase family, located in the mitochondrial matrix, and has been shown to physically bind to the mitochondrial ADP / ATP translocase. As a stress response protein, it maintains cell viability. Existing studies have reported that overexpression of AK4 can stabilize the HIF-1α protein by increasing the intracellular ROS level and induce metastasis in non-small cell gastric cancer. AK4 can promote the proliferation of bladder cancer cells and affect the expression of Ki67; downregulation of AK4 expression can inhibit the invasion of bladder cancer cells and significantly reduce the expression of MMP2 and MMP9. miR-3666 can directly target and downregulate STAT3 in ovarian cancer cells, and the transcriptional regulation of STAT3 downregulates AK4, thereby inhibiting the progression of ovarian cancer. AK4 is highly expressed in HER2-positive breast cancer tissues and promotes the proliferation of HER2-positive breast cancer cells. AK4 can also promote tamoxifen resistance in MCF-7 cells by inhibiting mitochondrial apoptosis, increasing ROS production, and enhancing p38 activation. In digestive tract tumors, SNHG11 can promote the migration and invasion of colorectal cancer cells by inducing the expression of the AK4 gene, a downstream target of HIF-1α. miR199a-3p is involved in the radioresistance of esophageal cancer, and AK4 is the target of miR199a-3p, which is positively correlated with the radioresistance of esophageal cancer. miR-199a-3p may promote multidrug resistance in osteosarcoma by upregulating its target gene AK4. LINC00662 promotes the stability of AK4 by binding to the HNRNPC protein, thereby playing its role in radiotherapy resistance in oral squamous carcinoma cells. However, the current expression status and specific function of AK4 in gastric cancer are still unclear. Summary of the Invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide siRNAs targeting the AK4 gene. Another technical problem to be solved by the present invention is to provide the application of siRNAs targeting the AK4 gene in the preparation of drugs for treating cancer.

[0005] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] An siRNA targeting the AK4 gene, the sequence of which is as follows:

[0007] ①AK4-siRNA-110526-1-F:

[0008] 5’-CcgggcCAGTCATTGAATTATACAACTCGAGTTGTATAATTCAATGACTGGCTTTTTg-3’,

[0009] AK4-siRNA-110526-1-R:

[0010] 5’-aattcaaaaagcCAGTCATTGAATTATACAACTCGAGTTGTATAATTCAATGACTGGC-3’;

[0011] and / or ②AK4-siRNA-110527-1-F:

[0012] 5’-CcggTGGCAAAGCAGTATATAGAGACTCGAGTCTCTATATACTGCTTTGCCATTTTTg-3’,

[0013] AK4-siRNA-110527-1-R:

[0014] 5’-aattcaaaaaTGGCAAAGCAGTATATAGAGACTCGAGTCTCTATATACTGCTTTGCCA-3’;

[0015] and / or ③AK4-siRNA-110528-1-F:

[0016] 5’-CcggACCCTCCTAGCGGAAGGGTATCTCGAGATACCCTTCCGCTAGGAGGGTTTTTTg-3’,

[0017] AK4-siRNA-110528-1-R:

[0018] 5’-aattcaaaaaACCCTCCTAGCGGAAGGGTATCTCGAGATACCCTTCCGCTAGGAGGGT-3’.

[0019] The target sequences corresponding to the siRNA targeting the AK4 gene are as follows:

[0020] ① AK4-siRNA-110526-1: 5’-gcCAGTCATTGAATTATACAA-3’;

[0021] and / or ② AK4-siRNA-110527-1: 5’-TGGCAAAGCAGTATATAGAGA-3’;

[0022] and / or ③ AK4-siRNA-110528-1: 5’-ACCCTCCTAGCGGAAGGGTAT-3’.

[0023] Use of the siRNA targeting the AK4 gene in inhibiting the expression of the AK4 gene.

[0024] Use of the siRNA targeting the AK4 gene in the preparation of a drug for inhibiting cell proliferation and cloning.

[0025] Use of the siRNA targeting the AK4 gene in the preparation of a drug for inhibiting cell migration.

[0026] Use of the siRNA targeting the AK4 gene in the preparation of a drug for promoting cell apoptosis.

[0027] Use of the siRNA targeting the AK4 gene in the preparation of a drug for promoting the sensitivity of cells to cisplatin.

[0028] Use of the siRNA targeting the AK4 gene in the preparation of a drug for treating cancer.

[0029] The biomarker for the diagnosis, treatment or prognosis judgment of gastric cancer is the AK4 gene.

[0030] The kit for the diagnosis, treatment or prognosis judgment of gastric cancer contains the AK4 gene sequence.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] 1) Based on the clinical treatment difficulty that the drug sensitivity of gastric cancer is prone to decline after cisplatin treatment, the present application first discloses an anti-gastric cancer gene therapy approach and invents a small interfering RNA fragment of AK4, which can be used in the preparation of drugs for anti-proliferation of gastric cancer and improving the drug sensitivity of cisplatin.

