Application of SRPK3 gene as target spot in screening medicine for treating lung cancer

Abstract

The invention discloses an application of an SRPK3 gene as a target spot in screening a medicine for treating lung cancer, and belongs to the technical field of biological medicines. An interference vector of the SRPK3 gene is constructed, the influence of the SRPK3 gene on the growth of tumor cells is studied, and the silent SRPK3 gene is verified to be capable of inhibiting the growth of the tumor cells by transfecting a human tumor cell line and combining a plurality of means such as cell proliferation experiments, flow cytometry and in-vivo verification of mice. The invention reveals that the SRPK3 can be used as a potential target spot for screening the lung cancer treatment medicines, so that the application of the SRPK3 gene as the target spot in screening the lung cancer treatment medicines is provided, and a foundation is laid for further developing the targeted treatment medicines aiming at the SRPK3.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of using the SRPK3 gene as a target in screening drugs for treating lung cancer. Background Art

[0002] Lung cancer is one of the malignant tumors with the highest incidence and mortality rates globally. According to the Global Cancer Observatory (GLOBOCAN) statistics, there were approximately 2.207 million newly diagnosed lung cancer cases and about 1.796 million newly added lung cancer death cases globally in 2020. Lung cancer can be divided into small cell lung cancer and non-small cell lung cancer, and non-small cell lung cancer can be further divided into squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Most small cell lung cancers are already in the advanced stage at the time of diagnosis, with a fast growth rate, high malignancy, and the median survival time of extensive-stage small cell lung cancer is about 15 months. Compared with small cell carcinoma, non-small cell lung cancer accounts for about 80% of all lung cancers. For lung cancers with gene mutations, targeted therapy can significantly extend the survival period, up to 3 - 4 years. Without a target, the average survival period is about 1 - 2 years. This shows that the research and development of targeted drugs have an obvious promoting effect on the extension of the survival period of lung cancer, and developing new treatment targets is also a research direction for conquering lung cancer.

[0003] SRPK3 (encoding X-linked serine / arginine protein kinase 3) is a protein kinase related to the regulation of RNA splicing in the nucleus. Existing studies have shown that SRPK3 plays an important role in regulating gene expression and the occurrence and development of tumors. However, there is currently a lack of in-depth research on its mechanism of promoting tumor cell proliferation. In the existing technology, although some literature on SRPK3 mentions its high expression in certain cancers, there is no clear evidence revealing its specific functional mechanism in the proliferation of lung cancer tumor cells. Therefore, there is a lack of theoretical support for the development of anti-tumor drugs targeting SRPK3. The present invention reveals the promoting effect of the SRPK3 gene on the growth of lung cancer cells through in vitro experiments, providing a new target and possible treatment strategies for lung cancer treatment. Summary of the Invention

[0004] In response to the current clinical demand for drugs for treating lung cancer targeting new targets, the purpose of the present invention is to provide the application of using the SRPK3 gene as a target in screening drugs for treating lung cancer. The present invention discovers and experimentally proves that the SRPK3 gene has a promoting effect on the growth of lung cancer cells, which is the first report, suggesting that SRPK3 can be used as a potential target for screening drugs for treating lung cancer.

[0005] The purpose of the present invention is achieved in the following way:

[0006] In the first aspect, the present invention provides the application of using the SRPK3 gene as a target in screening drugs for treating lung cancer.

[0007] Based on the above technical solution, further, the lung cancer includes lung adenocarcinoma cell lines A549, H1299, H358, and PC9, and preferably adenocarcinoma cell lines H1299, H358, and PC9.

[0008] In a second aspect, the present invention provides an inhibitor of the SRPK3 gene.

[0009] Based on the above technical solution, further, the inhibitor includes small interfering RNA of the SRPK3 gene.

[0010] Based on the above technical solution, further, the small interfering RNA of the SRPK3 gene includes shSRPK3-1, shSRPK3-2, and shSRPK3-3. The nucleotide sequence of the coding gene of shSRPK3-1 is shown in SEQ ID NO: 3-4, the nucleotide sequence of the coding gene of shSRPK3-2 is shown in SEQ ID NO: 7-8, and the nucleotide sequence of the coding gene of shSRPK3-3 is shown in SEQ ID NO: 11-12.

[0011] In a third aspect, the present invention provides the use of the above inhibitor of the SRPK3 gene in the preparation of a drug for treating lung cancer.

