An shRNA, lentiviral expression vector for targeted silencing of ALIX gene expression, and application thereof

Abstract

The present invention relates to the field of medical biotechnology, and particularly to an shRNA, a lentiviral expression vector and an application for targeted silencing of ALIX gene expression. The shRNA for targeted silencing of ALIX gene expression includes shALIX-1 and / or shALIX-2; the nucleotide sequence of the target site of shALIX-1 is as shown in SEQ ID NO.1, and the nucleotide sequence of the target site of shALIX-2 is as shown in SEQ ID NO.2. The shRNA designed by the present invention is packaged into lentivirus by a lentiviral vector and then infects human gastric cancer cells and monocytes, can bind to the mRNA of ALIX gene, efficiently interfere with its transcription and translation, reduce the expression of ALIX gene in gastric cancer cells and monocytes, and inhibit cell migration; significantly inhibit the growth rates of gastric cancer cells MGC803 and monocytes THP-1, which is of great significance for the treatment of gastric cancer.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical biotechnology, and in particular to an shRNA for targeting and silencing the expression of ALIX gene, a lentiviral expression vector and applications thereof. Background Art

[0002] Gastric cancer is one of the common malignant tumors, with its incidence ranking fifth among malignant tumors globally and having an extremely high mortality rate.

[0003] A large amount of research data shows that the clinical treatment effect of gastric cancer is closely related to the timing of its diagnosis. Early gastric cancer can be cured under the premise of active treatment. If it is advanced or late-stage gastric cancer, the five-year survival rate will be significantly reduced. At present, the main treatments for gastric cancer are endoscopic surgery and drug treatment. However, due to the lack of large-scale implementation of routine early screening for gastric cancer in China, the early diagnosis rate is low, the invasiveness is strong, the spread is fast, and the diagnostic and treatment effects of related drugs applied in clinical treatment are not good. Therefore, the discovery and application of new targeted markers are crucial for the diagnosis and treatment of gastric cancer.

[0004] Programmed cell death 6-interacting protein (PDCD6IP), also known as ALG2-interacting protein X (ALIX), is one of the multifunctional cytoplasmic and multi-domain scaffold proteins that have been relatively well studied in recent years. ALIX is ubiquitously expressed in cells and plays a series of important functions in endosomal pathways, virus budding, cytoskeletal dynamics, cell proliferation and apoptosis. Studies have shown that ALIX is highly enriched in exosomes of prostate cancer cell lines compared to non-cancerous prostate cell lines; ALIX can regulate tumor-mediated immunosuppression by controlling the activity of epidermal growth factor receptor (EGFR) and the presentation of programmed death ligand (PD-L1), and is closely related to the TNM stage and metastasis of pancreatic cancer. However, there is little research on ALIX in gastric cancer. Therefore, exploring its role is of great significance for the early diagnosis and treatment of gastric cancer.

[0005] RNA interference (RNAi) is a phenomenon in normal organisms that inhibits the expression of specific genes. It is a technology that achieves post-transcriptional gene silencing depending on specific short sequences of double-stranded RNA. It refers to the phenomenon that when double-stranded RNA (dsRNA) homologous to the coding region of endogenous mRNA is introduced into cells, the mRNA degrades, resulting in gene expression silencing. After exogenous dsRNA enters cells, the reverse strand of small interfering RNA (siRNA) and various nucleases form a RNA-induced silencing complex (RISC). RISC has the function of binding and cleaving mRNA and mediates the process of RNA interference. Currently, the commonly used RNAi technologies in laboratories mainly include siRNA oligonucleotide vectors and shRNA lentiviral plasmid expression vectors. Short hairpin ribonucleic acid (shRNA) can be maintained stably through virus-mediated transfection and can reduce off-target effects. RNAi has specificity and high efficiency. This technology has become an important tool for studying gene functions and will play an important role in the treatment of viral diseases, genetic diseases, and tumor diseases.