[0033] 2) Compared with the control group, after knocking down AK4 in HGC cells, the proliferation and colony formation ability of gastric cancer cells were significantly decreased, the migration ability of gastric cancer cells was reduced, apoptosis of gastric cancer cells was promoted, and knocking down AK4 increased the sensitivity of gastric cancer cells to cisplatin. It is shown that the small interfering RNA disclosed for the first time in this application has the important functions of inhibiting the protein expression of AK4 in gastric cancer cells, inhibiting the proliferation and migration and invasion of cancer cells, and increasing the sensitivity of gastric cancer to cisplatin drugs. Therefore, the present invention provides a new technical means for developing new targets for tumor treatment, and the siRNA of AK4 provided has very important application value and prospects for clinical treatment of tumors and development of targeted drugs. Description of the Drawings

[0034] Figure 1 It is a graph for analyzing the expression and prognosis of AK4 in a gastric cancer tissue microarray (A is a representative picture of low expression of AK4 in gastric cancer tissue (magnification, ×40 and ×200); B is a representative picture of high expression of AK4 in gastric cancer tissue (magnification, ×40 and ×200); C is the expression of AK4 detected by immunohistochemistry in adjacent tissues of cancer (magnification, ×40 and ×200); D is the ROC curve and AUC value of AK4 expression in 248 gastric cancer patients);

[0035] Figure 2 It is a graph for the relationship between AK4 expression and the survival and prognosis of gastric cancer patients (A is the relationship between the expression of AK4 in the TCGA database and the overall survival (OS), disease-specific survival (DSS) and disease-free survival (DFS) of patients; B is the analysis of AK4 and overall survival in 248 gastric cancer patients; C is the combined prognosis analysis of the expression level of AK4 and clinical indicators);

[0036] Figure 3 It is a graph for detecting the expression level of AK4 in gastric cancer cells by Western blot;

[0037] Figure 4 It is a graph for AK4 promoting the proliferation of gastric cancer cells (A is the verification of the knockdown efficiency of AK4; B is the verification of the overexpression efficiency of AK4; C is the CCK-8 cell proliferation experiment to detect the change of cell proliferation ability after knocking down the expression level of AK4 in HGC cells; D is the CCK-8 cell proliferation experiment to detect the change of cell proliferation ability after overexpressing the expression level of AK4 in AGS cells; E is the colony formation experiment to detect the change of colony formation ability after knocking down the expression level of AK4 in HGC cells; F is the colony formation experiment to detect the change of colony formation ability after overexpressing the expression level of AK4 in AGS cells; P < 0.05, P < 0.01, P < 0.001, ns indicates no statistical significance);

[0038] Figure 5 Figure showing AK4 promotes the migration of gastric cancer cells (A: Scratch assay results and wound healing statistical chart of AK4 knockdown; B: Scratch assay results and wound healing statistical chart of AK4 overexpression; C: Transwell assay results and statistical chart of the number of penetrated cells of AK4 knockdown; D: Transwell assay results and statistical chart of the number of penetrated cells of AK4 overexpression; P < 0.05, P < 0.01, P < 0.001, ns indicates no statistical significance);

[0039] Figure 6 Figure showing AK4 inhibits the apoptosis of gastric cancer cells (A: Expression of apoptotic proteins in HGC cells with AK4 knockdown; B: Expression of apoptotic proteins in AGS cells with AK4 overexpression);

[0040] Figure 7 Figure showing the expression of AK4 in gastric cancer cells after hypoxia stimulation (A: HGC cells were cultured under 1% O2 for 0, 12, 24, and 48 hours, and the expression levels of AK4 and HIF-1α proteins were detected by WB; B: AGS cells were cultured under 1% O2 for 0, 12, 24, and 48 hours, and the expression levels of AK4 and HIF-1α proteins were detected by WB);

[0041] Figure 8 Figure showing the effect of hypoxia and regulation of AK4 expression on the chemosensitivity of cisplatin (A: IC50 values of cisplatin in HGC and AGS cells; B: Changes in the IC50 of cisplatin in HGC cells under normoxic and hypoxic conditions were detected; C: Changes in the IC50 of cisplatin in HGC cells with AK4 knockdown and its normal control group under normoxic conditions were detected; D: Changes in the IC50 of cisplatin in AGS cells under normoxic and hypoxic conditions were detected; E: Changes in the IC50 of cisplatin in AGS cells with AK4 overexpression and its normal control group under normoxic conditions were detected);

[0042] Figure 9 Figure showing the regulation of the expression of the drug efflux gene MDR1 by AK4 (A: Expression of MDR1 in HGC cells with AK4 knockdown; B: Expression of MDR1 in AGS cells with AK4 overexpression). Detailed implementation manners

[0043] The present invention will be further described below in conjunction with specific embodiments. In the following embodiments, the operations not described in detail are all conventional biological experimental operations, which can be referred to in the molecular biology experimental manual and existing published journal literatures, etc., or carried out according to the operation steps in the instruction manual of the kit.

[0044] The human gastric cancer cell lines SGC-7901, KATO-III, AGS, Ncl-N87, and HGC used in this application were all purchased from the cell bank of the Shanghai Institute of Biological Sciences.