[0012] Based on the above technical solution, further, the lung cancer includes lung adenocarcinoma cell lines A549, H1299, H358, and PC9, and preferably adenocarcinoma cell lines H1299, H358, and PC9.

[0013] The beneficial effects of the present invention compared with the prior art are as follows:

[0014] 1. The present invention reveals that SRPK3 has the function of promoting the growth of lung cancer: The present invention first confirms the report of SRPK3 in lung cancer cells and its role in promoting proliferation and inhibiting apoptosis, enriching the understanding of the tumorigenesis mechanism.

[0015] 2. Provide a new target for tumor treatment: As a potential therapeutic target, the SRPK3 gene is expected to prevent the proliferation and spread of tumor cells by inhibiting its function.

[0016] 3. Promote the research and development of anti-tumor drugs: The present invention provides an experimental basis for the development of SRPK3-targeted inhibitors, which helps to promote the research and development of new drugs for tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention, the drawings involved in the embodiments will be briefly introduced below.

[0018] Figure 1Figure showing the expression of SRPK3 in A549, H1299, H358, PC9, and 16HBE cell lines detected by RT-PCR / Western blot method.

[0019] Figure 2 Schematic diagram of the RNAi primer design for the target gene.

[0020] Figure 3 Schematic diagram of the pLKO.1-puro vector map and the shRNA insertion site.

[0021] Figure 4 Schematic diagram of the colony PCR identification.

[0022] Figure 5 Figure showing the SRPK3 interference efficiency results detected by qRT-PCR and Western blot in the SRPK3 gene interference lentivirus transfected H358 and PC9 cell lines;

[0023] Figure 6 Figure showing the cell viability results of lung cancer H358 and PC9 cells after SRPK3 gene interference detected by CCK8;

[0024] Figure 7 Figure showing the cell proliferation of lung cancer H358 and PC9 cells after SRPK3 gene interference detected by the plate cloning experiment.

[0025] Figure 8 Figure showing the apoptosis results of lung cancer H358 and PC9 cells after SRPK3 gene interference detected by flow cytometry.

[0026] Figure 9 Figure showing the cell proliferation and apoptosis detected by the plate cloning experiment and flow cytometry in the H358 cell line with SRPK3 interference treated with 10 μM of the AKT activator SC79.

[0027] Figure 10 Figure showing the protein expression of AKT1 and p-AKT1 in the cells of the H358 cell line with SRPK3 interference treated with 10 μM of the AKT activator SC79 detected by Western blot.

[0028] Figure 11 Figure showing the tumor appearance and volume results after the experiment of the nude mouse subcutaneous tumor model constructed with the SRPK3 gene interference (shSRPK3-1) and the empty vector in the H358 stable transfected cell line of lung cancer. Detailed implementation method

[0029] The present invention will be described in detail below in conjunction with embodiments. However, the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, without creative efforts, obtaining other similar embodiments will fall within the protection scope of the present invention.

[0030] Example 1

[0031] Lung adenocarcinoma cell lines A549, H1299, H358, PC9 and human bronchial epithelial cells 16HBE were used to detect the expression of SRPK3 by qRT-PCR and Western blot.

[0032] 1. Detect the expression of SRPK3 in A549, H1299, H358, PC9, and 16HBE cell lines by RT-PCR method.

[0033] 1) Discard the culture medium, and directly add 1 mL of Total RNA Extraction Reagent to a 3.5 cm diameter culture plate, cover and repeatedly pipette to lyse the cells.

[0034] 2) Vigorously shake the homogenized sample and place it at room temperature for 5 minutes to completely dissociate ribosomes.

[0035] 3) Add 1 / 5 volume of chloroform (such as adding 0.2 mL of chloroform to every 1 mL of Total RNA Extraction Reagent) to the above lysis solution. Tighten the centrifuge tube cap, vigorously shake for 15 sec, and let it stand at room temperature for 2 - 3 min. Subsequently, centrifuge at 4°C, 12000g for 10 - 15 min.

[0036] 4) Carefully aspirate the upper aqueous phase into a new centrifuge tube, and add 1 / 2 volume of isopropanol (such as adding 0.5 mL of isopropanol to every 1 mL of Total RNA Extraction Reagent). Invert and mix well, then let it stand at room temperature for 10 min. Subsequently, centrifuge at 4°C, 12000g for 10 min.

