shRNA targeting RhoJ for silencing, its uses, recombinant plasmid and lentiviral vector

Abstract

The present invention provides a shRNA for targeted silencing of RhoJ, its use, a recombinant plasmid and a lentiviral vector, which relate to the field of biomedical technologies. The nucleotide sequence of the sense strand of the shRNA for targeted silencing of RhoJ is as shown in SEQ ID No.1 or SEQ ID No.3, and the nucleotide sequence of the antisense strand of the shRNA is as shown in SEQ ID No.2 or SEQ ID No.4. The shRNA for targeted silencing of RhoJ of the present invention can significantly reduce the expression of RhoJ in renal clear cell carcinoma A498 cells, and inhibit the proliferation, invasion and migration of renal clear cell carcinoma A498 cells.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and particularly relates to an shRNA for targeting and silencing RhoJ, its use, a recombinant plasmid, and a lentiviral vector. Background Art

[0002] RNA interference (RNAi) is a gene silencing phenomenon mediated by double-stranded RNA (dsRNA). When there is double-stranded RNA complementary to the target mRNA sequence in the cell, a series of intracellular reactions will be triggered. The nuclease Dicer in the cell will recognize and cleave the double-stranded RNA, processing it into small interfering RNA (siRNA), which is usually about 21 - 23 nucleotides in length. These siRNAs will bind to a complex called RNA-induced silencing complex (RISC). In RISC, one strand of the siRNA (the guide strand) will guide RISC to recognize and bind to the target mRNA complementary to it, and then the nuclease activity in RISC will cleave the target mRNA, causing it to degrade, thereby resulting in the silencing of the gene expression, that is, the protein cannot be normally translated. In the past few years, as a new gene therapy, RNAi technology has been widely used in the field of gene therapy research for diseases. The occurrence of many tumors is related to the abnormal activation of oncogenes. For example, in breast cancer, the overexpression of the HER-2 (human epidermal growth factor receptor-2) gene is associated with poor prognosis. Through RNA interference technology, siRNA targeting the HER-2 gene mRNA can be designed and introduced into breast cancer cells to effectively reduce the expression of the HER-2 gene. This can not only inhibit the proliferation of cancer cells but also enhance the sensitivity of cancer cells to chemotherapeutic drugs. The growth and metastasis of tumors depend on new blood vessels to provide nutrients and oxygen. Vascular endothelial growth factor (VEGF) is a key factor promoting angiogenesis. Using RNA interference technology to inhibit the expression of the VEGF gene can reduce tumor angiogenesis, thereby inhibiting the growth and metastasis of tumors. In animal experiments, when siRNA targeting the VEGF gene was delivered into tumor model animals, a significant decrease in tumor vessel density and a slowdown in tumor growth rate were observed.

[0003] As a member of the small GTPase family, since RhoJ was discovered in 2000, its role in vascular endothelial cells has mainly focused on regulating intercellular adhesion, cell proliferation and migration. And it has been proven to play an important promoting role in angiogenesis, tumor invasion and metastasis in glioblastoma, melanoma, gastric adenocarcinoma, and triple-negative breast cancer, and promotes tumor cell DNA repair through multiple pathways to induce tumor chemotherapy resistance.

[0004] The existing technology still needs to be improved and developed, and more effective Rho kinase inhibitors need to be developed to improve their specificity for Rho kinase and selectivity for different tissue cells. Summary of the invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides a shRNA for targeted silencing of RhoJ and its use, recombinant plasmid and lentiviral vector. The technical solution is as follows:

[0006] A shRNA for targeted silencing of RhoJ, wherein the nucleotide sequence of the sense strand of the shRNA is shown as SEQ ID No. 1 or SEQ ID No. 3, and the nucleotide sequence of the antisense strand of the shRNA is shown as SEQ ID No. 2 or SEQ ID No. 4.

[0007] A recombinant plasmid, comprising a vector and a target gene, wherein the vector is PLKO.1, and the target gene is the positive strand and the antisense strand of the shRNA targeting silencing RhoJ, wherein the PLKO.1 is double-digested by AgeI enzyme and ECORI enzyme.