[0045] This application selected 248 gastric cancer patients who underwent surgical treatment in the Affiliated Tumor Hospital of Nantong University from March 2012 to June 2015 as research samples. The cut-off date for the follow-up work was October 22, 2020. Inclusion criteria for all selected gastric cancer patients: 1) Received radical gastrectomy and had not received treatment in the forms of radiotherapy, chemotherapy, targeted therapy, and immunotherapy before surgery; 2) Complete clinical data collection; 3) Clear postoperative pathological diagnosis; 4) Complete follow-up data collection; 5) Graded using the WHO (2008) standard. Approved by the Ethics Committee of Nantong Tumor Hospital.

[0046] Example 1

[0047] 1. Immunohistochemical staining

[0048] (1) Baking the slides: Preheat the oven to a constant 85°C, and put the prepared 248 gastric cancer tissue microarrays into the baking machine, with a baking time of not less than 30 min.

[0049] (2) Deparaffinization: Take out the baked tissue microarrays and immerse them successively in two cylinders of different xylene reagents, with an infiltration time of 15 min in each cylinder. The soaking time can be appropriately extended according to the wax residue situation of the tissue microarrays to ensure complete deparaffinization.

[0050] (3) Hydration: Immerse the completely deparaffinized tissue microarrays successively in 100%, 95%, 80%, and 70% ethanol, with an immersion time of 3 minutes for each step to restore their original hydrated state. Subsequently, put the chip tissue into pure water and PBS solution and wash it three times respectively.

[0051] (4) Antigen heat repair: Place the tissue microarrays in the citrate repair solution, put the repair solution containing the microarrays into a pressure cooker filled with water, and perform antigen repair using the high-pressure and high-temperature environment. After the pressure cooker starts to steam, start timing for 3 min. After the time is up, turn off the power supply. Wait for the pressure of the pressure cooker to return to normal naturally, take out the microarrays, let them cool naturally at room temperature, and then wash the microarrays with PBS solution, with a washing time of 5 min each time, for a total of three times.

[0052] (5) Blocking endogenous peroxidase: Gently shake the microarrays to remove excess moisture, use an immunohistochemical pen to accurately circle the gastric cancer tissue, incubate with 3% H2O2 for 20 min, and note that the tissue should be fully covered during the incubation process. After incubation, immerse the microarrays in PBS solution and place them on a shaker for slow washing, with a washing time of 5 min each time, for a total of 3 times. Avoid drying the slides throughout the process.

[0053] (6)Incubate the primary antibody: Gently shake off the excess liquid on the chip, place it flat in a humidity box, dilute the AK4 antibody with the primary antibody diluent at a ratio of 1:200, add 200 μL of the diluted antibody solution to each chip, and incubate overnight at 4 °C in the refrigerator.

[0054] (7)Incubate the secondary antibody: Take out the chip the next day and warm it to room temperature for 30 min. Then, wash the chip with PBS three times, 5 min each time. Subsequently, drip an appropriate amount of the secondary antibody onto the slide to ensure that the secondary antibody can fully cover the tissue on the chip. After incubating at room temperature for 20 min, immerse the chip in PBS again and place it on a shaker for washing three times, 5 min each time.

[0055] (8)Color development: Prepare the DAB color development solution (1 mL of diluent + 30 μL of DAB concentrate) in the fume hood, drip the prepared DAB color development solution onto the slide, observe the color development situation by the sink. After the brown color appears, terminate the color development reaction with tap water.

[0056] (9)Counterstain: Evenly drip the hematoxylin staining solution onto the sample area on the slide to ensure that the cell nuclei can be fully stained. The staining time is about 30 s. After reaching the predetermined staining time, rinse the slide with running water.

[0057] (10)Dehydration: Gently wipe off the traces of the histochemical pen. First, soak the chip in 70% ethanol for 3 min, and then sequentially dehydrate it with 80%, 90%, and 100% ethanol, each concentration also soaking for 3 min. After dehydration, immerse the chip in two tanks of xylene for 8 min each.

[0058] (11)Cover slipping: Take an appropriate amount of neutral resin and drip it onto the center of the slide. Gently pick up the cover slip and place it steadily on the slide with the dropped neutral resin, so that the resin between the cover slip and the slide is evenly distributed. Pay attention to removing air bubbles during the process. Place the whole slide in the fume hood to dry and then observe it under the microscope.

[0059] (12)Scoring: The double-blind principle was adopted, and two experienced pathologists scored the AK4 tissue chip. Scoring criteria: The staining intensity was negative, weak, medium, and strong, which were scored 0, 1, 2, and 3 points respectively; the proportion of positive cells in each field of view was recorded as 0, 1, 2, 3, and 4 points according to 0%, <25%, 25%-50%, 50%-75%, and >75% respectively. The specific score of each sample was obtained by multiplying the staining intensity and the proportion of positive cells. Finally, those with a score lower than 6 were included in the AK4 low-expression group, and vice versa for the high-expression group.

