Application of a traditional Chinese medicine gecko microRNA in preparation of a medicine for treating anti-tumor metastasis

Abstract

The application discloses application of a traditional Chinese medicine gecko microRNA in preparation of an anti-tumor metastasis treatment drug. Through extensive and in-depth research, the application discloses that the gecko-miR-2862 mimic can inhibit invasion and migration of liver cancer cells through a scratch test and a Transwell test; the gecko-miR-2862 mimic is transfected into cancer cells to first disclose that the gecko-miR-2862 mimic can target and inhibit mRNA of a cancer gene SRC in the cancer cells and protein expression of genes related to the SRC signal path, and further discloses the treatment effect of the gecko-miR-2862 mimic on tumor metastasis. The application provides a direction and a technical basis for research and development of a new drug for resisting tumor metastasis treatment, and further develops a new traditional Chinese medicine gecko microRNA as an anti-tumor metastasis treatment drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and medical technology, and particularly relates to application of a traditional Chinese medicine gecko microRNA in preparation of a therapeutic drug for resisting tumor metastasis. BACKGROUND

[0002] The occurrence of malignant tumors is a complex multi-step process, and its malignant progression is related to multiple molecular mechanisms such as cell proliferation, apoptosis, autophagy, epithelial-mesenchymal transition, cell invasion and metastasis, angiogenesis, etc. Clinically, tumor metastasis is the main cause of patient condition deterioration and death. Some malignant tumors are highly occult, and patients are often in the advanced stage when they are found, missing the best treatment opportunity, which has brought great challenges to clinical treatment. Exploring and developing efficient and low-toxicity anti-tumor metastasis drugs and treatment strategies to improve patient clinical symptoms, prolong survival and improve quality of life have become the focus and difficulty to be solved at present.

[0003] MicroRNA is a kind of small molecule non-coding RNA, usually composed of 18-25 nucleotide sequences, which does not have the function of translating protein, but can play a negative regulatory role at the post-transcriptional level by recognizing specific mRNA 3'UTR (3'untranslated region) sites to cut or inhibit translation of target genes, thereby exerting specific biological regulatory effects. MicroRNA plays a crucial role in different stages of liver cancer and is involved in the growth, apoptosis, differentiation, invasion and metastasis of liver cancer cells.

[0004] Clinically, the traditional Chinese medicine gecko has shown good anti-tumor activity in liver cancer, lung cancer, esophageal cancer and other tumors, but the mechanism of action of microRNA in gecko against tumors has not been reported. Studies have shown that exogenous microRNA in traditional Chinese medicine can enter the mammalian body and has good bioavailability and biological activity, and can cross-regulate the corresponding animal target genes to inhibit the growth and metastasis of mammalian tumors. In addition, nucleic acid drugs represented by microRNA have the characteristics of obvious therapeutic effect, strong specificity and low drug toxicity. In view of the role of traditional Chinese medicine gecko microRNA in the process of tumor occurrence and development and the important role of microRNA as an effective active ingredient of traditional Chinese medicine gecko in clinical treatment, the development of a new traditional Chinese medicine gecko microRNA for preparing a therapeutic drug for resisting tumor metastasis has very important significance for promoting the modernization of traditional Chinese medicine and the development of new drugs, improving the quality of life and prolonging the survival of tumor patients. SUMMARY

[0005] In view of the problems in the prior art, the present application aims to provide application of a traditional Chinese medicine gecko microRNA in preparation of a therapeutic drug for resisting tumor metastasis.

[0006] To achieve the above object, the application adopts the following technical scheme:

[0007] The application provides the use of a gek-miR-2862 up-regulator in the preparation of a therapeutic drug for resisting tumor metastasis, wherein, according to the microRNA naming principle, gek in the gek-miR-2862 is the abbreviation of the Latin Gekko of gecko, and miR represents miRNA, the nucleotide sequence (such as SEQ ID NO. 1) of the gek-miR-2862 is 5'-CGGGGAGGUGGAGCCUGGG-3', wherein 5'-GGGGAGG-3' is the seed region sequence of the gek-miR-2862.

