PPP6R1 Gene, Its RNAi Lentiviral Interference System and Application

Abstract

The present invention discloses the PPP6R1 gene, its RNAi lentiviral interference system and applications, relating to the field of biomedical technologies. The present invention discloses that the PPP6R1 gene / protein is a target for the precise treatment of colorectal cancer, and is used to prepare a drug for treating colorectal cancer, for inhibiting the proliferation activity, migration ability and invasion ability of colorectal cancer cells. The constructed RNAi lentiviral interference system of the PPP6R1 gene has the sequences: PPP6R1-sh1: 5'-CGCCATGTTTTGGAAGTTTGACC-3', PPP6R1-sh2: 5'-ATCCAAAACCCTGTTGTGAAACA-3'. It is used in the application for preparing a drug for treating colorectal cancer, for inhibiting the proliferation activity, migration ability and invasion ability of colorectal cancer cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and more specifically, relates to the PPP6R1 gene, its RNAi lentiviral interference system, and applications. Background Art

[0002] Immunotherapy for colorectal cancer can, to a certain extent, slow down the progression of the disease, but its efficacy is mainly limited to patient groups with microsatellite instability characteristics. Although there have been a large number of research results on the evolution mechanism and prognosis of colorectal cancer, including bioinformatics databases mainly based on transcriptome data, currently, there is still an urgent need for key protein markers that can be used for diagnosis and treatment in clinical practice.

[0003] The PPP6R1 gene, also known as SAPS1, is an important regulatory subunit of protein phosphatase 6 (PPP6). Protein phosphatase 6 is a phosphatase widely present in cells and is involved in regulating various biological processes, including the cell cycle, signal transduction, and gene expression, etc. As a regulatory subunit of PPP6, SAPS1 can regulate the catalytic activity of PPP6, thereby affecting these biological processes. At the molecular mechanism level, the SAPS1 protein binds to the catalytic subunit (PPP6C) of PPP6 to form the active form of the holoenzyme. This binding can regulate the phosphatase activity of PPP6C, and thus affect the phosphorylation state of its substrates. Phosphorylation is a key protein modification method that can regulate the activity, stability, and subcellular localization of proteins, etc. Therefore, SAPS1 can indirectly affect the functions of numerous proteins during the process of regulating the activity of PPP6C.

[0004] Currently, the specific relationship between PPP6R1 and colorectal cancer is still unclear, and its expression pattern and regulatory mechanism in colorectal cancer also need further research. Summary of the Invention

[0005] Aiming at the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide an RNAi lentiviral interference system for the PPP6R1 gene. Another technical problem to be solved by the present invention is to provide the application of the RNAi lentiviral interference system for the PPP6R1 gene in the preparation of drugs for treating colorectal cancer.

[0006] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] Application of the PPP6R1 gene in the preparation of drugs for treating colorectal cancer.

[0008] The treatment of colorectal cancer is to inhibit the proliferation activity of colorectal cancer cells.

[0009] The treatment of colorectal cancer is to inhibit the migration ability of colorectal cancer cells.

[0010] The treatment of colorectal cancer mentioned above is to inhibit the invasion ability of colorectal cancer cells.

[0011] The biomarker for the diagnosis, treatment or prognosis judgment of colorectal cancer is the PPP6R1 gene.

[0012] The kit for the diagnosis, treatment or prognosis judgment of colorectal cancer contains the PPP6R1 gene sequence.

[0013] The application of the RNAi lentiviral interference system of the PPP6R1 gene in the preparation of drugs for the treatment of colorectal cancer, and the sequence of the RNAi lentiviral interference system is as follows:

[0014] PPP6R1-sh1: 5'-CGCCATGTTTTGGAAGTTTGACC-3',

[0015] PPP6R1-sh2: 5'-ATCCAAAACCCTGTTGTGAAACA-3'.