[0008] The method for constructing the recombinant plasmid comprises the following steps:

[0009] 1) synthesizing the sense strand and antisense strand of the shRNA, and annealing the sense strand and antisense strand of the shRNA;

[0010] 2) Double digestion of PLKO.1 with AgeI and EcoRI enzymes; and

[0011] 3) The vector digested with PLKO.1 and the annealing product obtained in step 1) are ligated by enzyme to obtain the recombinant plasmid.

[0012] A shRNA lentiviral vector, comprising a sense strand and an antisense strand of the shRNA, wherein the nucleotide sequence of the sense strand of the shRNA is shown as SEQ ID No. 1 or SEQ ID No. 3, and the nucleotide sequence of the antisense strand of the shRNA is shown as SEQ ID No. 2 or SEQ ID No. 4.

[0013] The method for constructing the shRNA lentiviral vector comprises the following steps:

[0014] (1) fully mixing the recombinant plasmid, the Pax2 plasmid and the Vsvg plasmid; and

[0015] (2) Add the transfection reagent Jetprime, buffer, and the mixed plasmid obtained in step (1) to 293T cells, culture, and collect the supernatant to obtain the shRNA lentiviral vector.

[0016] A method for targeted silencing of RhoJ, comprising infecting target cells with the shRNA lentiviral vector described above to obtain RhoJ-silenced cells.

[0017] RhoJ-silenced cells constructed by the method described above.

[0018] Use of the shRNA for targeted silencing of RhoJ, wherein the shRNA is used to prepare a biological agent for inhibiting the expression of the RhoJ gene.

[0019] Use of the shRNA for targeted silencing of RhoJ, wherein the shRNA is used to prepare a drug for treating clear cell renal cell carcinoma.

[0020] The beneficial effects brought by the technical solution provided by the present invention at least include:

[0021] Worldwide, renal cell carcinoma (RCC) ranks fourteenth among all malignant tumor incidences. And the incidence has regional differences, with higher incidences in Europe and North America, and lower incidences in other regions such as Africa and Asia. Therefore, its prevention and treatment are of great significance. Existing drugs mainly include chemotherapy drugs, targeted drugs, and immune checkpoint inhibitors. However, the efficacy of these drugs for renal cell carcinoma is poor. Drugs approved for the treatment of advanced renal cell carcinoma in China include pazopanib, sunitinib, axitinib, sorafenib, everolimus, interleukin-2, IFN-α, etc. However, neoadjuvant therapy is still exploratory, and there is no large-scale phase III randomized trial to support systemic therapy in this situation.

[0022] The development of RNAi technology has not only greatly promoted the human post-genome project, but also can be applied to high-throughput screening of drug target genes, promoting gene therapy and new drug development, and opening up new ways for the treatment of diseases such as cancer and genetic diseases. In the past few years, RNAi technology, as a new gene therapy, has been widely used in the field of gene therapy research for diseases. Compared with other treatment means, RNAi technology has stronger targeting for pathogenic genes and lower toxicity and side effects. Therefore, it is of great significance to develop more RNA interference fragments for renal cell carcinoma.

[0023] The shRNA for targeted silencing of RhoJ of the present invention can significantly reduce the expression of RhoJ in clear cell renal cell carcinoma A498 cells and inhibit the proliferation, invasion, and migration of clear cell renal cell carcinoma A498 cells. Brief Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0025] Figure 1 It is the RhoJ knockdown efficiency graph in the A498 cell line of renal cell carcinoma provided in Embodiment 3 of the present invention;

[0026] Figure 2 It is the result graph of the EdU proliferation experiment provided in Embodiment 4 of the present invention;

[0027] Figure 3 It is the statistical graph of the result of the EdU proliferation experiment provided in Embodiment 4 of the present invention;

[0028] Figure 4 It is the result graph of the scratch experiment provided in Embodiment 4 of the present invention;

[0029] Figure 5 It is the statistical graph of the result of the scratch experiment provided in Embodiment 4 of the present invention;

[0030] Figure 6 It is the schematic diagram of the PLKO.1-RhoJ-sh1 vector;

[0031] Figure 7 It is the schematic diagram of the PLKO.1-RhoJ-sh2 vector;

[0032] Figure 8 It is the result graph of the Transwell invasion and migration experiment provided in Embodiment 5 of the present invention;

[0033] Figure 9 It is the statistical graph of the result of the Transwell invasion and migration experiment provided in Embodiment 5 of the present invention. Detailed Embodiments

[0034] The following will describe the technical solutions in the present invention in conjunction with the drawings.