[0006] First, shRNA is inserted into the lentiviral vector to form a recombinant lentiviral plasmid. The lentiviral plasmid and other auxiliary plasmids form lentiviruses with the help of 293T cells, and finally, the cells are transfected with the lentiviruses to exert the silencing effect of shRNA. Under the action of endonuclease, shRNA is cleaved into nucleotide chains, which consist of a sense strand and an antisense strand. The antisense strand binds to specific enzymes to form the RNA-induced silencing complex RISC (the RISC complex contains elements such as siRNA, exonuclease, endonuclease, and helicase). The nucleotide double strand will be depolymerized into two single strands by the activated RISC. Subsequently, the antisense strand recognizes and binds to its homologous target mRNA. Under the guidance of the antisense strand, the activated RISC will cleave a specific position of the target mRNA, and at the same time, the cleaved mRNA will be specifically degraded by the enzymes in the RISC complex, thereby blocking the transmission of genetic information of mRNA.

[0007] Targeted drugs designed for cancer cell-related genes have been gradually applied in clinical practice. However, there is currently no shRNA specifically designed for the ALIX gene of gastric cancer cells using shRNA technology. In view of this, the present invention is specifically proposed. Summary of the Invention

[0008] The present invention provides an shRNA, a lentiviral expression vector and an application for targeted silencing of ALIX gene expression. The designed shRNA molecule is packaged into lentivirus by a lentiviral vector and infects human gastric cancer cells and monocytes, can bind to the mRNA of the ALIX gene, efficiently interfere with its transcription and translation, reduce the expression of the ALIX gene in gastric cancer cells and monocytes, and inhibit cell migration; significantly inhibit the growth rates of gastric cancer cells MGC803 and monocytes THP-1, which has great significance for the treatment of gastric cancer and solves the problems existing in the prior art.

[0009] The present invention provides one of the following technical solutions:

[0010] An shRNA for targeted silencing of ALIX gene expression, the shRNA includes shALIX-1 and / or shALIX-2; the nucleotide sequence of the target site of shALIX-1 is shown in SEQ ID NO.1, and the nucleotide sequence of the target site of shALIX-2 is shown in SEQ ID NO.2. Through screening experiments, it is found that this shRNA can effectively inhibit the expression of the ALIX gene.

[0011] Furthermore, the forward strand sequence of shALIX-1 is shown in SEQ ID NO.3, and the reverse strand sequence is shown in SEQ ID NO.4.

[0012] Furthermore, the forward strand sequence of shALIX-2 is shown in SEQ ID NO.5, and the reverse strand sequence is shown in SEQ ID NO.6.

[0013] The present invention provides the second of the following technical solutions:

[0014] A lentiviral expression vector containing the shRNA as described above.

[0015] Furthermore, the lentiviral expression vector is obtained by cloning shALIX-1 and / or shALIX-2 into a lentiviral expression vector. The lentiviral expression vector can be pLKO.1.

[0016] The construction method of the lentiviral expression vector as described above includes the following operation steps:

[0017] S1. Use primer annealing to synthesize the shALIX-1 sequence and / or shALIX-2 sequence;

[0018] S2. Clone the synthesized shALIX-1 and / or shALIX-2 sequences into a lentiviral expression vector.

[0019] Furthermore, the above construction method further includes the following steps:

[0020] The lentiviral expression vector expressing shRNA targeting and silencing ALIX constructed is transfected into competent Escherichia coli cells, inoculated into LB liquid medium containing ampicillin (Amp) overnight, and after obtaining bacterial precipitate, an endoribonuclease is added for enzymatic digestion, and the positive plasmid after enzymatic digestion is verified by sequencing.

[0021] The present invention provides the following technical solution three:

[0022] Use of the above shRNA in the preparation of a drug for inhibiting the expression of ALIX gene.

[0023] Use of the above shRNA in the preparation of a biological agent for treating / preventing gastric cancer.

[0024] The present invention also provides the following technical solution four:

[0025] A preparation for treating gastric cancer, comprising the shRNA targeting and silencing the expression of ALIX gene as described above.

[0026] Furthermore, the preparation for treating gastric cancer further includes a pharmaceutically acceptable carrier or excipient, diluent or vehicle.

[0027] The above "pharmaceutically acceptable" means that when the molecular entity and the composition are appropriately administered to an animal or a human, they do not produce adverse, allergic or other untoward reactions. A "pharmaceutically acceptable carrier or excipient" should be compatible with the active ingredient, i.e., can be blended with it without significantly reducing the effect of the drug under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; tabletting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffer solutions, etc. These substances are used as needed to help the stability of the preparation or to enhance the activity or its bioavailability or to produce an acceptable taste or odor in the case of oral administration.