[0037] 5) Carefully discard the supernatant, add an equal volume of 75% ethanol (prepared with DEPC water, such as adding 1 mL of 75% ethanol to every 1 mL of Total RNA Extraction Reagent). Vortex to wash thoroughly, and flick the bottom of the tube to suspend the precipitate. Centrifuge at 4°C, 7500g for 5 min, discard the supernatant, and pay attention not to lose the RNA precipitate.

[0038] 6) Let it air dry at room temperature for 5 - 10 min. Add 30 - 100 μL of RNase-free water to dissolve the RNA. After complete dissolution, take a small amount for detection, and store the remaining solution at -80°C.

[0039] 7) Subsequently, PCR reverse transcription, amplification, and machine detection were carried out, and the results are as Figure 1 shown.

[0040] 2. Detect the expression of SRPK3 in A549, H1299, H358, PC9, and 16HBE cell lines by Western blot

[0041] 1) Total cell protein extraction: Cells were lysed with RIPA lysis buffer on ice, centrifuged at 12000 rpm at 4 °C for 10 min, and the supernatant was collected, which was the total protein solution.

[0042] 2) Protein concentration determination: The protein concentration was measured using a BCA protein concentration assay kit. The protein solution was added to 5* reducing protein loading buffer at a ratio of 4:1 and denatured in a boiling water bath for 15 min, then stored at -20 °C in the refrigerator for later use;

[0043] 3) SDS-PAGE electrophoresis: Different gel concentrations were selected according to the molecular weight of the target protein. Low-concentration gels were used for high-molecular-weight proteins, and high-concentration gels were used for low-molecular-weight proteins for separation.

[0044] 4) Loading and electrophoresis: The prepared PAGE gel was placed in the electrophoresis tank, and the loading amount per well was about 25 μg of protein. Subsequently, electrophoresis was carried out. When the dye reached the bottom of the gel, the power supply was cut off to stop electrophoresis, and the next step of membrane transfer was carried out.

[0045] 5) Immune reaction: The transferred membrane was placed in an incubation tank containing TBST. Subsequently, the diluted SRPK3 antibody and secondary antibody were added, and incubated on a shaker overnight at 4 °C. Then, ECL chemiluminescence method was used for detection, and ImageJ software was used for analysis. The results are as Figure 1 shown.

[0046] The above results indicate that SRPK3 is highly expressed in tumor cell lines H1299, H358, and PC9.

[0047] Example 2

[0048] Construct SRPK3 gene interference (Design 3 sites) Lentivirus was transfected into H358 and PC9 cell lines, and the interference efficiency of SRPK3 was detected by qRT-PCR and Western blot ( Figures 2 - 5 );

[0049] Experimental steps:

[0050] 1. Acquisition of shSRPK3 gene:

[0051] 1.1 Design of interference target for the target gene

[0052] Design specific RNAi sequences targeting the target gene SRPK3 (NM_014370.4) through an online design software (DSIR), while referring to the following principles: The length of the RNAi sequence is controlled within 20 - 23 nt; there is no reverse complementary sequence; the GC content is controlled within 30% - 50%; the 3' end of the sense strand has relatively low stability, etc.; avoid the appearance of multiple consecutive G or C, C16 (the 16th base is C), non-G13, etc. Add a loop sequence and add sticky ends with Age I and EcoR I restriction sites at both ends. The RNAi primers are designed as Figure 2 as shown.

[0053] Table 1 Design results of interference targets

[0054]

[0055] 1.2 Synthesis of interfering shRNA

[0056] Send the designed shRNA to GenePharma for synthesis.

[0057] 1.3 Annealing of the synthesized shRNA double-strand

[0058] Dissolve the obtained DNA oligo fragment in water to 20 μM. Add 5 μl of each of the dissolved sense and antisense DNA oligo fragments and 5 μl of 10x annealing Buffer to the annealing system. After mixing, place it in a 95°C water bath for 5 min, then take it out at 70°C and let it cool to room temperature naturally.

[0059] 1.4 Vector linearization and map

[0060] The vector is the pLKO.1-puro plasmid. The vector digestion reaction system is shown in Table 2.

[0061] Table 2 Vector digestion reaction system

[0062] Purified DNA plasmid (pLKO.1 - puro, 1 μg / μl) 2 μl 10× buffer 5 μl Agel I (10 U / μl) 2 μl EcoR I (10 U / μl) 2 μl <![CDATA[H 2 O]]> 39 μl Total 50 μl

[0063] Place the above mixed reactants in a 37°C water bath for 2 h.