[0008] Preferably, the gek-miR-2862 up-regulator refers to a substance for increasing the content of the gek-miR-2862.

[0009] Preferably, the gek-miR-2862 is a modified gek-miR-2862, and the modification modes include but are not limited to glycosylation modification, methylation modification, hydrocarbon group modification, nucleic acid modification and the like.

[0010] Preferably, the gek-miR-2862 up-regulator can inhibit the target gene SRC, and further inhibit the invasion and migration of hepatoma cells.

[0011] Preferably, the gek-miR-2862 up-regulator is selected from the group consisting of the gek-miR-2862 and a gek-miR-2862 mimic.

[0012] The second aspect of the application provides the use of a gek-miR-2862 mimic in the preparation of a therapeutic drug for resisting tumor metastasis, wherein the gek-miR-2862 mimic is a microRNA for simulating the activity of the gek-miR-2862, and is named as gek-miR-2862mimics, the gek-miR-2862 mimic is composed of a sense strand and an antisense strand, the nucleotide sequence (such as SEQ ID NO. 2) of the sense strand is 5'-CGGGGAGGUGGAGCCUGGG-3', and the nucleotide sequence (such as SEQ ID NO. 3) of the antisense strand is 3'-GCCCCUCCACCUCGGACCC-5'.

[0013] -CGGGGAGGUGGAGCCUGGG-3', and the nucleotide sequence (such as SEQ ID NO. 3) of the antisense strand is 3'-GCCCCUCCACCUCGGACCC-5'.

[0014] Preferably, in the above use, the gek-miR-2862 mimic can inhibit the target gene SRC, and further inhibit the invasion and migration of cancer cells.

[0015] The third aspect of the present application provides a medicine for treating anti-tumor metastasis, comprising an effective dose of a gek-miR-2862 mimic and a nucleic acid transfection agent, wherein the gek-miR-2862 mimic is composed of a sense strand and an antisense strand, the nucleotide sequence of the sense strand is shown as SEQ ID NO. 2, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 3.

[0016] Preferably, the nucleic acid transfection agent is one or a combination of two or more of a lipid-based transfection agent, a nucleic acid, a nano-material package, an exosome for delivery, and a viral vector for nucleic acid delivery.

[0017] Preferably, the dosage form of the medicine is any one of a solid preparation, a liquid preparation, and a semi-solid preparation, for example, the solid preparation can be a tablet, a capsule, a granule, etc., the liquid preparation can be an injection, etc., and the semi-solid preparation can be a cream, etc.

[0018] Preferably, the tumor type is a liver tumor.

[0019] The present application has the following beneficial effects:

[0020] Through extensive and in-depth research, the present application reveals that the gek-miR-2862 mimic can inhibit the invasion and migration of cancer cells through scratch test and Transwell test; the present application first reveals that the gek-miR-2862 mimic can target and inhibit the mRNA of SRC in cancer cells and the protein expression of SRC pathway-related genes, and further clarifies the therapeutic effect of the gek-miR-2862 mimic on tumor metastasis. The present application provides a direction and technical basis for developing a new medicine for treating anti-tumor metastasis, and further develops a new Chinese medicine gecko microRNA as a therapeutic medicine for anti-tumor metastasis. This has a very important significance for promoting the modernization of traditional Chinese medicine and the development of new drugs, improving the quality of life and prolonging the survival period of tumor patients. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0022] Figure 1A Schematic diagram of the effect of the gek-miR-2862 mimic on the migration of liver cancer cells in the scratch test;

[0023] Figure 1BFigure 1. Schematic diagram of Transwell experiment for the effect of gek-miR-2862 mimic on the migration and invasion of hepatoma cells

[0024] Figure 2A Figure 2. Schematic diagram of the construction of pGL6-miR-SRC-3'UTR-WT and pGL6-miR-SRC-3'UTR-Mut luciferase reporter plasmids

[0025] Figure 2B Figure 3. Schematic diagram of the results of the dual-luciferase reporter system for detecting SRC

[0026] Figure 3A Figure 4. Results of the effect of gek-miR-2862 mimic on the mRNA expression level of SRC in hepatoma cells

[0027] Figure 3B Figure 5. Results of the effect of gek-miR-2862 mimic on the protein expression level of SRC pathway-related genes in hepatoma cells. DETAILED DESCRIPTION

[0028] In the following description, for purposes of explanation and not limitation, specific details are set forth such as particular architectures, technologies, techniques, etc. in order to provide a thorough understanding of the embodiments of the present application. However, it will be apparent to those skilled in the art that the present application can be practiced in other embodiments that depart from these specific details.