[0016] The RNAi lentiviral interference system of the PPP6R1 gene mentioned above inhibits the expression of PPP6R1 protein in colorectal cancer cells with the PPP6R1 gene as the target.

[0017] The RNAi lentiviral interference system of the PPP6R1 gene mentioned above knocks down the PPP6R1 gene in colorectal cancer cells with the PPP6R1 gene as the target.

[0018] The RNAi lentiviral interference system of the PPP6R1 gene mentioned above silences the PPP6R1 gene in colorectal cancer cells with the PPP6R1 gene as the target.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1) The present invention discloses that the PPP6R1 gene / protein is a target for the precise treatment of colorectal cancer, and the drugs prepared for the treatment of colorectal cancer are used to inhibit the proliferation activity, migration ability and invasion ability of colorectal cancer cells.

[0021] 2) The RNAi lentiviral interference system of the PPP6R1 gene constructed by the present invention, the sequence thereof is: PPP6R1-sh1: 5'-CGCCATGTTTTGGAAGTTTGACC-3', PPP6R1-sh2: 5'-ATCCAAAACCCTGTTGTGAAACA-3'. The application in the preparation of drugs for the treatment of colorectal cancer is used to inhibit the proliferation activity, migration ability and invasion ability of colorectal cancer cells.

[0022] 3) After the RNAi lentiviral interference system constructed by the present invention for the PPP6R1 gene was transfected into cells, compared with the control group (please add the construction method of the control group during the experiment), the relative protein expression level of PPP6R1 in the cells treated with the RNAi lentiviral interference system was significantly reduced. This result indicates that the protein expression of PPP6R1 in SW1116 cells and DLD-1 cells was effectively inhibited.

[0023] 4) After the RNAi lentiviral interference system constructed by the present invention for the PPP6R1 gene was transfected into cells, after knocking down PPP6R1 (sh-PPP6R1), the proliferation viability, migration ability and invasion ability of SW1116 cells and DLD-1 cells were significantly reduced. Description of the Drawings

[0024] Figure 1 Expression map of PPP6R1 protein in colorectal cancer tissues (A is the volcano map of the protein content difference detected by DIA-MS in 6 pairs of colorectal cancer patient tissues and their corresponding adjacent tissues; B is the fluorescence staining map of PPP6R1 in colorectal cancer tissues and their corresponding adjacent tissues; C is the statistical chart of fluorescence intensity; D is the relationship map between the high and low groups of PPP6R1 protein expression and the prognosis of colorectal cancer patients)

[0025] Figure 2 Expression and virus infection efficiency verification map of PPP6R1 protein in each cell line (A is the expression map of PPP6R1 protein in NCM460 cells, HCT116 cells, LoVo cells, SW480 cells, DLD-1 cells, SW1116 cells, HCT15 cells, CaCo2 cells and SW620 cells; B is the expression map of PPP6R1 protein in DLD-1 and SW1116 cells after knocking down the PPP6R1 gene; C-D is the expression map of PPP6R1 protein in CaCo2 and HCT15 cells after knocking down the PPP6R1 gene);

[0026] Figure 3 Structural diagram of the lentiviral vector;

[0027] Figure 4 Effect diagram of knocking down or overexpressing the PPP6R1 gene on the proliferation function of colorectal cancer cells;

[0028] Figure 5 Effect diagram of knocking down or overexpressing the PPP6R1 gene on the migration function of colorectal cancer cells;

[0029] Figure 6 Effect diagram of knocking down or overexpressing the PPP6R1 gene on the invasion function of colorectal cancer cells. Detailed Embodiments

[0030] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are all conventional means well known to those skilled in the art.

[0031] The main reagents, consumables, and instruments used in the following embodiments are as follows:

[0032] The cancer tissues and adjacent normal tissues of 6 colorectal cancer patients were randomly retrieved from the clinical samples stored in the biobank of the Affiliated Hospital of Nantong University.