[0064] 1.5 Recovery of linearized vector

[0065] 1) After recovering the target fragment, add 600 μL of solubilizing solution and solubilize it in a 60°C dry bath.

[0066] 2) After the gel melts, add 200 μL of isopropanol to it and cool it to room temperature.

[0067] 3) After the solubilized solution cools to room temperature, add the solubilized solution to the adsorption column, let it stand for 1 min, centrifuge at 10000 g for 1 min, and discard the effluent.

[0068] 4) Add 650 μL of Washing Buffer to the adsorption column, centrifuge at 10,000 g for 1 min, and discard the effluent.

[0069] 5) Centrifuge at 10,000 g for 2 min to remove the residual WB.

[0070] 6) Place the adsorption column in a clean 1.5 mL centrifuge tube, open the lid to allow the ethanol to evaporate; add 35 μL of ddH₂O preheated to 60 °C, let stand at room temperature for 1 min, and centrifuge at 10,000 g for 1 min to elute the DNA. 2 O, let stand at room temperature for 1 min, centrifuge at 10,000 g for 1 min to elute the DNA.

[0071] 7) Add the DNA eluted from the 1.5 mL centrifuge tube back into the adsorption column, repeat step (6) once, and more than 80% of the DNA can be collected.

[0072] 8) Take 5 μL of the recovered DNA for gel loading detection, and reserve the remaining 30 μL for ligation.

[0073] 1.6 Ligate the recovered linearized vector with the annealed shRNA. The ligation reaction system is shown in Table 3.

[0074] Table 3 Ligation reaction system

[0075] Reagent Self - ligation control (μL) Ligation group (μL) Annealed double - stranded DNA 100 ng / μl - 5 Linearized vector 40 ng / μL 5 5 10× T4 DNA ligase buffer 2 2 T4 DNA ligase 1 1 <![CDATA[dd H 2 O]]> Up to 20 Up to 20

[0076] Incubate at 22 °C for 1 h, immediately transform or store at 4 °C for later transformation.

[0077] 1.7 Transformation (for reference on transformation operation: Molecular Cloning: A Laboratory Manual, 2nd Edition, pages 55 - 56)

[0078] 1) Take a tube of competent cells DH5α (100 μl per tube, stored at -80 °C) and place it on ice. After it dissolves, add 10 μl of the ligation solution, gently rotate to mix the contents, and place it in ice for 30 min.

[0079] 2) Place the tube in a preheated 42 °C water bath for heat shock for 90 s.

[0080] 3) Quickly transfer the tube to an ice bath to cool the cells for 2 - 3 min.

[0081] 4) Add 500 μl of antibiotic-free LB culture medium to each tube, then transfer the tubes to a shaker at 37 °C and incubate at 200 rpm for 1 h to resuscitate the bacteria.

[0082] 5) Take 200 μl of the transformed bacterial solution and spread it on an LB agar plate (containing the corresponding antibiotic for the expression vector).

[0083] 6) Invert the petri dish and incubate at 37 °C in a constant temperature incubator for 16 h.

[0084] 1.8 Positive clone identification

[0085] Pick the transformants grown on the plate and resuspend them in 10 μl of LB culture medium. Take 1 μl from it as a template for colony PCR identification. The schematic diagram of colony PCR identification is as shown in Figure 3 shown, and the PCR reaction system is shown in Table 4.

[0086] Table 4 PCR reaction system (total 25 μL)

[0087] Reagent Volume (μL) <![CDATA[10×Buffer(with Mg 2+ )]]> 2.5 dNTP 2.5 mmol / L each 0.5 Primer (+) (10 μmol / L) 0.5 Primer (-) (10 μmol / L) 0.5 Taq enzyme 0.2 Template 0.5 <![CDATA[H 2 O]]> 20.3

[0088] Table 5 PCR reaction conditions

[0089]

[0090] 1.9 Endotoxin-free plasmid extraction

[0091] 1) In a 50-ml culture flask, inoculate the E. coli bacterial solution carrying the plasmid to be isolated (pLKO.1-shRNA) into 15 ml of LB medium containing ampicillin and culture it;

[0092] 2) Transfer the bacterial solution to a 10-ml centrifuge tube and centrifuge at 5000×g for 10 min at room temperature;

[0093] 3) Pour off the supernatant, and add 500 μl of RNaseA Solution I to the precipitated bacteria (mix well with a vortex oscillator to completely suspend the precipitated bacteria);