[0035] In the embodiments of the present invention, the meaning expressed by "and / or" can be both, or either of them can be selected.

[0036] Two RhoJ-shRNA knockdown sequences were constructed. Their DNA template sequences are shown in Table 1 and Table 2:

[0037] Table 1

[0038]

[0039] Synthesized by Beijing Bomed Company

[0040] Table 2

[0041]

[0042] Synthesized by Beijing Bomed Company

[0043] The experimental materials are shown in Table 3 below:

[0044] Table 3

[0045]

[0046] The primer sequences of the U6 promoter are shown in Table 4:

[0047] Table 4

[0048]

[0049] The U6 promoter is upstream of the shRNA sequence. Primers are designed for qPCR detection to verify whether the sequence and the plasmid are successfully combined.

[0050] Example 1

[0051] 1. Anneal the sense strand (RhoJ-shRNA knockdown sequence) and the antisense strand (RhoJ-shRNA antisense sequence), with a system of 20 µl.

[0052] 4 µl sense strand + 4 µl antisense strand + 2 µl NEB buffer + 10 µl ddH 2 O. The PCR reaction conditions are as follows: 95 °C for 2 min, one cycle, decreasing by 0.1 °C every eight seconds to 25 °C, 700 cycles, and then cooling to 4 °C.

[0053] 2. Digest the vector, with a system of 50 µl.

[0054] 1 µg of PLKO.1 vector + 1 µl of AgeI enzyme + 1 µl of ECORI enzyme + 5 µl of buffer, supplemented with ddH 2 O to 50 µl. Digest at 37 °C for 3 h.

[0055] 3. Recover nucleic acids by gel electrophoresis. Prepare a 1% agarose gel by adding 1% agarose to 1×TAE. Microwave for 1 - 2 min until the liquid is clear. After cooling, add nucleic acid dye (pay attention to the management of the contamination area). Perform agarose gel electrophoresis on the digested product, and add the empty PLKO.1 vector as a positive control when loading the sample.

[0056] 4. Ligation: 100 ng of the vector digested with PLKO.1, 1 µl of the annealing product obtained in step 1), 1 µl of T4 ligase, and 1 µl of buffer. Make up to 10 µl with ddH 2 O. Incubate at room temperature for 30 - 60 min for ligation.

[0057] 5. Transformation: Perform plasmid transformation using DH5α competent cells.

[0058] 6. Plating: Add 6 µl of Amp+ to 6 ml of solid LB medium, pour it into a 6-cm sterile Petri dish, and let it cool and solidify. Add the transformed product and gently shake to evenly distribute the bacterial solution on the surface of the solid medium. Incubate upright for 10 - 30 min, then invert the Petri dish and culture it in an incubator at 37°C for 16 h.

[0059] 7. Colony picking and PCR identification: Pick a single isolated round colony into 9 µl of ddH 2 O and mix well. Take 1 - 2 µl of the bacterial solution, 0.3 µl of the U6 promoter primer forward strand, 0.3 µl of the U6 promoter primer reverse strand, and 10 µl of Taq PCR StarMix. Make up to 20 µl with ddH 2 O. The PCR reaction conditions are as follows: 95°C for 5 minutes, 94°C for 30 s, 55°C for 30 s, 72°C for 60 s, for 30 cycles, 72°C for 5 minutes, and cool down to 4°C for storage. Perform agarose gel electrophoresis on the PCR product to observe whether the plasmid construction is successful.

[0060] Example 2: Lentiviral packaging of the RhoJ lentiviral interference vector:

[0061] One day before transfection, seed 293T cells in a 100 mm plate (10 mL DMEM) and culture for 12 - 24 h. When the cell density reaches 40 - 50%, for each transfection system, the plasmid mixture required is 5 μg of packaging plasmids (Pax2 and Vsvg, Sigma) and 5 μg of PLKO.1-puro lentiviral recombinant plasmid (i.e., the plasmid constructed in Example 1). Mix the above plasmids well; for each group, 20 μL of JetPrime transfection reagent + 500 μL of Buffer are needed. Use the shNC viral vector targeting non-target gene sequence as a control. Mix the transfection reagent and the plasmid mixture well respectively and let stand at room temperature for 10 min. Slowly add 1 mL of the mixture drop by drop to a 100 mm culture dish, and culture in a 37 °C incubator for 8 - 12 h, then change to fresh complete culture medium; to increase the virus concentration, 8 mL of culture medium can be used when changing the medium. After culturing for another 48 h, collect the supernatant and replace it with fresh complete culture medium in the culture dish. Centrifuge the collected supernatant at 3500 rpm for 10 min to obtain the corresponding virus solution. Aliquot about 700 μL of the virus solution into each sterile 1.5 mL EP tube on average. After 72 h, process in the same way and collect the virus supernatant.