[0033] Opal 7-color immunohistochemistry kit (PerkinElmer, USA).

[0034] Anti-human PPP6R1 antibody (17819-1-AP, Proteintech, Wuhan, China).

[0035] Anti-CK antibody, colorectal cancer cell lines (HCT116, LoVo, SW480, DLD-1, SW1116, HCT15, SW620), intestinal epithelial cell line (NCM460), and human embryonic kidney cell line (HEK293T) were purchased from Nanjing Kebai Biotechnology Co., Ltd.

[0036] RPMI-1640 medium, DMEM high-glucose medium, and fetal bovine serum (Gibco, USA).

[0037] The main instruments used in the following embodiments are as follows:

[0038] Multispectral pathology scanning system: PerkinElmer, USA.

[0039] Inverted fluorescence microscope: Carl Zeiss, Germany.

[0040] Gel imaging system: Tianneng, China.

[0041] Multifunctional microplate reader: Thermo, USA.

[0042] Example 1

[0043] 1. Fabricate tissue microarrays

[0044] 1) Preparation of pathological tissue sections

[0045] Take fresh colorectal cancer tissue blocks (with a thickness of 0.5 cm) resected surgically and fix them in a pre-prepared 10% formalin solution. Subsequently, dehydrate them through alcohol with gradient concentrations until they are cleared with xylene. Immerse the cleared tissue blocks in melted paraffin, and after the paraffin has completely penetrated the tissue blocks, perform embedding. After it cools and solidifies, perform serial sectioning (with a thickness of 5 - 8 μm), and place the sections in an incubator at 45°C for drying.

[0046] 2) HE staining procedure

[0047] After treating the sections with distilled water, place them in hematoxylin aqueous solution for staining. Subsequently, perform differentiation treatment using hydrochloric acid alcohol and ammonia water. After rinsing with running water for 1 hour, briefly soak the sections in distilled water. Then, dehydrate them through alcohol gradients of 70%, 75%, and 90%, each lasting for 5 minutes. Next, place the sections in eosin staining solution for staining, lasting for 2 - 3 minutes. The stained sections are dehydrated again through gradient alcohol and cleared with xylene. Finally, drop neutral balsam, cover with a coverslip for mounting, and observe under a microscope to determine the tumor area.

[0048] 3) Tissue microarray fabrication

[0049] Mix paraffin and beeswax in a ratio of 1:1 to fabricate blank recipient wax blocks. Design a tissue array of 10×7 holes on the wax blocks, with a total of 350 points. Use a tissue microarrayer to take out the marked representative tumor areas (with a diameter of 2 mm) from the donor wax blocks, taking 1 core from each case. Transfer the taken tissue cores to the holes of the recipient wax blocks, and add corresponding non-tumor intestinal tissues as controls. Heat and fuse the tissue array blocks in an incubator at 55°C for 10 minutes, then take them out before they melt quickly and cool to room temperature to make the recipient wax blocks tightly combine with the donor tissues. Freeze the tissue microarray at 4°C for 4 hours, then use a fully automatic tissue slicer for correction at a speed of 20 mm / revolution until all tissue cores are completely exposed. Then, use the slicer to section the tissue array blocks, float the serial sections in cold water to unfold naturally, and then transfer them to warm water at 45°C for spreading for 2 minutes. After the sections are unfolded, stick them on glass slides treated with anti - detachment agent and let them dry. Finally, bake the sections in an environment at 60°C for 3 minutes, and then continue to bake at 58°C for 16 hours.

[0050] 2. Immunofluorescence staining

[0051] 1) Place the cut paraffin tissue microarray on a baking instrument, first bake it at 70°C for 1 hour, and then bake it at 60°C for 1 hour.

[0052] 2) Immerse the dried tissue microarray into xylene twice successively, with each immersion lasting for 5 minutes. After taking it out, perform gradient alcohol dehydration treatment in sequence: 100% ethanol for 5 minutes, 95% ethanol for 5 minutes, and 75% ethanol for 5 minutes. Finally, rinse the tissue microarray with distilled water.