[0029] Example 1

[0030] I. Study of the effect of gek-miR-2862 on the migration and invasion of hepatoma cells

[0031] 1. Experimental materials and methods

[0032] 1.1 Experimental materials

[0033] Human hepatoma HepG2 and Huh7 cell lines were purchased from Zhejiang Meisen Cell Technology Co., Ltd. gek-miR-2862 mimic (mimic, SEQ ID NO. 2: 5'- UUGUACUACACAAAAGUACUG-3', SEQ ID NO. 3: 3'- AAACAUGAUGUGUUUUCAUGAC-5'), and negative control (NC, sequence: SEQ ID NO. 4: 5'- UUUGUACUACACAAAAGUACUG-3', SEQ ID NO. 5: 3'- AAACAUGAUGUGUUUUCAUGAC-5') were synthesized by Guangzhou Ribobio Biotechnology Co., Ltd.

[0034] -CCGGGAGGUGGAGCCUGGG-3', SEQ ID NO. 3: 3'- AAACAUGAUGUGUUUUCAUGAC-5'), and negative control (NC, sequence: SEQ ID NO. 4: 5'- UUUGUACUACACAAAAGUACUG-3', SEQ ID NO. 5: 3'- AAACAUGAUGUGUUUUCAUGAC-5') were synthesized by Guangzhou Ribobio Biotechnology Co., Ltd.

[0035] -CCGGGAGGUGGAGCCUGGG-3', SEQ ID NO. 3: 3'- AAACAUGAUGUGUUUUCAUGAC-5'), and negative control (NC, sequence: SEQ ID NO. 4: 5'- UUUGUACUACACAAAAGUACUG-3', SEQ ID NO. 5: 3'- AAACAUGAUGUGUUUUCAUGAC-5') were synthesized by Guangzhou Ribobio Biotechnology Co., Ltd.

[0036] 1.2 Experimental methods

[0037] 1.2.1 Observation of the effect of gek-miR-2862 mimics on the migration of liver cancer cells by scratch test

[0038] Specific steps: (1) Before inoculating cells, use Marker pen to draw 3 horizontal lines on the back of the 6-well plate at an interval of 2 cm. Prepare a cell suspension containing 2.5 x 10 5 cells per milliliter using culture medium without antibiotics, and inoculate the cell suspension into the 6-well plate at 2 ml per well. After 24 hours, the cell density is more than 60%; (2) Transfect the gek-miR-2862 negative control (NC) and gek-miR-2862 mimics into HepG2 and Huh7 liver cancer cells, and add the following amounts of mixture to each well of the 6-well plate: 500 μl Opti-MEM + 2.5 μl amount of liposome + 10 μl mimic (before adding, aspirate the same volume of culture medium); (3) When the cell confluence rate reaches 100%, use a 200 μl pipette tip to draw a straight line vertically to the bottom, gently rinse with PBS to remove floating cells, and then add serum-free culture medium and place it in a 5% CO2, 37°C incubator. Observe and record the cell migration in the scratch under an inverted microscope at 0, 24, and 48 h, record the scratch area, and test the scratch area using Image J software to calculate the cell migration rate. Cell migration rate (%) = (scratch area at 0 - area at the time point to be tested) / scratch area at 0 x 100%, and the results are shown in Figure 1A .