[0094] 4) Transfer the bacterial suspension to a new 2-ml centrifuge tube, and at the same time add 500 μl of Solution II, gently invert 4 - 6 times to fully lyse the bacteria;

[0095] 5) Add 250 μl of neutralization buffer (pre-cooled buffer N3), immediately gently invert several times to mix well until white flocculent precipitate appears; centrifuge at 13000×g for 10 min at room temperature;

[0096] 6) Carefully transfer the supernatant to a clean 2-ml centrifuge tube, add 0.1 volume of ETR buffer, invert 10 times to mix and incubate on ice for 10 min;

[0097] 7) Incubate the lysate from the previous step at 42 °C for 5 min, and the lysate will become turbid again. Centrifuge at 13000×g for 3 min at room temperature, and the ETR Solution will form a blue liquid layer at the bottom of the centrifuge tube;

[0098] 8) Transfer the above aqueous phase to a new 1.5 ml centrifuge tube, add 0.5 volume of absolute ethanol, gently invert the tube up and down 6 - 7 times to mix, and let it stand at room temperature for 2 min;

[0099] 9) Transfer the solution (take 700 μl) from the previous step to a clean HiBind DNA Minicolumn II, which is assembled onto a 2 ml collection tube, and centrifuge at 10000×g for 1 min at room temperature to allow the solution to pass through the adsorption column;

[0100] 10) Discard the waste liquid after passing through the column, put the adsorption column back into the collection tube, add the remaining solution that has not passed through the column to the adsorption column, and repeat the operation of the previous step;

[0101] 11) Wash the column with 500 μl Buffer HB and centrifuge at 10000×g for 1 min;

[0102] 12) Discard the waste liquid after passing through the column, put the adsorption column back into the collection tube, add 700 μl of DNA Wash Buffer diluted with ethanol to elute the adsorption column, centrifuge at 10000×g for 1 min, discard the liquid after passing through the column, and put the adsorption column back into the tube;

[0103] 13) Repeat the above elution step again;

[0104] 14) Discard the waste liquid after passing through the column, put the adsorption column back into the collection tube, and centrifuge the HiBind DNAMinicolumn II at 130000×g for 2 min again to remove the residual liquid in the adsorption column;

[0105] 15) Put the adsorption column into a new clean 1.5 ml microcentrifuge tube, directly add 60 μl of elution buffer, let it stand for 1 min, and then centrifuge at 13000×g for 1 min to elute the DNA.

[0106] 2. Recombinant interfering lentivirus packaging

[0107] 2.1 Extraction of pLKO.1 - shSRPK3 (pLKO.1 - shRNA), psPAX2, and pMD2G plasmids

[0108] Using OMGEA's Endo - free Plasmid Mini Kit I for extraction, the specific steps are as follows:

[0109] 1) Inoculate Escherichia coli containing pLKO.1 - shSRPK3, psPAX2, and pMD2G into centrifuge tubes containing 100 mL of LB (containing ampicillin) with 30 mL each, and shake - culture overnight at 37°C and 250 r / min;

[0110] 2) Take 10 mL of the bacterial solution and place it in a 10 mL centrifuge tube. Centrifuge at 5000 g for 10 min at room temperature;

[0111] 3) Discard the supernatant, add 500 μL of solution I / RNase A to the tube, and shake vigorously to fully lyse the precipitated bacteria.

[0112] 4) Add 500 μL of solution II to the above tube, invert the tube 7 - 10 times up and down, and let it stand at room temperature for 2 min;

[0113] 5) Add 250 μL of pre - cooled N3, gently invert the centrifuge tube up and down until a white flocculent precipitate appears; centrifuge at 12000 g for 10 min at 4°C;

[0114] 6) Pipette the supernatant into a 1.5 mL centrifuge tube, add ETR at 0.1 times the volume, invert the centrifuge tube 7 - 10 times up and down, incubate on ice for 10 min, incubate at 42°C for 5 min, and centrifuge at 12000 g for 3 min at 25°C;

[0115] 7) Pipette the supernatant into a new 2.0 mL centrifuge tube, add 1 / 2 times the volume of absolute ethanol, invert the centrifuge tube several times, and let it stand at room temperature for 2 min;

[0116] 8) Take 700 μL of the mixture from the previous step into the HiBind TM DNA Mini ColumnⅡ adsorption column, and centrifuge at 10000 g for 1 min at room temperature;