[0060] The results are as Figure 1As shown, AK4 is mainly localized in the cytoplasm. Immunohistochemical images of 248 patients were scored and divided into a high-expression group of 46 cases and a low-expression group of 202 cases of AK4 ( Figure 1 A-C). The ROC curve of 248 patients was plotted to evaluate the effectiveness of the prognostic model, and the AUC values at 1, 3, and 5 years were 0.632, 0.577, and 0.508 respectively ( Figure 1 D). The higher the AUC value, the stronger the predictive ability of the gene.

[0061] As shown in Table 1, the expression of AK4 was closely related to the degree of differentiation (P < 0.01).

[0062] As shown in Table 2, the results of COX univariate and multivariate regression analysis showed that the prognosis of gastric cancer was closely related to tumor size (P < 0.001), depth of tumor invasion (P < 0.001), presence or absence of lymph node metastasis (P < 0.001), presence or absence of cancer thrombus (P < 0.001), presence or absence of nerve invasion (P < 0.001), degree of differentiation (P = 0.020), and AK4 expression level (P = 0.004). Therefore, lymph node metastasis (P = 0.010) and AK4 expression level (P = 0.005) were selected as independent prognostic indicators for gastric cancer patients.

[0063] Table 1 Relationship between AK4 expression and clinicopathological parameters in 248 gastric cancer patients

[0064]

[0065] Note: Indicates P < 0.05.

[0066] Table 2 Univariate and multivariate analysis of OS in gastric cancer patients

[0067]

[0068] Note: Indicates P < 0.05.

[0069] The results are as Figure 2 shown. Using the TCGA database to explore the relationship between AK4 expression and the prognosis of gastric cancer, the results showed that the higher the AK4 expression, the shorter the overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS) of the patients ( Figure 2 A). Kaplan-Meier curve analysis of 248 patients showed that high expression of AK4 represented poor prognosis, which was consistent with the database analysis results ( Figure 2B). In addition, a combined prognostic analysis was performed on the expression level of AK4 and clinical indicators. The results showed that in gastric cancer patients with T3-T4 stage, poor differentiation, vascular invasion and non-vascular invasion, high expression of AK4 indicated poor prognosis ( Figure 2 C).

[0070] 2. Western blot (WB)

[0071] 1) Preparation of protein samples

[0072] Tissue protein: Weigh 0.1 g of gastric cancer tissue and its paired adjacent cancer tissue. Immerse the tissue in paraformaldehyde, and after treatment, place it into an EP tube. Use a pipette to take an appropriate amount of protein lysate and mix it well with the tissue. Homogenize the tissue with a homogenizer and let it stand on ice for 30 min. Centrifuge at 4°C and 12,000 rpm for 10 min, then aspirate the supernatant and transfer it into a new labeled EP tube. Add an appropriate amount of protein loading buffer, mix well by pipetting, place it in a dry bath incubator, heat at 100°C for 10 min, centrifuge again at 4°C and 12,000 rpm for 10 min, use a spectrophotometer to detect and record the protein concentration, and store the EP tube in an -80°C refrigerator.

[0073] Cell protein: Place the cell culture flask (with a cell confluence of approximately 80%) on ice. After removing the culture medium, aspirate PBS solution and gently wash the cells 3 times. Add an appropriate amount of pre-prepared lysis buffer and let the cells lyse completely in the lysis buffer. After lysis, use a cell scraper to scrape off all the cells, and use a pipette to collect the lysis buffer and cell debris into a labeled EP tube. After collection, place the EP tube in a dry bath incubator, heat at 100°C for 10 minutes, and then quickly return the EP tube to ice for cooling. Centrifuge at 4°C and 12,000 rpm for 10 min, take the supernatant, use a spectrophotometer to detect and record the protein concentration, and finally store the EP tube in an -80°C refrigerator to ensure the stable preservation of cell protein.

[0074] 2) SDS-PAGE gel electrophoresis

[0075] Wash two glass plates for preparing gels, ensuring that the surfaces are free of impurities. Then place them in an electrothermal constant temperature drying oven for drying. Take out the dried glass plates, fix them and check for leaks. Prepare the lower separating gel according to the ratio and inject it between the two glass plates. Quickly add isopropanol to press the gel. After the separating gel has solidified for about 30 minutes, rinse it with running water and blot the excess water with absorbent paper. Prepare the upper stacking gel and inject it between the glass plates in the same way. Insert the Bio-rad comb, avoiding the generation of bubbles, and wait for it to solidify for about 20 minutes. After the gel has completely solidified, place the gel plate into the groove containing an appropriate amount of electrophoresis buffer, and prepare for electrophoresis. Vertically remove the Bio-rad comb, add the marker and the sample to be detected into the wells, correctly connect the electrodes, set the voltage to 80 - 130 V, and then perform electrophoresis. During electrophoresis, closely monitor the migration of the protein sample. When it reaches the bottom layer of the gel, stop electrophoresis in a timely manner.