[0053] 3) Place the tissue microarray on a high-temperature resistant section rack and immerse it in AR6 repair solution with a pH of 6.0. Perform high-temperature antigen repair. First, heat it at 100% power for 2.5 minutes, and then heat it at 20% power for 15 minutes.

[0054] 4) After it naturally cools down to room temperature, take out the microarray from distilled water and rinse it 3 times with PBS, 2 minutes each time. Then, use an immunohistochemistry pen to mark the approximate tissue area on the tissue microarray and add 200 μL of primary antibody blocking solution and block for 10 minutes.

[0055] 5) Add 200 μL of rabbit anti-human PPP6R1 monoclonal antibody working solution (dilution ratio is 1:100) to the tissue microarray and incubate it overnight at 4°C.

[0056] 6) The next day, take out the tissue microarray and let it warm up for half an hour. After recovering the primary antibody, rinse it 3 times with PBS, 2 minutes each time. Then, take out the microarray and spin it dry.

[0057] 7) Add 200 μL of secondary antibody working solution to the tissue microarray and incubate it at room temperature for 10 minutes. Then, rinse it 3 times with PBS, 2 minutes each time. Then, take out the microarray and spin it dry.

[0058] 8) According to the wavelength of the fluorescent dye to be prepared, add an appropriate amount of fluorescent dye to the tissue microarray. Incubate it at room temperature for 10 minutes in the dark. After that, rinse it 3 times with PBS, 2 minutes each time.

[0059] 9) If it is necessary to continue incubating the second antibody, repeat the high-temperature antigen repair step. If no more antibody incubation is needed, after drying and making it transparent, perform a mounting treatment with DAPI.

[0060] 10) Images of each sample were captured at 20x magnification using Vectra 3 imaging software. The images were deeply analyzed and scored by inForm 26.1.0 (Perkin Elmer) software, and the positive or negative threshold for each cell was set. Subsequently, the percentage of cells in each region was calculated and scored (range 0 - 100). The final staining score of PPP6R1 was obtained by multiplying the staining intensity by the stained area of positive cells. Using X-tile software, the cut-off point of PPP6R1 expression score was determined according to the survival time and survival status. The scoring rules were as follows: 0 - 42.50 was low or no expression, while 42.51 - 100 was high expression. All data were processed by statistical software SPSS V.25.0. Measurement data were expressed in the form of mean ± standard deviation, and one-way ANOVA was used for comparison between groups. To explore the relationship between PPP6R1 expression and the prognosis of colorectal cancer patients, Kaplan-Meier survival analysis was performed. All test results were judged to be statistically significant with P < 0.05 as the criterion.

[0061] The results were as Figure 1 shown, and staining at specific cell sites was regarded as a positive manifestation ( Figure 1 A). Compared with non-tumor intestinal tissues, the expression level of PPP6R1 was higher in colorectal cancer tissues ( Figure 1 B, C). Among tumor patients, those with high expression of PPP6R1 had a shorter survival time and a worse prognosis ( Figure 1 D).

[0062] Example 2

[0063] 1. Colorectal cancer cell lines

[0064] The colorectal cancer cell lines DLD-1 cells, SW1116 cells, HCT116 cells, HCT15 cells, and CaCo2 cells were cultured in RPMI-1640 complete medium; LoVo cells, SW480 cells, and SW620 cells were cultured in DMEM high-glucose complete medium; and the intestinal epithelial cell line NCM460 cells were cultured in RPMI-1640 complete medium. The temperature was maintained at 37°C and the CO2 humidity was 5% in the incubator. Conventional passage culture was performed, and the medium was changed every 2 - 3 days. Cells in the logarithmic growth phase were selected for experiments.