[0062] Example 3

[0063] 1. Cell culture: Culture in DMEM complete medium containing 5% FBS, collect cells with good growth status, centrifuge and count, and seed 6×10 5 cells per well in a 60 mm dish, and culture at 37 °C with 5% CO 2 for 24 h.

[0064] 2. shRhoJ lentivirus infection of A498:

[0065] Seed A498 cells in a 6-well plate at a density of 1.5×10 5 cells per well. After culturing for 12 - 24 h, infect A498 with a mixture of 500 μL DMEM + 500 μL shRhoJ lentivirus solution + 0.5 μL polybrene (final concentration 10 μg / μL). Use the shNC lentiviral expression vector and lentivirus targeting non-target gene sequence as a control for the shRhoJ lentiviral expression vector and lentivirus to infect A498.

[0066] 3. 72 h after the cells are infected with the virus, add culture medium containing puromycin at a final concentration of 1 μg / μL and continue to culture the cells until a cell line stably infected with shRhoJ lentivirus is selected.

[0067] 4. Western blot to detect the protein expression level:

[0068] Collect protein samples. After protein quantification, load 40 μg of the protein sample and perform SDS-PAGE electrophoresis at a constant voltage of 100 V. Transfer the membrane: at a constant current of 200 mA for 1.5 h. After transfer, place the membrane in a 5% non-fat milk blocking solution and block for 1 h, then wash the membrane twice with TBST. Add the primary antibody (diluted 1:1000) prepared with 3% non-fat milk, place it on a shaker, and incubate overnight at 4°C. The next day, wash the membrane three times with TBST, 5 min each time, add the secondary antibody (diluted 1:5000) prepared with 3% non-fat milk, hybridize at room temperature for 1 h, and then develop the color (or perform chemiluminescent autoradiography).

[0069] The experimental results are as Figure 1 shown. Among them, sh1 is shRNA1, sh2 is shRNA2, and shNC is the blank control group. From Figure 1 it can be seen that the expression level of RhoJ protein in the knockdown group is significantly lower than that in the control group.

[0070] Example 4

[0071] 1. shRhoJ can inhibit the proliferation of A498

[0072] EdU cell proliferation experiment: Use the EdU cell proliferation detection kit for EdU labeling and perform the experiment according to the kit instructions. Briefly, inoculate about 1×10 4 cells into a 48-well plate. After culturing for 24 h, perform the transfection experiment immediately. The next day, culture under hypoxia or normoxia for 24 h, and then add 20 μM EdU and continue to culture for 4 h. After taking out the cells, fix them with 4% paraformaldehyde at room temperature for 30 min, permeabilize them with 0.5% Triton X-100 for 10 min, wash the cells with PBS, and then add 150 μL of 1× Apollo staining reaction solution to each well of the cells and react for 30 min. Stain the DNA with 1× Hochest 33342 (150 μL per well) for 5 min and take pictures under a fluorescence microscope.

[0073] Result analysis:

[0074] The experimental results are as Figure 2 and Figure 3 shown. From Figure 2 and Figure 3 it can be seen that the EdU staining intensity in the RhoJ-sh1 and RhoJ-sh2 groups is significantly less than that in the control group, indicating that knocking down the RhoJ gene significantly inhibits the proliferation of A498 cells.

[0075] 2. shRhoJ inhibits the migration of A498

[0076] Scratch assay: Inoculate 1×10 5Seed sh1, sh2, and shNC-A498 cells in a 6-well plate and allow them to adhere for 24 hours. Then, use a 200-µl pipette tip to draw a vertical line in the center, and observe the degree of scratch closure at 0 h and 24 h.