[0076] 3) Transfer membrane

[0077] Immerse the PVDF membrane in methanol and activate it for about 30 s. Then place it in ddH2O to remove the residual methanol and depolarize it. Prepare the "sandwich" materials for membrane transfer. Place the sponge pads and filter papers on both sides of the transfer membrane clip. Place the gel on the filter paper on the black clip side, cover it with the activated PVDF membrane, and make sure there are no bubbles between the membrane and the gel before closing the clip. Finally, place the assembled clip into the transfer membrane apparatus pre-filled with buffer, ensure that the positive and negative poles of the clip are aligned with the corresponding interfaces of the transfer membrane apparatus, and connect the power supply. Set the current to 300 mA and perform the membrane transfer operation for about 90 minutes.

[0078] 4) Blocking

[0079] Take out the PVDF membrane that has been subjected to membrane transfer and place it in the blocking buffer prepared with 5% skim milk powder. Place the blocking buffer with the membrane on a shaker and shake it slowly to allow the blocking solution to fully contact the membrane. The entire blocking process lasts for 2 hours.

[0080] 5) Incubate with primary antibody

[0081] Take out the blocked PVDF membrane and dilute the primary antibodies according to the ratio using the primary antibody diluent according to the antibody instructions: AK4 - 1:1000), HIF - 1α - 1:1000), MDR1 - 1:1000), Bax (1:1000), Bcl - 2 (1:1000), Bcl - xl (1:1000), β - actin (1:1000), Tubulin (1:10000) and GAPDH (1:5000). Add the prepared antibodies to the PVDF membrane and incubate it overnight in a 4°C refrigerator.

[0082] 6) Incubate with secondary antibody

[0083] Take out the PVDF membrane incubated with the primary antibody in the refrigerator, warm it at room temperature for about 30 min, and then wash the membrane 3 times with TBST buffer for 5 min each time. Then, add the pre-prepared secondary antibody to the washed PVDF membrane and incubate it at room temperature for 2 h. After incubation, wash the membrane 3 times with TBST buffer for 10 min each time.

[0084] 7) Development

[0085] Prepare the developer in proportion, take an appropriate amount of the developer with a pipette and apply it to the PVDF membrane, and develop and save the image data in the Bio-RAD imaging system.

[0086] The results are as Figure 3 shown. AK4 is highly expressed in HGC and Ncl-N87, relatively lowly expressed in AGS and KATO-III, and hardly expressed in SGC-7901 cells.

[0087] Example 2

[0088] 1. Design siRNA targeting the AK4 gene

[0089] Design siRNA according to the sequence of the AK4 gene. The target sequences corresponding to the three siRNAs are as follows:

[0090] AK4-siRNA-(110526-1): 5’-gcCAGTCATTGAATTATACAA-3’;

[0091] AK4-siRNA-(110527-1): 5’-TGGCAAAGCAGTATATAGAGA-3’;

[0092] AK4-siRNA-(110528-1): 5’-ACCCTCCTAGCGGAAGGGTAT-3’.

[0093] The sequences of the three designed double-stranded DNA oligos are as follows:

[0094] AK4-siRNA-(110526-1)-F:

[0095] 5’-CcgggcCAGTCATTGAATTATACAACTCGAGTTGTATAATTCAATGACTGGCTTTTTg-3’,

[0096] AK4-siRNA-(110526-1)-R:

[0097] 5’-aattcaaaaagcCAGTCATTGAATTATACAACTCGAGTTGTATAATTCAATGACTGGC-3’;

[0098] AK4-siRNA-(110527-1)-F:

[0099] 5’-CcggTGGCAAAGCAGTATATAGAGACTCGAGTCTCTATATACTGCTTTGCCATTTTTg-3’,

[0100] AK4-siRNA-(110527-1)-R:

[0101] 5’-aattcaaaaaTGGCAAAGCAGTATATAGAGACTCGAGTCTCTATATACTGCTTTGCCA-3’;

[0102] AK4-siRNA-(110528-1)-F:

[0103] 5’-CcggACCCTCCTAGCGGAAGGGTATCTCGAGATACCCTTCCGCTAGGAGGGTTTTTTg-3’,

[0104] AK4-siRNA-(110528-1)-R:

[0105] 5’-aattcaaaaaACCCTCCTAGCGGAAGGGTATCTCGAGATACCCTTCCGCTAGGAGGGT-3’.

[0106] Negative control transfected with AK4-siRNA (Scramble sequence: TTCTCCGAACGTGTCACGT)

[0107] 2. Cell culture

[0108] Four gastric cancer cell lines (SGC-7901, KATO-III, Ncl-N87, HGC) were cultured using complete medium, which contained 10% fetal bovine serum, 1% double antibiotics (penicillin, streptomycin) and 89% RPMI-1640 basal medium. The AGS cell line was completely cultured using a basal medium containing 10% fetal bovine serum, 1% double antibiotics (penicillin, streptomycin) and 89% F-12K. The cells were cultured in an incubator under suitable growth conditions (37 °C, 5% CO2). In the preparation stage of cell experiments, we needed to turn on the ultraviolet lamp and irradiate the workbench for 30 minutes to ensure a sterile environment. And the medium, trypsin and PBS solution were taken out in advance and placed at room temperature for rewarming treatment.