[0065] 2. Extraction of total cellular protein

[0066] 1) Target cells were accurately taken out from the incubator at a constant temperature of 37°C.

[0067] 2) Remove the culture medium, and then wash the cells twice with pre-cooled PBS. After thoroughly removing the PBS, carefully aspirate the residual solution using a pipette, ensuring not to dilute the cellular proteins.

[0068] 3) Add an appropriate amount of RIPA cell lysis buffer according to the specifications of the cell culture flask and the cell growth density. Subsequently, gently collect the cells using a cell scraper and transfer them to a clean EP tube.

[0069] 4) Let the collected cellular proteins stand on ice to ensure sufficient lysis for 20 - 30 minutes.

[0070] 5) Centrifuge the samples at 4°C at a speed of 12,000 r for 15 minutes.

[0071] 6) After centrifugation, take the supernatant and accurately determine the concentration of the cellular proteins using the BCA method. Subsequently, add an appropriate amount of loading buffer, and pipette thoroughly to mix evenly. Boil at 95°C for 5 minutes, aliquot, and store in an -80°C refrigerator for subsequent use.

[0072] 3. Western blot

[0073] 1) Prepare a polyacrylamide gel, including a 5% stacking gel and a 12.5% separating gel.

[0074] 2) Ensure that the glass plates are clean and stain-free. After tilting to dry, assemble the glass plates. Subsequently, add the separating gel to 2 cm from the upper end of the glass plates and immediately seal it with isopropanol. Let it stand for 30 minutes to ensure that the separating gel is completely solidified. After the separating gel solidifies, gently remove the upper layer of isopropanol, then add the stacking gel to the top of the glass plates and immediately insert the comb. Let it stand for another 30 minutes until the stacking gel is completely solidified.

[0075] 3) Place the prepared gel into the electrophoresis tank and fill it with electrophoresis buffer. Subsequently, load Protein Maker and the extracted protein samples, and then add the remaining electrophoresis solution. Connect the power supply, set the initial voltage to 80V. After the Proteinmarker is separated, increase the voltage to 100V. After electrophoresis, take out the gel and accurately cut the target band.

[0076] 4) Cut a PVDF membrane of appropriate size, polarize it in methanol for about 30 seconds first, and then put it into the membrane transfer solution. At the same time, soak the sponge and filter paper in the membrane transfer solution for about 20 minutes. Install the membrane transfer device in the following order: cathode carbon plate, sponge, filter paper, gel, PVDF membrane, filter paper, sponge, anode carbon plate. Place the membrane transfer device in the transfer tank, add ice packs and enough membrane transfer solution. Connect the power supply and perform wet transfer at a constant current of 300 mA for 0.5 hours. The whole process needs to be carried out in an ice box.

[0077] 5) After the membrane transfer is completed, put the PVDF membrane into the blocking solution (made by dissolving 5 g of non-fat milk powder in 100 mL of TBST), and block it at room temperature on a shaker at a speed of 80 r / min for 2 hours.

[0078] 6) After the blocking is completed, prepare the primary antibody dilution solution with the blocking solution according to the dilution ratio of the primary antibody. Uniformly drip the diluted primary antibody on the PVDF membrane, and then incubate it overnight at 4 °C.

[0079] 7) The next day, wash the membrane 3 times with TBST, 15 minutes each time. After the membrane washing is completed, prepare the secondary antibody dilution solution with TBST according to the dilution ratio of the secondary antibody. Uniformly drip the diluted secondary antibody on the PVDF membrane and incubate it at room temperature for 1.5 hours. After the incubation is completed, wash the membrane again 3 times with TBST, 15 minutes each time.

[0080] 8) After the membrane washing is completed, blot the moisture on the PVDF membrane with filter paper, and then lay it flat at the corresponding position of the imager. Mix the A liquid and B liquid of the ECL luminescent solution in equal proportions, and uniformly drip it on the PVDF membrane. Use the gel imaging system to take pictures and save the images.

