ShRNA (short hairpin ribonucleic acid) of targeted silence RhoJ, application of shRNA, recombinant plasmid and lentiviral vector

Abstract

The invention provides shRNA (short hairpin Ribonucleic Acid) of targeted silence RhoJ and application thereof, a recombinant plasmid and a lentiviral vector, and relates to the technical field of biological medicines. The nucleotide sequence of the positive-sense strand of the shRNA of the targeted silent 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 of the targeted silence RhoJ can significantly reduce RhoJ expression of renal clear cell carcinoma A498 cells and inhibit proliferation, invasion and migration of the renal clear cell carcinoma A498 cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a shRNA for targeted silencing of RhoJ and its use, recombinant plasmid and lentiviral vector. Background Art

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

[0003] RhoJ is a member of the small GTPase family. Since its discovery in 2000, its role in vascular endothelial cells has mainly focused on regulating intercellular adhesion, cell proliferation and migration. It has also been shown to play an important role in promoting angiogenesis, tumor invasion and metastasis in glioblastoma, melanocytoma, gastric adenocarcinoma and triple-negative breast cancer, and promotes tumor cell DNA repair in multiple ways 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 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.

[0016] A method for targeted silencing of RhoJ comprises using the shRNA lentiviral vector to infect target cells to obtain RhoJ silenced cells.

[0017] The RhoJ silenced cells were constructed using the method described above.

[0018] The use of the shRNA that targets and silences RhoJ is to use the shRNA to prepare a biological agent that inhibits the expression of the RhoJ gene.

[0019] The use of the shRNA that targets and silences RhoJ is to prepare a drug for treating renal clear cell carcinoma.

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

[0021] Worldwide, renal cell carcinoma (RCC) ranks 14th in the incidence of all malignant tumors. And the incidence rate has regional differences, with a higher incidence in Europe and North America and a lower incidence in other regions such as Africa and Asia. Therefore, its prevention and treatment is of great significance. Existing drugs mainly include chemotherapy drugs, targeted drugs and immune checkpoint inhibitors. However, these drugs are not effective for renal cell carcinoma. 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 treatment in this case.

[0022] The development of RNAi technology has not only greatly promoted the human post-genome project, but can also be used for high-throughput screening of drug target genes, promote gene therapy and new drug development, and open up new ways to treat cancer, genetic diseases and other 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 methods, RNAi technology has stronger targeting of pathogenic genes and lower toxic side effects, so it is of great significance to develop more RNA interference fragments for renal cell carcinoma.

[0023] The targeted silencing RhoJ shRNA 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 is a diagram of RhoJ knockdown efficiency in the renal cell carcinoma A498 cell line provided in Example 3 of the present invention;

[0026] Figure 2 is a graph showing the results of an EdU proliferation experiment provided in Example 4 of the present invention;

[0027] Figure 3 is a statistical graph of the EdU proliferation experiment results provided in Example 4 of the present invention;

[0028] Figure 4 is a scratch test result diagram provided in Example 4 of the present invention;

[0029] Figure 5 is a statistical graph of scratch test results provided in Example 4 of the present invention;

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

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

[0032] Figure 8 is a diagram showing the results of a Transwell invasion and migration experiment provided in Example 5 of the present invention;

[0033] Fig. 9 It is a statistical graph of the Transwell invasion and migration experiment results provided in Example 5 of the present invention. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is described below in conjunction with the accompanying drawings.

[0035] In the embodiment of the present invention, the meaning of "and / or" can be both, or one of the two can be selected.

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

[0037] Table 1

[0038]

[0039] Synthesized by Beijing Bomade Company.

[0040] Table 2

[0041]

[0042] Synthesized by Beijing Bomade Company.

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

[0044] Table 3

[0045]

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

[0047] Table 4

[0048]

[0049] The U6 promoter is upstream of the shRNA sequence, and primers are designed for qPCR detection to verify whether the sequence and 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) in 20µl.

[0052] 4µl positive strand + 4µl antisense strand + 2µl NEB buffer + 10µl ddH2O. PCR reaction conditions are as follows: 95℃ for 2min, one cycle, decreasing 0.1℃ to 25℃ every eight seconds, 700 cycles, and then cooling to 4℃.