[0039] 1.2.2 Observation of the effect of gek-miR-2862 mimics on the migration of liver cancer cells by Transwell migration test

[0040] Specific steps: (1) Prepare a cell suspension containing 2.5 x 10 5Cell suspension, 2 ml per well, 24 hours later, cell density is more than 60%; (2) Transfection of gek-miR-2862 negative control (NC) and gek-miR-2862 mimic into HepG2 and Huh7 liver cancer cells, 6-well plate, 500 μl Opti-MEM + 2.5 μl liposome + 10 μl mimic per well (before adding, the same volume of medium is sucked out); (3) Place the Transwell chamber in a 24-well plate, add 600 μl to the lower chamber and 50 μl serum-free medium to the upper chamber to activate the chamber basement membrane, and place it in a 5% CO2, 37°C incubator overnight; (4) After 24 hours of transfection, digest and centrifuge the cells in the 6-well plate, resuspend with serum-free medium, add 200 μl (2 x 10 5 cells per well) cell suspension to the upper chamber, and at the same time add 600 μl complete medium to the lower chamber, use the serum concentration difference between the upper and lower chambers to induce the migration of cells in the upper chamber to the lower chamber, pay attention to avoid bubbles; (5) After 24 hours of incubation, take out the 24-well plate, add 4% paraformaldehyde to the upper and lower chambers for 20 minutes, rinse with PBS twice; 0.1% crystal violet staining for 20 minutes, rinse with PBS twice, then gently wipe the chamber basement membrane with a cotton swab to remove non-migrated cells; (6) Place the chamber in a well-ventilated area and let it dry naturally, then randomly select five fields of view under an inverted microscope and take pictures, use Image J image processing software to calculate the number of migrated cells, the results are shown in Figure 1B .

[0041] 1.2.3 Observation of the effect of gek-miR-2862 mimic on liver cancer cell invasion by Transwell invasion experiment

[0042] Specific steps: (1) Prepare the equipment and reagents required for preparing Matrigel matrix glue, such as Tip head, EP tube, DMEM, etc., in advance, then mix Matrigel matrix glue stock solution with DMEM at a volume ratio of 1:6 to prepare a mixed solution, then add the mixed solution to the upper chamber of the Transwell chamber (100 μl per well), and place it in a 5% CO2, 37°C incubator for 30 minutes. The remaining experimental steps are the same as those of the Transwell migration experiment, and the results are shown in Figure 1B .

[0043] 2. Experimental results

[0044] From Figure 1A The results show that the scratch test results show that 100 nM gek-miR-2862 mimic has a significant inhibitory effect on the migration ability of HepG2 and Huh7.

[0045] From Figure 1BThe results show that the Transwell migration and invasion experiment results show that the transfection of 100 nM concentration of gek-miR-2862 mimic has obvious inhibitory effect on the migration and invasion of Huh7 and HepG2 cells.

[0046] II. Prediction and verification of the target gene of gek-miR-2862

[0047] 1. Experimental materials and methods

[0048] 1.1 Experimental materials

[0049] Online databases: miRDB (http: / / mirdb.org / ), RNAhybrid (https: / / bibiserv.cebitec.uni-bielefeld.de / rnahybrid) and miRanda (http: / / www.microrna.org / microrna / home.do); double-fluorescent reporter system SRC vector, synthesized by Keli (Wuhan) Biotechnology Co., Ltd.; double-fluorescent luciferase reporter gene detection kit, purchased from China Biyun Tian Biotechnology Co., Ltd.

[0050] 1.2 Experimental methods

[0051] 1.2.1 Bioinformatics prediction of the target gene of gek-miR-2862

[0052] Through bioinformatics method, the target gene of gek-miR-2862 was predicted by using online databases miRDB, RNAhybrid and miRanda, and the intersection of the target genes predicted by the above three databases was taken. The oncogene SRC was selected as the candidate target gene of gek-miR-2862.