[0081] Extract the proteins of NCM460 cells, HCT116 cells, LoVo cells, SW480 cells, DLD-1 cells, SW1116 cells, HCT15 cells, CaCo2 cells and SW620 cells according to the above method, and detect the expression of PPP6R1 in each cell by Western blot to screen for high and low expression cells.

[0082] The results are as Figure 2 shown in A. The expression of PPP6R1 protein is relatively high in SW1116 and DLD-1 colorectal cancer cells, and relatively low in CaCo2 and HCT15 colorectal cancer cells.

[0083] Example 3

[0084] 1. Construct an RNAi lentiviral interference system for the PPP6R1 gene

[0085] For the PPP6R1 gene sequence (Gene ID: 22870, Protein ID: Q9UPN7), design an RNAi lentiviral interference system that specifically targets the PPP6R1 gene. The sequences used to construct the lentivirus-mediated RNAi interference system are as follows:

[0086] PPP6R1-sh1: 5'-CGCCATGTTTTGGAAGTTTGACC-3',

[0087] PPP6R1-sh2: 5'-ATCCAAAACCCTGTTGTGAAACA-3'.

[0088] Add a stem-loop structure (CTCGAG) to the above sequences, supplement the palindromic sequence and restriction enzyme sites, obtain the interference sequence fragments through the annealing and purification processes of the designed sequences, and ligate them to the RNAi lentiviral interference system vector using DNA ligase.

[0089] 2. Lentivirus infection of cells and screening of stable strains

[0090] Co-transfect 2.5 μg of the recombinant vector plasmid with 2 μg of psPAX2 and 1 μg of pMD2.G into HEK293T cells and collect the supernatant after 48 hours, which is the successfully packaged lentiviral particles. Determine the lowest usage amount of the virus with an infection efficiency greater than 90% as the appropriate virus infection concentration by gradient dilution of the virus stock solution at a ratio of 1:2. Take the adherent target cells (SW1116 cells and DLD-1 cells) with a density of 60%, replace them with the virus diluted with complete medium for culture treatment, which is the lentivirus infection process. Replace the culture medium 12 - 16 hours after infection and continue culturing. 72 - 96 hours after infection, observe the fluorescence expression by inverted fluorescence microscopy, and add 1 μg / mL of puromycin to the infected cells for drug treatment to eliminate the influence of uninfected cells.

[0091] 3. Expression of PPP6R1 protein in monoclonal strains after knocking down the PPP6R1 gene

[0092] Using the limiting dilution method, dilute the cells to 1000 cells / mL, then dilute them at a dilution gradient of 1:10 and seed them into 10 96-well plates to prepare monoclonal strains, with a final cell concentration of 1 cell / well, and then culture them normally. After one week, observe the growth of the monoclonal strains, and transfer the well-grown monoclonal strains to 48-well plates for expansion culture after about two weeks. Subsequently, transfer the monoclonal strains to 24-well plates and 12-well plates for further expansion culture in turn. After each monoclonal strain is expanded in two 12-well plates, take out the cells from one well, perform cell lysis to extract proteins, and use Western blot to detect the relative protein expression of PPP6R1 in the monoclonal strains after knocking down the PPP6R1 gene.

[0093] The results are as Figure 2 shown in B. Compared with the control group (the original vector was the control group), the relative protein expression level of PPP6R1 was significantly decreased in the cells treated with the RNAi lentiviral interference system. These results indicate that the protein expression of PPP6R1 in SW1116 cells and DLD-1 cells was effectively inhibited.

[0094] Example 4

[0095] 1. Construction of an overexpression vector for the PPP6R1 gene

[0096] Using the head-to-tail primer amplification method and relying on gene recombination technology, the PPP6R1 expression fragment was recombined onto the pCDH-CMV-MCS-EF1-EGFP-2A-puro vector ( Figure 3 ) to obtain the open reading frame region of PPP6R1 mRNA.