[0053] 2. Enzyme digestion of vector, 50µl system.

[0054] PLKO.1 vector 1µg + AgeI enzyme 1µl + ECORI enzyme 1µl + buffer 5µl, add ddH2O to 50µl. Digest at 37℃ for 3h.

[0055] 3. For nucleic acid gel recovery, prepare 1% agarose gel and add 1% agarose to 1×TAE. Microwave for 1-2 minutes until the liquid is clear. After cooling, add nucleic acid dye (pay attention to the management of contaminated areas). Perform agarose gel electrophoresis on the products after enzyme digestion, and add PLKO.1 empty load as a positive control when spotting.

[0056] 4. Enzyme ligation: 100ng of vector digested with PLKO.1, 1µl of annealed product obtained in step 1), 1µl of T4 ligase, and 1µl of buffer. Add ddH2O to 10µl. Ligation at room temperature for 30-60min.

[0057] 5. Transformation: Plasmid transformation is performed into DH5a competent cells.

[0058] 6. For plating, add 6µl Amp+ to 6ml solid LB medium, pour into a 6cm sterile culture dish and cool until solidified. Add the transformed product and shake slowly to evenly distribute the bacterial solution on the surface of the solid culture medium. Place upright for 10-30 minutes, then invert the culture dish and place in a 37℃ incubator for 16 hours.

[0059] 7. Pick bacteria, PCR identification, pick a single isolated round colony and mix it in 9µl ddH2O, take 1-2µl of bacterial solution + 0.3µl U6 promoter primer F chain + 0.3µl U6 promoter primer R chain + 10µl Taq PCR StarMix, add ddH2O to 20µl. PCR reaction conditions are as follows: 95℃5 minutes, 94℃30s, 55℃30s, 72℃60s, 30 cycles, 72℃5 minutes, cool to 4℃ for storage. PCR products are subjected to agarose gel electrophoresis to observe whether the plasmid construction is successful.

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

[0061] The day before transfection, 293T cells were inoculated in a 100mm plate (10mL DMEM) and cultured for 12-24h. When the cell density reached 40-50%, the plasmid mixture required for each group of the transfection system was 5μg packaging plasmid (Pax2 and Vsvg, Sigma) and 5μg PLKO.1-puro lentiviral recombinant plasmid (i.e., the plasmid constructed in Example 1), and the above plasmids were fully mixed; each group required a transfection reagent of 20μL JetPrime+500μL Buffer. The experiment used the shNC viral vector targeting the non-target gene sequence as a control. The transfection reagent and the plasmid mixture were fully mixed and allowed to stand at room temperature for 10min. A volume of 1mL of the mixture was slowly added dropwise to a 100mm culture dish, and after culturing in a 37°C incubator for 8-12h, fresh complete culture medium was replaced; in order to increase the concentration of the virus, 8mL of culture medium can be changed when changing the liquid. After another 48 hours of culture, collect the supernatant and replace it with fresh complete culture medium in a culture dish. Incubate at 3500rpm for 10 minutes. Collect the supernatant, which is the corresponding virus solution. Dispense about 700 μL per tube into a 1.5 mL sterilized EP tube. After 72 hours, treat 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, count by centrifugation, and count at 6×10 5 Each well was inoculated into a 60 mm dish and cultured at 37°C, 5% CO2 for 24 h.

[0064] 2. Infection of A498 with shRhoJ lentivirus:

[0065] A498 cells were seeded in 6-well plates at a cell density of 1.5×10 5 Cells / well. After 12-24h of culture, A498 cells were infected with a mixture of 500μL DMEM + 500μL shRhoJ lentivirus solution + 0.5μL polybrene (polybrene, final concentration of 10μg / μL). A498 cells were infected with shNC lentivirus expression vectors and lentivirus targeting non-target gene sequences as controls for shRhoJ lentivirus expression vectors and lentivirus.

[0066] 3. 72 hours after the cells were infected with the virus, puromycin culture medium with a final concentration of 1 μg / μL was added to continue culturing the cells until a cell line stably infected with shRhoJ lentivirus was selected.