[0109] 3. Transfection of AGS cells with AK4 overexpression plasmid

[0110] Plating: Take AGS cells in the logarithmic growth phase and evenly inoculate them in a six-well plate at a density of 1×10 5 cells per well. After culturing for about 24 h, the cells reached a suitable adherent state and their abundance reached 30% - 40%.

[0111] Plasmid transfection: The AK4 overexpression plasmid was purchased from GeneChem Co., Ltd. During the transfection process, the transfection operation was strictly carried out according to the instructions of the jetPRIME polyplus transfection reagent. After transfection for 4 - 6 h, the medium was changed, and the cells were collected 48 hours after transfection for subsequent experiments.

[0112] 4. Infection of HGC cells with AK4 knockdown lentivirus

[0113] The synthesized oligos were respectively dissolved in oligo annealing buffer to 20 μM, and 30 μL of each complementary single strand was taken and mixed. Then the oligo mixture was heated in a water bath at 95 °C for 5 min, and then the lid of the water bath was opened and allowed to cool naturally to room temperature in the air to form double-stranded oligo fragments. 1 μL was taken for the subsequent ligation reaction, and the rest was stored at 20 °C.

[0114] The expression vector was digested with restriction enzymes. The enzyme digestion reaction system was: 2 μg of plasmid, 5 μL of 10× reaction Buffer, 1 μL of each restriction enzyme, and deionized water was added to make up 50 μL, and incubated in a 37 °C water bath for more than 2 h. The enzyme digestion products were detected by agarose gel electrophoresis to check the digestion effect, and the target vector band was cut and recovered using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver. 3.0.

[0115] The ligation product obtained by ligating the double-stranded oligo fragment with the linearized expression vector was transformed into competent Escherichia coli prepared with calcium chloride. The identified positive clone transformants were inoculated into an appropriate amount of LB liquid medium containing antibiotics and cultured at 37 °C for 12 - 16 h. An appropriate amount of the bacterial solution was taken for sequencing. The sequencing results were compared and analyzed with the target gene sequence.

[0116] The bacterial solution with correct sequencing was transferred to 10 mL of LB liquid medium containing antibiotics and cultured overnight at 37 °C. Plasmid extraction was performed using the Tiangen endotoxin-free plasmid mini extraction midiprep kit: The overnight cultured bacterial solution was collected in a labeled 5 mL centrifuge tube and centrifuged at 12000 rpm for 2 min to collect the bacteria; after discarding the supernatant, 250 μL of cell resuspension was added, and the mixture was shaken thoroughly to suspend the bacterial mass evenly; after the bacterial cells were lysed and clarified, neutralizing liquid was added, and after protein precipitation, the mixture was centrifuged on ice. The plasmid was extracted using the recovery column.

[0117] Plating: HGC cells in the logarithmic growth phase were evenly inoculated in a six-well plate at a density of 1×10 5 cells per well. After culturing for about 24 h, the cells reached an appropriate adherent state, and their abundance reached 30% - 40%.

[0118] Lentiviral infection: According to the virus MOI value (the MOI value for the HGC cell line is 50), the required virus volume was calculated accordingly. An appropriate amount of virus and infection reagent were added to the culture plate, shaken evenly, and then cultured in an incubator for about 12 h. Puromycin was used to screen for stable gastric cancer cell lines. During this period, the cell morphology was observed dynamically, and the cell culture medium was replaced in a timely manner to ensure good cell state. After screening for more than three generations, cell proteins were extracted, and the expression abundance of AK4 was detected by WB to confirm the infection efficiency.

[0119] Example 3

[0120] 1. CCK-8 proliferation

[0121] The AGS cells after overexpression of AK4 and the HGC cells after lentiviral infection with AK4 knockdown were respectively diluted to a density of about 1×10 6 cells per well and inoculated into a 96-well plate. The plate was placed in a constant-temperature cell incubator for culture. 10 μL of CCK-8 reagent was added to each well at 24 h, 48 h, 72 h, and 96 h after culture, and the absorbance values at each time point were observed and recorded.

[0122] The processed gastric cancer cells were seeded in a 6-well plate at a density of approximately 200 cells / well and placed in an incubator. After 2 weeks of culture, the cell culture medium was aspirated, and the cells were gently rinsed 3 times with PBS. The cells were fixed with paraformaldehyde for 15 min, and then stained with 0.5% crystal violet reagent for 30 min. After air-drying, pictures were taken and saved.

[0123] After transfection of gastric cancer cells with AK4 overexpression lentivirus and three interfering lentiviruses of AK4 for 72 h, puromycin screening was performed. Total proteins of each group of cells were extracted and collected, and protein quantification of each group was performed using a BCA kit (purchased from Beyotime). Electrophoresis buffer was prepared, and the total amount of loaded protein in each group was 40 μg. Electrophoresis separation was performed on a 10% SDS-PAGE gel. The PVDF membrane after transfer was taken out, and the membrane was placed in a blocking buffer prepared with 5% skim milk powder. The blocking buffer containing the membrane was placed on a shaker and slowly shaken to allow the blocking solution to fully contact the membrane. The entire blocking process lasted for 2 h.