[0097] 2. Screening of overexpressing cells and stable cell lines

[0098] 2.5 μg of the recombinant vector plasmid was co-transfected with 2 μg of psPAX2 and 1 μg of pMD2.G into HEK293T cells, and the supernatant was collected after 48 hours, which was the successfully packaged lentiviral particles. By performing gradient dilution of the virus stock solution at a ratio of 1:2, the lowest usage amount of the virus with an infection efficiency greater than 90% was determined as the appropriate virus infection concentration. The adherent target cells (CaCo2 cells and HCT15 cells) with a density of 60% were taken, and the medium was replaced with the virus diluted with complete medium for culture treatment, which was the lentiviral infection process. The culture medium was replaced 12 - 16 hours after infection and continued to be cultured. 72 - 96 hours after infection, the fluorescence expression was observed through an inverted fluorescence microscope, and 1.0 μg / mL of puromycin was added to the infected cells for drug treatment to eliminate the influence of uninfected cells.

[0099] 3. Expression of PPP6R1 protein in monoclonal cell lines after overexpression of the PPP6R1 gene

[0100] Using the limited dilution method, the cells were diluted to 1000 cells / mL, and then seeded into 10 96-well plates at a dilution gradient of 1:10 to prepare monoclonal strains. The final cell concentration was 1 cell / well, and then they were cultured normally. After one week, the growth of the monoclonal strains was observed, and the well-growing monoclonal strains were transferred to 48-well plates for expansion culture after about two weeks. Subsequently, the monoclonal strains were successively transferred to 24-well plates and 12-well plates for further expansion culture. After each monoclonal strain was expanded in 2 12-well plates, the cells in one well were taken out for lysis to extract proteins, and the relative protein expression of PPP6R1 in the monoclonal strains after overexpressing the PPP6R1 gene was detected by Western blot method.

[0101] The results are as Figure 2 shown in C. Compared with the control group (the original vector was the control group), the relative protein expression level of PPP6R1 in the cells after the overexpression treatment was significantly increased. This result indicates that the protein expression of PPP6R1 in CaCo2 cells and HCT15 cells was effectively upregulated.

[0102] Example 5

[0103] 1. Cell proliferation / clonogenic ability (monoclonal formation assay)

[0104] The CaCo2 cells and HCT15 cells overexpressing the PPP6R1 gene and the SW1116 cells and DLD-1 cells with knocked-down PPP6R1 gene were grouped and digested, centrifuged at 800 rpm at room temperature for 5 minutes; the supernatant was discarded, and the cell pellet was resuspended by centrifuging at 800 rpm at room temperature using fresh complete medium. Then, the cell number was accurately determined using a hemocytometer; at a constant cell density, 2×10 3Cells were evenly distributed in each well of a six-well culture plate. At the same time, two auxiliary wells were set for each group as controls to ensure the accuracy and reliability of the experimental results. After 4 - 8 hours, when the cells adhered to the surface of the culture plate, they were gently rinsed several times with PBS to remove non-adherent cells. Subsequently, 2 mL of fresh complete medium was replaced in each well to provide the growth environment required by the cells, and the cells were cultured for another 10 - 14 days; the medium was removed by centrifugation at 800 rpm at room temperature for 5 minutes, and the cells were washed several times with PBS. Then, the cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes to fix the cell morphology and structure. After removing the fixing solution, 1 mL of crystal violet staining solution was added to each culture dish and stained for 10 - 15 minutes. Subsequently, the staining solution was recovered, and the culture dish was washed with PBS until the staining solution was completely removed. Finally, the culture dish was left to dry at room temperature. Each culture dish was photographed one by one using a professional camera, and the number of cell clone colonies in it was counted. The data were processed and analyzed using Graphpad Prism 8 software to generate corresponding charts.