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

[0068] Collect protein samples, quantify protein, take 40μg of protein samples, and perform SDS-PAGE electrophoresis at 100v constant voltage. Transfer: 200mA constant current for 1.5h. After transfer, place the membrane in 5% skim milk powder blocking solution for 1h, and then wash the membrane twice with TBST. Add the primary antibody prepared with 3% skim milk powder (1:1000 dilution), place it in a shaker, and keep it at 4℃ overnight. The next day, wash the membrane three times with TBST, 5min each time, add the secondary antibody prepared with 3% skim milk powder (1:5000 dilution), and hybridize at room temperature for 1h before color development (or chemiluminescence autoradiography reaction).

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

[0070] Example 4

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

[0072] EdU cell proliferation assay: EdU labeling was performed using an EdU cell proliferation assay kit and the experimental procedure was performed according to the kit instructions. 4 Cells were placed in 48-well plates and transfected after 24 hours of culture. The next day, the cells were cultured under hypoxia or normoxia for 24 hours, and then 20μM EdU was added to continue culturing for 4 hours. After the cells were removed, they were fixed with 4% paraformaldehyde at room temperature for 30 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes, washed with PBS, and then 150μL 1×Apollo staining reaction solution was added to each well of cells for 30 minutes. DNA was stained with 1×Hochest 33342 (150μL per well) for 5 minutes and photographed under a fluorescence microscope.

[0073] Result analysis:

[0074] The experimental results are as follows Figure 2 and Figure 3 As shown. Figure 2 and Figure 3 It can be seen that the EdU staining intensity of the RhoJ-sh1 and RhoJ-sh2 groups was significantly lower than that of the control group, indicating that knocking down the RhoJ gene significantly inhibited the proliferation of A498 cells.

[0075] 2. shRhoJ inhibits A498 migration

[0076] Scratch test: 1×10 5 sh1, sh2, and shNC-A498 cells were plated in 6-well plates and allowed to adhere for 24 hours. A vertical line was then drawn in the middle using a 200µl pipette tip, and the degree of wound closure was observed at 0h and 24h.

[0077] Result analysis:

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

[0079] Example 5 Transwell experiment:

[0080] Migration experiment: sh1, sh2 and shNC-A498 cells were mixed with serum-free MEM medium and plated into 20,000 cells per upper chamber (Corning). 600 μL of complete medium was added to the lower chamber. After 24 hours of culture, the upper chamber was removed, washed with PBS, fixed with 4% paraformaldehyde for 10 minutes, and then washed with PBS. Then, crystal violet was used for staining for 5 minutes, rinsed with running water, and photographed under a microscope after drying.

[0081] Invasion assay: First, dilute the high-concentration matrix gel with MEM complete medium to the appropriate concentration, and evenly add 40 μL of matrix gel to each upper chamber, and place it in a 37°C incubator to dry. Mix sh1, sh2, and shNC-A498 cells with serum-free MEM medium, and place 20,000 cells per upper chamber (Corning). Add 600 μL of complete medium to the lower chamber. After 48 hours of culture, remove the upper chamber, wash with PBS, fix with 4% paraformaldehyde for 10 minutes, and then wash with PBS. Then use crystal violet staining for 5 minutes, rinse with running water, and take pictures under a microscope after drying.

[0082] Experimental results: The experimental results are as follows Figure 8 and Fig. 9 As shown. Figure 8 and Fig. 9 It can be seen that the number of permeable cells in the RhoJ-sh1 and RhoJ-sh2 groups was significantly lower than that in the control group in the invasion and migration experiments, 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 herein, those skilled in the art should understand that the embodiments of the present invention may be modified without departing from the spirit of the present invention. The above embodiments are merely exemplary and should not be used as a limitation on the scope of the present invention.

Claims

1. A shRNA for targeted silencing of RhoJ, characterized in that: 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.

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 sense strand and antisense strand of the shRNA targeting silencing 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 strand and antisense strand of the shRNA according to claim 1, 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.

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. A method for targeted silencing of RhoJ, characterized in that: The method comprises using the shRNA lentiviral vector according to claim 4 to infect target cells, thereby obtaining RhoJ silenced cells.

7. The RhoJ silenced cells constructed according to the method of claim 6.

8. The use of shRNA for targeted silencing of RhoJ according to claim 1, characterized in that: The shRNA is used to prepare a biological agent for inhibiting the expression of the RhoJ gene.

9. 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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