[0053] 1.2.2 Verification of whether SRC is the target gene of gek-miR-2862 by double-fluorescent reporter system

[0054] Specific steps:

[0055] (i) Construction of pGL6-SRC vector

[0056] The SRC target gene was synthesized by ELK biotechnology company. The SRC primers were designed according to Table 1 below, and then the PCR reaction system was configured according to Table 2 below, and the PCR amplification reaction conditions were set as shown in Table 3. After the vector overlap sequence was added, the enzyme-digested vector was recombined with the enzyme-digested vector according to Table 4 below, and the recombination reaction was carried out at 37°C for 30 min, and then ice bath for 5 min. Then use high-efficiency DH5a competent cells for transformation, pick single colonies for sequencing detection after 1 day, and store the single colonies with correct sequencing and endotoxin-free plasmid. The mutual site of gek-miR-2862 and target gene SRC is shown in Figure 2A .

[0057] Table 1 SRC primer design

[0058]

[0059] When configuring the PCR reaction system according to Table 2 below, all operations must be carried out on ice. Melt the required reagents on ice, centrifuge briefly before opening the cap to concentrate the reagents at the bottom of the tube, and avoid loss and contamination when opening the cap.

[0060] Table 2. PCR reaction system

[0061]

[0062] Table 3. PCR amplification reaction condition setting

[0063]

[0064]

[0065] Table 4. Recombination reaction system

[0066]

[0067] (ii) Grouping and cell preparation

[0068] Specific steps:

[0069] Grouping: divided into the following seven groups:

[0070] A. NC

[0071] B. pGL6-miR-SRC-3'UTR-WT + pRL-TK

[0072] C. mimics NC + pGL6-miR-SRC-3'UTR-WT + pRL-TK

[0073] D. gek-miR-2862 mimics + pGL6-miR-SRC-3'UTR-WT + pRL-TK

[0074] E. pGL6-miR-SRC-3'UTR-Mut + pRL-TK

[0075] F. mimics NC + pGL6-miR-SRC-3'UTR-Mut + pRL-TK

[0076] G. gek-miR-2862 mimics + pGL6-miR-SRC-3'UTR-Mut-3'UTR + pRL-TK

[0077] Cell preparation: (1) One day before transfection, centrifuge and digest HepG2 cells, resuspend the cells with culture medium without antibiotics, configure into a single cell suspension of 1 x 10 5 cells / ml. Inoculate in 24-well plates at 500ul per well. After 24 hours, the cell confluence is 30-50%. (2) Take 50ul of Opti-MEM low serum medium and mix gently with 0.2ug of pGL6-SRC, pRL-TK; take 0.5ul of Lipofectamine 2000 and mix with 50ul of Opti-MEM low serum medium, incubate at room temperature for 5 minutes, then mix and incubate for another 20 minutes. (3) Add 100ul of transfection solution to each well of cells, gently shake in a "cross" method. After 24 hours of culture at 37℃, perform detection.

[0078] (iii) Dual luciferase reporter gene detection

[0079] (1) Lysis of cells: Mix the reporter gene cell lysis solution thoroughly, and after aspirating the cell culture solution, the reporter gene cell lysis solution can be added directly, add 100ul of cell lysis solution, resuspend the cells, lyse thoroughly for 5 minutes, then centrifuge and carefully aspirate the supernatant into a new 1.5ml EP tube, store at -80℃ for standby;

[0080] (2) Thaw the luciferase detection reagent and Renilla luciferase detection buffer, and reach room temperature, and the Renilla luciferase detection substrate (100X) is placed on ice bath or ice box for standby;

[0081] (3) According to the amount of 100ul required for each sample, take an appropriate amount of Renilla luciferase detection buffer, and add Renilla luciferase detection substrate (100X) according to 1:100ul to prepare Renilla luciferase detection working solution;

[0082] (4) According to the instrument operation manual, open the fluorescence detector, set the measurement interval to 2 seconds and the measurement time to 10 seconds. When measuring each sample, take 100 μl of sample, add 100 μl of firefly luciferase detection reagent, mix well, and measure the relative light unit (RLU). The reporter gene cell lysate is used as a blank control. After completing the above firefly luciferase measurement, add 100 μl of Renilla luciferase detection working solution, mix well, and measure the RLU. Divide the RLU value obtained by the firefly luciferase measurement by the RLU value obtained by the Renilla luciferase measurement under the condition of Renilla luciferase as an internal reference.