[0077] Result analysis:

[0078] The experimental results are as Figure 4 and Figure 5 shown. As can be seen from Figure 4 and Figure 5 , the scratch closure ratios of the RhoJ-sh1 and RhoJ-sh2 groups at 24 hours were significantly lower than those of the control group, indicating that knocking down the RhoJ gene significantly inhibited the migration of A498 cells.

[0079] Example 5 Transwell assay:

[0080] Migration assay: Mix sh1, sh2, and shNC-A498 cells with serum-free MEM medium and seed 20,000 cells per upper chamber (Corning). Add 600 μL of complete medium to the lower chamber. After culturing for 24 h, remove the upper chamber, wash with PBS, fix with 4% paraformaldehyde for 10 min, and then wash with PBS. Subsequently, stain with crystal violet for 5 min, rinse thoroughly with running water, air dry, and take microscopic pictures.

[0081] Invasion assay: First, dilute high-concentration Matrigel with complete MEM medium to an appropriate concentration, and then evenly add 40 μL of Matrigel to each upper chamber. Place it in an incubator at 37 °C until dry. Mix sh1, sh2, and shNC-A498 cells with serum-free MEM medium and seed 20,000 cells per upper chamber (Corning). Add 600 μL of complete medium to the lower chamber. After culturing for 48 h, remove the upper chamber, wash with PBS, fix with 4% paraformaldehyde for 10 min, and then wash with PBS. Subsequently, stain with crystal violet for 5 min, rinse thoroughly with running water, air dry, and take microscopic pictures.

[0082] Experimental results: The experimental results are as Figure 8 and Figure 9 shown. As can be seen from Figure 8 and Figure 9 , the number of cells that passed through in the RhoJ-sh1 and RhoJ-sh2 groups was significantly lower than that of the control group in both invasion and migration assays, indicating that knocking down RhoJ significantly inhibited the invasion and migration of renal clear cell carcinoma A498 cells.

[0083] Although several embodiments of the present invention have been given in this article, those skilled in the art should understand that the embodiments in this article can be changed without departing from the spirit of the present invention. The above embodiments are only exemplary and should not be used to limit the scope of the present invention.

Claims

1. A shRNA targeting silencing RhoJ, characterized in that: The nucleotide sequence of the sense strand of the shRNA is shown as SEQ ID No. 1, and the nucleotide sequence of the antisense strand is shown as SEQ ID No. 2; or, the nucleotide sequence of the sense strand of the shRNA is shown as SEQ ID No. 3, and the nucleotide sequence of the antisense strand is shown as SEQ ID No.

4.

2. A recombinant plasmid, characterized in that: The recombinant plasmid comprises a vector and a target gene, wherein the vector is PLKO.1, and the target gene is the positive strand and the antisense strand of the shRNA for targeted silencing of RhoJ according to claim 1, wherein the PLKO.1 is double-digested by AgeI enzyme and ECORI enzyme.

3. The method for constructing a recombinant plasmid according to claim 2, characterized in that: The following steps are involved: 1) synthesizing the sense strand and antisense strand of the shRNA, and annealing the sense strand and antisense strand of the shRNA; 2) Double digestion of PLKO.1 with AgeI and EcoRI enzymes; and 3) Enzyme ligation is performed on the vector digested with PLKO.1 and the annealing product obtained in step 1) to obtain the recombinant plasmid according to claim 2.

4. A shRNA lentiviral vector, characterized in that: The shRNA lentiviral vector comprises the sense chain and antisense chain of the shRNA according to claim 1, wherein the nucleotide sequence of the sense chain of the shRNA is shown as SEQ ID No. 1, and the nucleotide sequence of the antisense chain is shown as SEQ ID No. 2; or, the nucleotide sequence of the sense chain of the shRNA is shown as SEQ ID No. 3, and the nucleotide sequence of the antisense chain is shown as SEQ ID No.

4.

5. The method for constructing the shRNA lentiviral vector according to claim 4, characterized in that: The following steps are involved: (1) The recombinant plasmid described in claim 2, the Pax2 plasmid and the Vsvg plasmid are thoroughly mixed; (2) Add the transfection reagent Jetprime and buffer and the mixed plasmid obtained in step (1) to 293T cells, culture them, and collect the supernatant to obtain the shRNA lentiviral vector.

6. The use of shRNA for targeted silencing of RhoJ according to claim 1, characterized in that: The shRNA is used to prepare a drug for treating renal clear cell carcinoma.

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