[0028] Furthermore, the dosage form of the above preparation can be made into injections, oral liquids, tablets, capsules, dripping pills, sprays.

[0029] The present invention also provides the following technical solution five:

[0030] A targeted - silenced ALIX gene, whose target sequence is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0031] Advantages of the present invention:

[0032] 1. Based on the mRNA sequence of the ALIX gene, the present invention designs and synthesizes shRNA molecules, and constructs oligonucleotide sequences of shRNA. Among them, the two pairs of shRNA sequences such as SEQ ID NO.1 and SEQ ID NO.2, after being packaged into lentiviruses through lentiviral vectors and infecting human gastric cancer cells and mononuclear macrophages, can bind to the mRNA of the ALIX gene, efficiently interfere with its transcription and translation, reduce the expression of the ALIX gene in gastric cancer cells and mononuclear macrophages, and inhibit cell migration, showing obvious effects.

[0033] 2. The above - designed shRNA sequences of the present invention can significantly inhibit the growth rate of gastric cancer cell MGC803, which is of great significance for the treatment of gastric cancer. It provides a theoretical basis for a new gastric cancer treatment strategy centered on ALIX - related preparations, and is expected to be applied to the preparation of anti - cancer gene drugs with high efficiency, strong specificity and small side effects, especially for the research and development of gastric cancer gene drug preparations, with huge social and economic benefits. Description of the Drawings

[0034] Figure 1 It is the Western blot (WB) detection diagram of the expression level of the ALIX gene of MGC803 cells by shALIX of the present invention;

[0035] Figure 2 It is the WB diagram of the expression level of the ALIX gene of THP - 1 cells by shALIX of the present invention;

[0036] Figure 3 It is the WB diagram of the expression level of the ALIX gene of MGC803 cells by the siRNA sequence of the comparative example in Example 4;

[0037] Figure 4 It is the WB diagram of the expression level of the ALIX gene of MGC803 cells by four different shALIX sequences constructed in Example 5;

[0038] Figure 5 It is the diagram of the influence of the shALIX sequence on the proliferation of MGC803 cells in Example 6.

[0039] Among them, Figure 1 A shows the cell state of MGC803 cells before and after ALIX gene knockdown by shALIX, Figure 1 B shows the WB detection result diagram of the expression level of MGC803 cells before and after ALIX gene knockdown by shALIX;

[0040] Figure 2 In A, the state of THP-1 cells before and after ALIX gene knockdown by shALIX is shown. Figure 2 In B, the WB detection result graph of the expression level of THP-1 cells before and after ALIX gene knockdown by shALIX is shown;

[0041] Figures 1 - 5 In this figure, GAPDH is the internal reference protein, NC is the control group infected with the pLKO.1 empty vector, and shALIX-1# and shALIX-2# are the experimental groups infected with pLKO.1-shALIX-1# and pLKO.1-shALIX-2# respectively. Detailed implementation mode

[0042] To clearly illustrate the technical characteristics of this solution, the present invention will be elaborated in detail below through specific implementation modes in combination with the accompanying drawings.

[0043] Some sources of the materials and reagents used in the following examples are as follows: The sequences of shALIX-1 and shALIX-2 were synthesized by Yixin Biotechnology (Shanghai) Co., Ltd., the plasmids pLKO.1, psPAX2 and pMD2.G were purchased from Youbao Biotechnology, and the transfection reagent Lipofectamine 2000 was purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0044] For those conditions not specified in the following examples, all are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase. Unless otherwise specified, the present invention adopts the existing technologies in this field.

[0045] Example 1 Construction of lentiviral vector

[0046] According to the mRNA sequence of the human ALIX gene (NM_013374) obtained from the GenBank database (http: / / www.ncbi.nlm.nih.gov / genbank), and based on the shRNA design principle, shRNAs targeting the human ALIX gene were designed, and their target sequences are shown in Table 1 respectively, for silencing the human ALIX gene.

[0047] The nucleotide sequences of the target sites of the shRNAs and the sequences of the forward and reverse strands for preparing the shRNAs are shown in Table 1.