[0083] Statistical analysis and data processing were performed using GraphPad Prism 8.0.2 statistical software. Data are expressed as mean ± standard deviation (SD). If the data meet the normal distribution and homogeneity of variance, the independent sample t test is used for comparison between two groups, the one-way analysis of variance (ANOVA) is used for comparison of means of multiple groups, the Pearson correlation test is used for correlation analysis, and P < 0.05 is considered statistically significant. The results are shown in Figure 2B .

[0084] 2. Experimental results:

[0085] From Figure 2A It can be seen from the results that by synthesizing the wild type and mutant sequences of the SRC gene, the pGL6-miR-SRC-3'UTR-WT and pGL6-miR-SRC-3'UTR-Mut luciferase reporter plasmids were constructed by cloning the SRC gene wild type and mutant sequences into the pGL6-miR vector backbone.

[0086] From Figure 2BThe results show that the dual-luciferase reporter system detection results show that, compared with the wild type recombinant plasmid (pGL6-miR-SRC-3'UTR-WT) +mimics NC group, the relative luciferase activity of the wild type recombinant plasmid (pGL6-miR-SRC-3'UTR-WT) +gek-miR-2862 mimics group is reduced, and the difference is statistically significant (P<0.05); and when the potential binding site of the SRC gene 3'UTR is mutated, the relative luciferase activity of the mutant plasmid (pGL6-miR-SRC-3'UTR-Mut) +gek-miR-2862 mimics group and the mutant plasmid (pGL6-miR-SRC-3'UTR-Mut) +mimics NC group has no significant change, and the difference is not statistically significant (P>0.05), which shows that the gek-miR-2862 and the mimics NC cannot inhibit the mutant plasmid luciferase activity. The above results show that the gek-miR-2862 can bind to the SRC gene 3'UTR, and can specifically act on the site to inhibit the expression of the SRC gene. Note: ** indicates that compared with the NC group, P<0.01.

[0087] III. Study on the effect of gek-miR-2862 mimics on the expression of oncogene SRC and downstream genes of SRC pathway in hepatocarcinoma cells

[0088] 1. Experimental materials and methods:

[0089] 1.1 Experimental materials:

[0090] Human hepatocarcinoma cells, gek-miR-2862 mimics and negative control are the same as in Example 1.

[0091] 1.2 Experimental method:

[0092] Specific steps:

[0093] (i) Transfecting gek-miR-2862 into HepG2 and Huh7 hepatocarcinoma cells: HepG2 and Huh7 hepatocarcinoma cells were cultured in a 6-well plate, and the gek-miR-2862 negative control (NC) and gek-miR-2862 mimics (mimics) were transfected into the HepG2 and Huh7 hepatocarcinoma cells. The amount of mixture added to each well of the 6-well plate was 500 μl Opti-MEM + 2.5 μl lipid, and 10 μl mimics (the same volume of culture medium was removed before adding). The cells were collected after 24 hours for determination of SRC mRNA and protein content.

[0094] (ii) Real-time quantitative PCR (RT-PCR) detection of SRC mRNA expression level:

[0095] ① Extraction of RNA:

[0096] Sample preparation: 200 μl Trizol was added to each well of 24-well plate, and pipette was used to aspirate several times to promote cell lysis, and then the sample was moved to a centrifuge tube.

[0097] Phase separation: incubation at room temperature for 5 minutes to facilitate complete separation of ribosomes in the homogenate sample. Add 40 μl of chloroform, cover the sample cover with hand, shake the tube for 15 seconds, incubate at room temperature for 3 minutes. 4,000 g centrifugation at 12,000 g for 15 minutes, after centrifugation, the mixture is separated into red lower layer (phenol-chloroform phase), middle phase and upper colorless water phase (RNA exists in this water phase).