[0105] The results are as Figure 4 shown. After knocking down the PPP6R1 gene, the colony formation ability of colorectal cancer cells decreased, that is, the proliferation activity decreased; after overexpressing the PPP6R1 gene, the colony formation ability of colorectal cancer cells increased, that is, the proliferation activity increased.

[0106] 2. Cell migration (Transwell chamber method)

[0107] After stable transfection treatment, the cells collected from each group were digested and centrifuged for subsequent experiments. Subsequently, the cells were resuspended with basal medium, and the cell density was adjusted to 5×10 4 / mL. In a 24-well plate, 800 μL of complete medium was added to each well, and the chamber was placed in for sufficient infiltration. Then, 100 μL of cell suspension was added to the upper chamber. After culturing under conventional conditions for 24 hours, the chamber was taken out, washed twice with 1×PBS, fixed with 4% paraformaldehyde for 20 minutes, and then washed twice again with 1×PBS. Next, 500 μL of crystal violet staining solution was added to the 24-well plate, the chamber was placed in it, taken out after staining for 10 minutes, and washed twice with 1×PBS. Finally, the chamber was inverted, and the cells that did not pass through the upper chamber were gently wiped off with a cotton swab.

[0108] The results are as Figure 5 shown. Through inverted microscope observation, it was found that after knocking down PPP6R1 (sh-PPP6R1), the migration ability of SW1116 cells and DLD-1 cells decreased significantly; after overexpressing PPP6R1 (PPP6R1OE), the migration ability of CaCo2 cells and HCT15 cells increased significantly.

[0109] 3. Cell invasion (Transwell chamber method)

[0110] Mix 50 μL of hydrogel with 350 μL of basal medium, and then add 50 μL of basal medium. Stir well. Subsequently, add the mixture to the upper chamber of the Transwell chamber, 100 μL for each upper chamber, taking care to avoid generating air bubbles. Next, digest and collect the cells in each group 48 hours after transfection, and then perform centrifugation for subsequent use. Resuspend the cells with basal medium and adjust the cell density to 5×10 4 / mL. Add 800 μL of complete medium to a 24-well plate and place the chamber into it for sufficient infiltration. Then, take 100 μL of the cell suspension and add it to the upper chamber. After culturing the cells under normal conditions for 24 - 48 hours, take them out, wash twice with 1×PBS, fix with 4% paraformaldehyde for 20 minutes, and wash twice again with 1×PBS. Add 500 μL of crystal violet staining solution to the 24-well plate and place the chamber into it. After staining for 10 minutes, take it out and wash twice again with 1×PBS. Subsequently, invert the chamber and gently wipe off the cells that did not pass through the upper chamber with a cotton swab. Finally, observe the experimental results using an inverted microscope.

[0111] The results are as Figure 6 shown. After knocking down PPP6R1, the invasion ability of SW1116 cells and DLD-1 cells decreased; after overexpressing PPP6R1, the invasion ability of CaCo2 cells and HCT15 cells increased.

Claims

1. PPP6R1 The invention relates to an application of a RNAi lentiviral interference system for the preparation of a drug for treating colorectal cancer, characterized in that: The sequence of the RNAi lentiviral interference system is as follows: PPP6R1-sh1: 5'-CGCCATGTTTTGGAAGTTTGACC-3', PPP6R1-sh2: 5'-ATCCAAAACCCTGTTGTGAAACA-3'.

2. The use according to claim 1, characterized in that: The PPP6R1 RNAi lentiviral interference system PPP6R1 Gene as a target to inhibit PPP6R1 protein expression in colorectal cancer cells.

3. The use according to claim 1, characterized in that: The PPP6R1 RNAi lentiviral interference system PPP6R1 Gene knockdown as a target in colorectal cancer cells PPP6R1 Gene.

4. The use according to claim 1, characterized in that: The PPP6R1 RNAi lentiviral interference system PPP6R1 Gene silencing as a target in colorectal cancer cells PPP6R1 Gene.

Images