[0124] Incubate with AK4 antibody overnight. The next day, after incubating with the HRP-labeled secondary antibody at room temperature for 2 h, develop the image.

[0125] The results are as Figure 4 shown. Among the three shRNA sequences targeting AK4, AK4 shRNA (sh2) had the most significant inhibitory efficiency ( Figure 4 A), while the overexpression lentivirus significantly upregulated the expression of AK4 in the gastric cancer cell line ( Figure 4 B). Compared with the control group, after knocking down AK4 in HGC cells, the proliferation and colony formation ability of the cells decreased significantly. On the contrary, after overexpressing AK4 in AGS cells, its proliferation and colony formation ability increased significantly compared with the control group ( Figure 4 C-F).

[0126] 2. Cell scratch and cell migration

[0127] 1) AGS cells after overexpressing AK4 and HGC cells infected with AK4 knockdown lentivirus were respectively seeded in a 6-well plate with lines drawn on the back in advance. When the cells adhered and grew to an overall confluence of approximately 80%, a 200 μL pipette tip was used to perform a vertical scratch operation on the cell surface. The detached cell debris was gently rinsed off with PBS, and the culture medium was changed to a basal medium. The changes in the cells in the scratched area were observed under a phase contrast microscope 48 hours before and after, and Image J software was used for image processing and data analysis of the scratched area.

[0128] 2) Cell migration

[0129] 5×10 4AGS cells after overexpression of AK4 and HGC cells after infection with AK4 knockdown lentivirus were mixed with 200 μL of serum-free medium and resuspended to ensure uniform cell distribution. Then the cell suspension was added to the Transwell chamber, and the chamber was placed into a 24-well plate pre-added with 500 μL of complete medium. After culturing for 48 h, the chamber was gently taken out using sterile forceps and gently washed in a beaker containing PBS. The cells were fixed with 4% paraformaldehyde for 20 min, then stained with 0.5% crystal violet reagent for 30 min. The non-migrated cells in the chamber were wiped off using a cotton swab, the chamber was air-dried at room temperature, and the cells were observed and photographed under a microscope.

[0130] The results were as Figure 5 shown. Compared with the control group, after knocking down AK4 in HGC cells, the scratch healing ability of the cells decreased significantly compared with the control group, indicating a reduction in cell migration ability ( Figure 5 A). On the contrary, in AGS cells overexpressing AK4, the scratch healing ability increased and the cell migration ability increased ( Figure 5 B). Similarly, the results of the Transwell migration assay showed that after knocking down AK4, the migration ability of gastric cancer cells decreased, and after overexpressing AK4, the migration ability of AK4 increased ( Figure 5 C-D).

[0131] 3. Cell apoptosis

[0132] AGS cells after overexpression of AK4 and HGC cells after infection with AK4 knockdown lentivirus were respectively seeded in 6-well plates. When the cells adhered and grew to an overall confluence of about 80%, protein lysate was added to extract proteins. Subsequently, protein quantification was performed using a BCA kit, and the protein expression levels of apoptosis indicators in each group were detected by immunoblotting.

[0133] The results were as Figure 6 shown. Compared with the control group, after knocking down AK4 in HGC cells, the expression of Bax increased, and the expressions of Bcl-2 and Bcl-xl decreased, promoting gastric cancer cell apoptosis ( Figure 6 A). On the contrary, in AGS cells overexpressing AK4, the expression of Bax decreased compared with the control group, while the expressions of Bcl-2 and Bcl-xl increased compared with the control group, inhibiting gastric cancer cell apoptosis ( Figure 6 B).

[0134] 4. Expression of intracellular AK4 after hypoxia stimulation

[0135] AGS cells overexpressing AK4 and HGC cells infected with AK4 knockdown lentivirus were cultured in an environment of 1% O2 for 0, 12, 24, and 48 h respectively, and the expressions of AK4 and hypoxia-inducible factor HIF-1α were detected by WB.

[0136] The results were as Figure 7 shown. After hypoxic stimulation of HGC cells, the expression level of HIF-1α protein increased, indicating a hypoxic state. At the same time, with the prolongation of hypoxia time, the expression level of AK4 protein gradually increased in HGC cells. Similarly, in AGS cells, the expression level of AK4 also showed an increasing trend with the increase of hypoxia time. The results indicated that the hypoxic environment increased the expression level of AK4.

[0137] 4. Drug sensitivity

[0138] AGS cells, HGC cells, AGS cells overexpressing AK4, and HGC cells infected with AK4 knockdown lentivirus were respectively prepared into a density of about 5×10 3 cells per well and inoculated into a 96-well cell culture plate. Each group was set with 8 replicate wells. After the cells adhered, the culture medium was replaced with a culture medium containing different concentrations of cisplatin injection (concentration range: 0 μg / mL - 64 μg / mL). After the cell plate was placed in an incubator for 24 h, 10 μL of CCK-8 reagent was added to each well, and the absorbance value of the cells was detected by an enzyme-linked immunosorbent assay (ELISA) reader. The experimental data were statistically analyzed and visualized using R 4.2.1 software. The R packages involved: the drc package [3.0 - 1] was used for analysis, and ggplot2 was used for visualization.