[0098] RNA precipitation: transfer the water phase to a new centrifuge tube and add isopropanol (0.5 ml isopropanol is used for every 1 ml of the initial homogenized Trizol reagent), to precipitate RNA from the water phase, incubate the sample at room temperature for 10 minutes, centrifuge at 4,000 g for 10 minutes. Usually the RNA precipitate is not visible before centrifugation, and a gel-like precipitate is formed on the side and bottom of the tube after centrifugation.

[0099] RNA washing: discard the supernatant, wash the RNA precipitate with 75% ethanol once (at least 1 ml of 75% ethanol is used for every 1 ml of the initial homogenized Trizol reagent), vortex mix, centrifuge at 4,000 g for 5 minutes.

[0100] RNA resuspension: discard the supernatant, air dry at room temperature for 10 minutes, add 40 μl of enzyme-free water to dissolve, incubate at 60°C in a metal bath for 10 minutes, store the extracted RNA at -80°C to avoid repeated freeze-thawing. Use a microplate reader to determine the concentration and purity of the total RNA extracted, and extract total RNA (concentration greater than 100 ng / ul, 260 / 280 ratio between 1.8-2.0) for subsequent experiments.

[0101] ② First-strand cDNA synthesis:

[0102] According to the following Table 5, configure the reaction system, all operations must be performed on ice. Melt the required reagents on ice, centrifuge briefly before opening to concentrate the reagents at the bottom of the tube, to avoid loss and contamination when opening. Incubate the reaction system at 42°C in a metal bath for 5 min, and place it on ice after the reaction is completed.

[0103] Reverse transcription reaction: Prepare the reverse transcription reaction system according to Table 6 below. All operations are performed on ice. After gently mixing the reverse transcription reaction system, centrifuge it, incubate it in a metal bath at 50°C for 15 min, and then incubate it in a metal bath at 75°C for 5 min to terminate the reaction. The reaction product can be directly used for RT-PCR. If it is not used immediately, it should be stored at -20°C, and stored at -80°C for a long time.

[0104] Table 5. Reaction System

[0105]

[0106] Table 6. Reverse Transcription Reaction System

[0107]

[0108] ③ Real-time quantitative PCR:

[0109] The synthesized first-strand cDNA was directly used for RT-PCR. A 20 μl reaction system was prepared according to Table 7 below. All operations were performed on ice. The resulting reaction system was centrifuged to avoid generating bubbles. The reaction program was set using a Roche PCR instrument according to the reaction conditions in Table 8 below.

[0110] Table 7. Reaction System

[0111]

[0112] Table 8. Setting of reaction conditions

[0113]

[0114] The primer sequences for the target SRC gene are as follows:

[0115] Forward primer: 5'-GCTGGCTTCTGCTGTTGA-3' (SEQ ID NO.8)

[0116] Reverse primer: 5'-GAGGATFFTCAGGTTGTGC-3' (SEQ ID NO.9)

[0117] According to 2 -△△ct The relative expression levels of each target gene were calculated using the formula. The experiment was repeated three times. The results are shown below. Figure 3A .

[0118] (iii) The expression levels of SRC and downstream related genes and proteins in the SRC pathway were detected by Western blotting.

[0119] Specific steps:

[0120] (1)Discard the culture medium, rinse twice with pre-cooled PBS, mix RIPA lysis buffer and phosphatase inhibitor at a ratio of 1:99, add 300 μl of the mixture to each well, and then repeatedly blow and beat with a pipette gun. Then, transfer the mixture to a centrifuge tube, incubate on ice for 20 min, and centrifuge at 4,000 rpm for 14,000 rpm. Transfer the supernatant to a new tube;

[0121] (2) Dilute the BSA standard according to the data in Table 9 below, and prepare the BCA working solution according to formula (I):

[0122] BCA working solution total amount = (number of standard samples + number of sample samples) x 3 x 200 μL (I)

[0123] Each sample has 3 replicate wells. According to the calculated total amount of BCA working solution, prepare BCA-A and BCA-B working solution at a volume ratio of 50:1, and mix well;

[0124] Table 9 BSA standard dilution system

[0125]