[0048] Table 1

[0049]

[0050] The above 4 double-stranded oligonucleotide short fragments were respectively ligated to the double-digested lentiviral vector pLKO.1. The ligation products were obtained by inoculating Escherichia coli containing the target plasmid into 30 - 50 mL of LB liquid medium containing Amp, placing it in a constant temperature shaker at 37 °C, at 200 rpm / min, and incubating for 12 - 16 hours; taking out the cultured bacterial solution from the shaker, centrifuging at 3500 - 5000 g for 10 min at room temperature; sucking off the supernatant and discarding the excess medium on the container wall to obtain bacterial pellets. Add 2.5 mL of Solution I (RNase A has been added) to the pellets, and resuspend the bacterial pellets by vortex oscillation; collect the bacteria to extract the plasmid, verify the plasmid by enzyme digestion, and send the digested positive plasmid to BGI for sequencing verification. The sequenced lentiviral plasmid shown to be successfully constructed was used for subsequent experiments.

[0051] By the above method, lentiviral vectors pLKO.1-shALIX-1# and pLKO.1-shALIX-2# containing the target plasmid were respectively constructed.

[0052] Example 2: Preparation of cell lentivirus and lentivirus transfection of cells

[0053] Package the lentivirus with reference to the Lipofectamine 2000 reagent instruction manual.

[0054] Using the two lentiviral vectors prepared in Example 1 to transfect cells as the experimental group, and at the same time using the pLKO.1 empty vector to transfect cells as the control group. Lipofectmin2000 was used to transfer the core plasmids (pLKO.1-shALIX-1#, pLKO.1-ALIX-2# or pLKO.1) and packaging plasmids (psPAX2 and pMD2.G) into 293T cells, prepare and collect the virus, and then use the lentivirus containing the target shALIX sequence to infect human gastric cancer cells MCG803 and monocytes THP-1, and screen out the resistant cells with puromycin for experiments.

[0055] Perform lentivirus packaging in 293T cells (the cell density is required to be about 90%). Add Lipofectamine-2000 transfection reagent to 150 μL of serum-free culture medium and let it stand for 5 min; add a total of 1.5 μg of the packaging plasmids psPAX2 and pMD2.G in a ratio of 3:1 and 1.5 μg of the core plasmid to 150 μL of serum-free culture medium;

[0056] Add the transfection mixture to the cell culture medium in a 35-mm cell culture dish and mix well. After 6 - 8 h, replace it with 2 mL of fresh complete DMEM medium (10% FBS); collect the virus after 36 - 48 h, centrifuge at 8000 g for 5 min, collect all the supernatant, filter it through a 0.45-μm filter membrane, and transfer the virus supernatant to a new collection tube, then store it at -20°C.

[0057] Infect MGC803 cells and THP-1 cells (the cell seeding density is controlled at about 50%) with the virus supernatant obtained by packaging with lentivirus. Determine the amount of virus to be added according to the virus titer, and at the same time add polybrene (diluted 1:1000) at 10 μg / μL to accelerate the entry of the virus into the cells through charge interaction. Perform a medium change after 12 h, and the virus will be expressed in the cells after 24 - 48 h. After 48 h of infection, replace it with fresh medium containing puromycin (1 μg / mL). Change the medium once every 2 days, and select positive clones for detection after about 7 days.

[0058] Example 3: Detection of the protein expression level of ALIX gene in MGC803 cells and THP-1 cells by Western blot

[0059] Collect the virus-infected MGC803 cells and THP-1 cells obtained in Example 2, extract the total protein, measure the protein concentration, run the gel and transfer the membrane after protein denaturation. After blocking the membrane with 5% skim milk for 2 hours, incubate overnight with ALIX antibody and GAPDH antibody, then incubate with goat anti-mouse antibody conjugated with horseradish peroxidase at room temperature for 1 hour. After antibody incubation, wash the membrane 3 times with TBS-T solution at room temperature for 5 min each time, and then develop the image using BeyoECL Plus (ultra-sensitive ECL chemiluminescence kit) on a ChemiDocTM XRS+ (Bio-Rad) imaging system. Detect the expression of the target protein ALIX in cells of different groups by WB. The experimental results are as Figure 1 、 Figure 2 shown.

[0060] As Figure 1 、 Figure 2 can be seen, compared with the control group NC, both shALIX-1# and shALIX-2# can significantly silence the expression of ALIX protein in MGC803 cells and THP-1 cells, and have a significant knockdown effect on the expression of ALIX in MCG803 cells and THP-1 cells.