[0139] The results were as Figure 8 shown in Fig. A. The IC 50 value of cisplatin in HGC cells was 6.52 μmol / mL, and the IC 50 value of cisplatin in AGS cells was 11.16 μmol / mL.

[0140] Under normoxic and hypoxic conditions, different concentrations of cisplatin were added to HGC cells with AK4 knockdown and AGS cells with AK4 overexpression, and their corresponding control cells, and then the CCK-8 kit was used to detect the proliferation viability of the cells and calculate the IC 50 value.

[0141] The results were as Figure 8 shown in Figs. B - E. The IC 50 value of the HGC control cells under normoxic conditions was 6.32 μmol / mL, and the IC 50 value increased to 14.26 μmol / mL under hypoxic conditions ( Figure 8 Fig. B); the IC 50The value was 7.57 μmol / mL. After knocking down AK4, the IC 50 value decreased to 3.61 μmol / mL ( Figure 8 C). The IC 50 value of AGS control cells under normoxic conditions was 11.04 μmol / mL, and the IC 50 value increased to 17.53 μmol / mL under hypoxic conditions ( Figure 8 D); in AGS control cells, the IC 50 value was 10.66 μmol / mL. After overexpressing AK4, the IC 50 value increased to 16.72 μmol / mL ( Figure 8 E). The above results indicate that knocking down AK4 increases the sensitivity of gastric cancer cells to cisplatin, while hypoxic conditions and overexpressing AK4 can both reduce the sensitivity of gastric cancer cells to cisplatin.

[0142] 5. AK4 regulates the expression of the drug efflux gene MDR1

[0143] The effect of AK4 expression on the expression level of the drug efflux protein MDR1 was detected by WB.

[0144] The results are as Figure 9 shown. In HGC cells with knocked-down AK4, the expression level of MDR1 also decreased ( Figure 9 A). On the contrary, in AGS cells with overexpressed AK4, the expression level of MDR1 increased ( Figure 9 B). The results indicate that AK4 affects the sensitivity of gastric cancer cells to cisplatin by promoting drug efflux.

Claims

1. An siRNA targeting the AK4 gene, with the sequence shown as follows: ① AK4-siRNA-110526-1-F: 5’-CcgggcCAGTCATTGAATTATACAACTCGAGTTGTATAATTCAATGACTGGCTTTTTg-3’, AK4-siRNA-110526-1-R: 5’-aattcaaaaagcCAGTCATTGAATTATACAACTCGAGTTGTATAATTCAATGACTGGC-3’; and / or ② AK4-siRNA-110527-1-F: 5’-CcggTGGCAAAGCAGTATATAGAGACTCGAGTCTCTATATACTGCTTTGCCATTTTTg-3’, AK4-siRNA-110527-1-R: 5’-aattcaaaaaTGGCAAAGCAGTATATAGAGACTCGAGTCTCTATATACTGCTTTGCCA-3’; and / or ③ AK4-siRNA-110528-1-F: 5’-CcggACCCTCCTAGCGGAAGGGTATCTCGAGATACCCTTCCGCTAGGAGGGTTTTTTg-3’, AK4-siRNA-110528-1-R: 5’-aattcaaaaaACCCTCCTAGCGGAAGGGTATCTCGAGATACCCTTCCGCTAGGAGGGT-3’.

2. The target sequence corresponding to the siRNA targeting the AK4 gene according to claim 1 is shown as follows: ① AK4-siRNA-110526-1: 5’-gcCAGTCATTGAATTATACAA-3’; and / or ② AK4-siRNA-110527-1: 5’-TGGCAAAGCAGTATATAGAGA-3’; and / or ③ AK4-siRNA-110528-1: 5’-ACCCTCCTAGCGGAAGGGTAT-3’.

3. Use of the siRNA targeting the AK4 gene according to claim 1 in inhibiting the expression of the AK4 gene.

4. Use of the siRNA targeting the AK4 gene according to claim 1 in the preparation of a drug for inhibiting cell proliferation and cloning.

5. Use of the siRNA targeting the AK4 gene according to claim 1 in the preparation of a drug for inhibiting cell migration.

6. Use of the siRNA targeting the AK4 gene according to claim 1 in the preparation of a drug for promoting cell apoptosis.

7. Use of the siRNA targeting the AK4 gene according to claim 1 in the preparation of a drug for promoting the sensitivity of cells to cisplatin drugs.

8. Use of the siRNA targeting the AK4 gene according to claim 1 in the preparation of a drug for treating cancer.

9. A biomarker for the diagnosis, treatment or prognosis determination of gastric cancer, characterized in that, is the AK4 gene.

10. A kit for the diagnosis, treatment or prognosis judgment of gastric cancer, characterized in that, Contains the AK4 gene sequence.

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