[0126] (3) Add 25 μL of prepared BSA standard and protein sample to be tested in the 96-well plate, and then add BCA working solution (200 μL / well). Incubate the 96-well plate in a 37°C constant temperature incubator for 30 min, and then cool to room temperature;

[0127] (4) Use the enzyme marker to detect the absorbance of the sample and the BSA standard at 570 nm, draw a standard curve, calculate the protein concentration in the sample, and quantify it;

[0128] (5) After quantification, normalize the protein concentration of each sample, and add SDS-PAGE protein loading buffer at a ratio of 4:1. Cook the protein in a metal bath at 100°C for 10 minutes;

[0129] (6) Prepare the protein pre-gel (4-12%), fix it in the slot (place the high side outside), remove the green comb and place it in the electrophoresis tank, add the electrophoresis solution, and add Marker and protein sample (10 μL / well) to each lane. First, set the electrophoresis instrument to 160V and run for 10 min, then set it to 120V and run for 30 min. Stop electrophoresis when the bromophenol blue band runs to the bottom of the glass plate;

[0130] (7) Wet transfer method is used for transferring film. The sponge is immersed in the transfer solution in advance and balanced for 10 min. The PVDF film is cut into appropriate size, activated with methanol for 1 min, and completely covered on the prepared glue. Avoid bubbles between the glue and the film. After clamping the clamp, put it into the electric transfer instrument, set the parameters as 400 mA, 30 min;

[0131] (8) The blocking solution (0.5 g of skimmed milk powder + TBST 10 ml) is configured, and the PVDF is soaked in the blocking solution, incubated in a constant temperature shaker at 37°C for 60 min. If the detected protein is a phosphorylated protein, replace the skimmed milk powder with BSA blocking solution;

[0132] (9) Discard the blocking solution, wash the film with TBST for 3 times, 10 min each time. Add the prepared primary antibody (purchased from Abeam company), and incubate overnight at 4°C. Recover the primary antibody, wash the film with TBST for 3 times, 5 min each time, add the secondary antibody (goat anti-rabbit IgG-HRP secondary antibody, purchased from China white shark biology), and incubate at 37°C in a constant temperature shaker for 60 min. Mix ECL A liquid and B liquid at a ratio of 1:1, place the PVDF in the mixed solution for 1 min, and then put it into the developing instrument to take a photo. The results are shown in Figure 3B .

[0133] 2、Experimental results:

[0134] HepG2 and Huh7 liver cancer cells are transfected with 100 nM gek-miR-2862 mimic and NC mimic, and after 48 hours of intervention, the expression of SRC mRNA and protein is detected by real-time fluorescence quantitative PCR and Western blot respectively. From Figure 3A It can be known from the results that after the liver cancer cells are intervened by the gek-miR-2862 mimic, the mRNA expression of SRC is obviously decreased. From Figure 3B It can be known from the results that after the liver cancer cells are intervened by the gek-miR-2862 mimic, the SRC and SRC pathway related gene protein expression is decreased (note: *** indicates that compared with the NC group, P<0.001, **** indicates that compared with the NC group, P<0.0001). That is, the SRC mRNA and protein expression level in the Huh7 and HepG2 cells and the SRC pathway related gene protein expression can be inhibited by the gek-miR-2862 mimic.

[0135] The present application is not limited to the above specific embodiments, and various modifications made by those skilled in the art without creative labor, all of which fall within the scope of the present application.

Claims

1. The application of the gek-miR-2862 mimic in the preparation of drugs for the treatment of liver tumor metastases, characterized in that, The gek-miR-2862 mimics are microRNAs that mimic the activity of gek-miR-2862, named gek-miR-2862 mimics. The gek-miR-2862 mimics consist of a sense strand and an antisense strand. The nucleotide sequence of the sense strand is 5'-CGGGGAGGUGGAGCCUGGG-3' (SEQ ID NO.2), and the nucleotide sequence of the antisense strand is 3'-GCCCCUCCACCUCGGACCC-5' (SEQ ID NO.3).

2. The application according to claim 1, characterized in that, The gek-miR-2862 mimic can inhibit the target gene SRC, further suppressing cancer cell invasion and migration.

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