[0061] Example 4:

[0062] Use the following three groups of siRNA sequences, perform Western blot detection according to the method of Example 3, and compare with the shRNA in Example 1. The experimental results are asFigure 3 As shown, it indicates that the following three groups of siRNA sequences cannot effectively silence the expression of ALIX in MGC803 cells.

[0063] siALIX-1F: CGAUUUGGUUAACAGAUCA (SEQ ID NO.7);

[0064] siALIX-1R: UGAUCUGUUAACCAAAUCG (SEQ ID NO.8).

[0065] siALIX-2F: GAUCGAGUUCCAGACCUUA (SEQ ID NO.9);

[0066] siALIX-2R: UAAGGUCUGGAACUCGAUC (SEQ ID NO.10).

[0067] siALIX-3F: GCUCAAGAUGGUGUGAUAA (SEQ ID NO.11);

[0068] siALIX-3R: UUAUCACACCAUCUUGAGC (SEQ ID NO.12).

[0069] Example 5

[0070] According to the method of Example 1, 4 groups of shRNA sequences targeting the human ALIX gene were constructed in the experiment. According to the method of Example 3, WB detection was performed on the other 2 groups of shRNA and the shRNA with the target sequence in Table 1. The results of the effect comparison are as Figure 4 shown, suggesting that compared with the shRNA of other sequences, the shRNA-1# and shRNA-2# of the present invention can achieve more significant and effective silencing of the expression of ALIX in MGC803 cells.

[0071] The target site nucleotide sequences of the other 2 groups of shRNA and the forward and reverse strand sequences for preparing the corresponding shRNA are shown in Table 2.

[0072] Table 2

[0073]

[0074] Example 6: Detection of the effect of shALIX on the proliferation of MGC803 cells by colony formation assay

[0075] Collect the virus-infected MGC803 cells obtained in Example 2, and use the pLKO.1 empty vector transfected cells as the control group.

[0076] Cells in the logarithmic growth phase with good growth status were digested with trypsin for 3 - 5 min, then medium was added to neutralize. After centrifugation at 1000 g for 3 - 5 min, the cells were rinsed twice with PBS, the supernatant was aspirated, and the cells were resuspended in complete medium for standby.

[0077] The cell suspension was inoculated into a culture plate at a gradient dilution as needed, gently mixed to resuspend the cells, and placed in a 37°C, 5% CO 2 cell incubator for 2 - 3 weeks.

[0078] The culture dish was observed macroscopically, and the culture was stopped when obvious cloned cell clusters appeared. The cells were washed with PBS, 4% paraformaldehyde was added, and left standing for 15 min. The fixing solution was removed, the cells were washed with PBS, GIMSA staining solution was added and left for 10 - 15 min, then repeatedly rinsed and air-dried.

[0079] The petri dish was inverted and photographed, and the number of cloned spots was calculated using Image J software.

[0080] The experimental results were as Figure 5 , compared with the control group, shALIX had a significant inhibitory effect on the proliferation of MGC803 cells (*P < 0.05).

[0081] The above specific implementation manners cannot be used as a limitation to the protection scope of the present invention. For those skilled in the art of this technology, any alternative improvement or transformation made to the implementation manners of the present invention falls within the protection scope of the present invention.

[0082] Where the present invention is not described in detail, it is all common knowledge for those skilled in the art of this technology.

Claims

1. A targeted silencing ALIX A shRNA for gene expression, characterized in that The shRNA includes shALIX-1 and / or shALIX-2; the target site nucleotide sequence of the shALIX-1 is shown in SEQ ID NO.1, and the target site nucleotide sequence of the shALIX-2 is shown in SEQ ID NO.2; the forward chain sequence for preparing the shALIX-1 is shown in SEQ ID NO.3, and the reverse chain sequence is shown in SEQ ID NO.4; the forward chain sequence for preparing the shALIX-2 is shown in SEQ ID NO.5, and the reverse chain sequence is shown in SEQ ID NO.

6.

2. A lentiviral expression vector comprising the shRNA according to claim 1.

3. The lentiviral expression vector according to claim 2, characterized in that The lentiviral expression vector is obtained by cloning shALIX-1 and / or shALIX-2 into a lentiviral expression vector.

4. Use of the shRNA according to claim 1 in the preparation of a biological agent for treating gastric cancer.

5. A preparation for treating gastric cancer, characterized in that: Comprising the targeted silencing as claimed in claim 1 ALIX shRNA for gene expression.

Images