[0117] 9) Repeat step (8) until all the mixture has passed through the adsorption column;

[0118] 10) Wash the adsorption column with 500 μL of Buffer HB;

[0119] 11) Wash the adsorption column with 700 μL of DNA wash Buffer; repeat this step once;

[0120] 12) Centrifuge at ≧13000 g for 3 min to spin - dry the residual alcohol in the adsorption column;

[0121] 13) Discard the collection tube, place a new 1.5 mL centrifuge tube under the adsorption column, add 80 μL of Endotoxin - Free Elution Buffer, let it stand at room temperature for 2 min, and centrifuge at ≧13000 g for 1 min to elute the DNA;

[0122] 14) Measure the concentration, and transfect cells after detecting by agarose gel.

[0123] 2.2 Cell culture

[0124] 1) Resuscitation of 293T cells

[0125] Take out the cryopreserved cells from the liquid nitrogen tank and quickly place them in a 37°C water bath, shaking constantly to allow the cells to pass through the ice crystal stage and thaw the cells; transfer the thawed cells into a 15 mL centrifuge tube with an arrow, add 10 mL of cell culture medium DMEM (containing 10% fetal bovine serum and 1% double antibody), and pipette evenly; centrifuge at 1000 r / min for 5 min and discard the supernatant; add 1 mL of cell culture medium DMEM (containing 10% fetal bovine serum and 1% double antibody), pipette to resuspend the cells, and then transfer them to a culture flask for culture, adding enough medium to about 3 mL; gently shake the culture flask to evenly distribute the cells, and place it in a 37°C, 5% CO 2 incubator for culture; observe the cell adhesion situation after 2 h, change the medium after 24 h and continue to culture, and pay attention to observing the cell growth status at any time.

[0126] 2) Subculture of cells

[0127] When the cells in the culture flask are confluent and the cell adhesion rate reaches over 90%, the cells need to be subcultured. The subculture steps are as follows: discard the cell culture medium in the culture flask, add pre-warmed PBS buffer at 37°C to wash the cells; discard the PBS and add pre-warmed 0.25% trypsin at 37°C and place it in the cell incubator for digestion. Observe the cell digestion situation under the microscope. When the cells begin to shrink, become round and small, discard the trypsin, add cell culture medium with twice the volume of trypsin to neutralize the trypsin and terminate the digestion; pipette the cells in the culture flask to form a single cell suspension as much as possible; collect the cell suspension into a centrifuge tube, centrifuge at 1000 r / min for 5 min, and discard the supernatant; add cell culture medium DMEM (containing 10% fetal bovine serum and 1% double antibody), pipette to resuspend the cells, transfer them to a new culture flask for continued culture, and supplement an appropriate amount of cell culture medium DMEM according to the size of the culture flask, and place it in a 37°C constant temperature incubator for culture. Generally, one flask of confluent cells can be subcultured into 2 flasks.

[0128] 2.3 Transfection of eukaryotic vectors mediated by liposomes

[0129] Transfer 293T cells into a 6-well plate. When the cell adhesion reaches over 90%, transfection can be carried out. The transfection ratio of the core plasmid and the packaging plasmid is as follows:

[0130] pLKO.1-shSRPK3: 1000 ng

[0131] psPAX2: 900 ng

[0132] pMD2G: 100 ng

[0133] The specific transfection steps are as follows:

[0134] Take two sterilized 2.0 mL centrifuge tubes and label them A and B respectively;

[0135] Add 250 μL of Opti-MEM I Reduced Serum and 5 μL of Translipid to A; add 250 μL of Opti-MEM I Reduced Serum and 2 μg of DNA to B. Let stand at room temperature for 5 min;

[0136] Mix A and B and let stand at room temperature for 20 min;

[0137] Take out the inoculated 293T cells (density 80%-90%) and replace the medium with serum-free medium.

[0138] Add the mixture of A and B, gently shake the culture dish to mix evenly, and place it in the incubator;

[0139] After 4-6 hours of transfection, replace the medium with complete medium and culture for 48 hours and 72 hours to collect the virus solution.

[0140] 2.4 Recombinant lentivirus infects 293T cells

[0141] Inoculate 293T cells in a 6-well plate, divided into blank control group, empty vector group, and recombinant lentivirus interference group. Add 2 μL of virus to each group; add the same amount of PBS to the blank control group. Observe the fluorescence after 48 h. If the fluorescence is okay, then perform large-scale virus packaging (storage of recombinant lentivirus with high titer and large volume).

[0142] Send the primer human U6 on the vector to General Biotechnology Co., Ltd. for sequencing. Compare the sequencing results with the original sequence. No base deletion, mutation, or frameshift indicates successful vector construction.

[0143] 3. Vector construction and transfection:

[0144] Use liposome reagent to transfect the SRPK3 interfering plasmid pLKO.1-shRNA vector into human tumor cell lines (H358 and PC9). The specific steps are as follows: Inoculate the cells in a 6-well plate, divided into blank control group (PBS, Control), empty vector group (shNC), and recombinant lentivirus interference group (shSRPK3-1, shSRPK3-2, shSRPK3-3). After culturing to 70-80% confluence, mix the SRPK3 interfering plasmid with liposome to form a complex, and add it to the cell culture medium for transfection. Replace the fresh medium after 24 hours, and detect the expression of SRPK3 after 48 hours (verified by qPCR or Western Blot, the steps are the same as above, and the results Figure 5 )

[0145] Example 3

[0146] Lung cancer H358 and PC9 cells after SRPK3 gene interference were used to detect cell viability by CCK8 (0 / 12 / 24 / 48 / 72 h), cell proliferation by plate cloning assay, and apoptosis by flow cytometry (48 h).

[0147] 1. CCK8 assay method for detecting cell viability ( Figure 6 ):

[0148] 1) H358 and PC9 cells in the logarithmic growth phase were digested with trypsin, counted under a microscope, and then made into a cell suspension of 3×10 4 cells / ml. 100 μl was taken and added to a 96-well culture plate respectively. For each type of cell, 3 identical wells were inoculated on each plate as duplicate wells, with 3×10 3 cells / well, and 100 μl of culture medium was used as a blank control, and cultured overnight at 37°C.

[0149] 2) Cells transfected with sh-NC, shSRPK3-1, and shSRPK3-2 were mixed with Cell Counting Kit-8 (CCK-8) and serum-free essential medium at a volume ratio of 1:10 at 0, 12, 24, and 48 h, and 100 μL was added to each well to be tested. Incubate in an incubator at 37°C and 5% CO 2 for 1 h;

[0150] 3) The absorbance at a wavelength of 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. Record the values of each plate.

[0151] 2. Plate cloning assay for detecting cell proliferation ( Figure 7 ):

[0152] 1) H358 and PC9 cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare a single-cell suspension.

[0153] 2) The cell suspension was diluted in gradient multiples and inoculated into culture dishes at an appropriate cell density (according to the proliferation ability). 1×10 3 cells were inoculated per 10 cm dish, and gently rotated to make the cells evenly dispersed. Incubate in an environment at 37°C, 5% CO 2 and saturated humidity for 3 weeks, and the medium was changed every 3 days during this period.

[0154] 3) Observe regularly. When visible clones appear in the culture dish, terminate the culture. Discard the supernatant, carefully wash 2 times with PBS. Add 5 mL of pure methanol or 1:3 acetic acid / methanol, fix for 15 min. Then remove the fixing solution, add an appropriate amount of crystal violet or Coomassie brilliant blue staining solution and stain for 10 - 30 min, and then slowly wash off the staining solution with running water and air dry.

[0155] 4) Invert the petri dish and take a photo. Manually count the clones directly with the naked eye, or count the number of clones with more than 10 cells under a microscope (low magnification).

[0156] 5) Finally, calculate the colony formation rate: Colony formation rate = (number of colonies / number of seeded cells) × 100%.

[0157] 3. Detection of apoptosis by flow cytometry ( Figure 8 )

[0158] 1) Aspirate the culture media of H358 and PC9 cells into a suitable centrifuge tube. Wash the adherent cells once with PBS, and add an appropriate amount of trypsin cell digestive solution (which may contain EDTA) to digest the cells. Incubate at room temperature until the adherent cells can be detached by gentle pipetting. Then aspirate the trypsin cell digestive solution, taking care to avoid over-digestion by trypsin.

[0159] 2) Add the cell culture media collected in step A, mix gently, transfer to a centrifuge tube, centrifuge at 1000g for 5 minutes, discard the supernatant, collect the cells, and gently resuspend the cells with PBS and count.

[0160] Note: Adding the cell culture media in step A can collect the floating apoptotic or necrotic cells on one hand, and on the other hand, the serum in the cell culture media can effectively inhibit or neutralize the residual trypsin; the residual trypsin will digest and degrade the subsequently added Annexin V-FITC, resulting in staining failure.

[0161] 3) Take 50,000 - 100,000 resuspended cells, centrifuge at 1000g for 5 minutes, discard the supernatant, and add 195 μL of Annexin V-FITC binding buffer to gently resuspend the cells.

[0162] 4) Add 5 μL of Annexin V-FITC, mix gently, and incubate at 4°C in the dark for 15 min.

[0163] 5) Add 5 μL of propidium iodide staining solution, mix gently, and incubate at 4°C in the dark for 5 min. At the same time, use a tube without adding Annexin V-FITC and PI as a negative control.

[0164] 6) Immediately perform flow cytometry detection. Annexin V-FITC emits green fluorescence, corresponding to the FITC detection channel of the flow cytometer, and propidium iodide (PI) emits red fluorescence, corresponding to the PE detection channel of the flow cytometer.

[0165] Example 4

[0166] The SRPK3 - interfered H358 cell line was treated with 10 μM of the AKT activator SC79 (S7863; Selleck). The cell proliferation was detected by the colony formation assay, and apoptosis was detected by flow cytometry. Figure 9), Detection of the expression of AKT1 and p-AKT1 by Western blot Figure 10 )

[0167] The implementation steps of the plate clone experiment, flow cytometry, and Western blot are the same as above.

[0168] Example 5

[0169] Construct stable transfected cell lines of lung cancer H358 with SRPK3 gene interference (shSRPK3-1) and empty vector, and construct subcutaneous tumors in nude mice (Per group of 6) , After the tumors grow, measure the tumor size every 3 days, and sacrifice the mice to obtain the tumors and weigh them after 3 weeks Figure 11 )

[0170] The specific process is as follows: Randomly divide 12 nude mice into 2 groups, and inoculate them with H358 / shNC and H358 / shSRPK3 stable transfected cell lines respectively. The specific process is as follows: Prepare a single cell suspension (1×10 7 cells / ml), and in an SPF-level environment, implant 200 μl of the single cell suspension subcutaneously into the axilla of the right forelimb of the nude mice. One group is the shNC group, and the other group is the shSRPK3 group. When inoculating, first grab the nude mice, disinfect them with alcohol cotton balls, and then inject them subcutaneously under the armpit. After inoculation, raise the nude mice of different groups in separate cages, feed them water and feed regularly, change the bedding regularly, and tumors will form after about 1-2 weeks. After the tumors form, measure the tumor size with a vernier caliper every 3 days. At the end of the experiment, sacrifice 6 randomly selected experimental mice in each group by cervical dislocation under anesthesia. Immediately remove the tumors after the nude mice are sacrificed. Use white paper as the background, arrange the complete tumor bodies of each group neatly in the order of grouping, place a steel ruler below, and take pictures for record. Weigh and measure the volume, fix the tumors in formalin for subsequent pathological section preparation.

[0171] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; modify the technical solutions recorded in the foregoing examples, or perform equivalent replacements on some or all of the technical features therein; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An application of the SRPK3 gene as a target in screening drugs for treating lung cancer.

2. The use according to claim 1, characterized in that: The lung cancer includes lung adenocarcinoma cell lines A549, H1299, H358, and PC9, and preferably adenocarcinoma cell lines H1299, H358, and PC9.

3. An inhibitor of SRPK3 gene.

4. The SRPK3 gene inhibitor according to claim 3, characterized in that The inhibitor includes small interfering RNA of SRPK3 gene.

5. The SRPK3 gene inhibitor according to claim 4, characterized in that The small interfering RNA of the SRPK3 gene includes shSRPK3-1, shSRPK3-2 and shSRPK3-3, the nucleotide sequence of the gene encoding shSRPK3-1 is shown in SEQ ID NO: 3-4, the nucleotide sequence of the gene encoding shSRPK3-2 is shown in SEQ ID NO: 7-8, and the nucleotide sequence of the gene encoding shSRPK3-3 is shown in SEQ ID NO: 11-12.

6. Use of the SRPK3 gene inhibitor according to any one of claims 3 to 5 in the preparation of a drug for treating lung cancer.

7. The use according to claim 6, characterized in that: The lung cancer includes lung adenocarcinoma cell lines A549, H1299, H358, and PC9, and preferably adenocarcinoma cell lines H1299, H358, and PC9.

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