Application of siPRMT3 in HIV infection

Abstract

The invention discloses application of siPRMT3 in HIV (human immunodeficiency virus) infection, a plurality of siRNAs (small interfering Ribonucleic Acid) capable of targeting PRMT3 are designed, the effect of the siRNAs for knocking down the PRMT3 in MT4 cells is evaluated, and the siRNAs capable of knocking down the PRMT3 in the MT4 cells are screened out; the method comprises the following steps: screening out siRNA from PRMT3, respectively using MT4 cells infected with HIV-1 virus and primary CD4 + T cells of an HIV infected patient, evaluating and verifying the influence of the screened siRNA to knock down the PRMT3 on HIV-1 virus replication and latent reactivation, finally finding that the siRNA capable of inhibiting the HIV-1 virus replication and latent reactivation by knocking down the PRMT3 is found, perfecting an evidence chain for treating HIV-1 by targeting the PRMT3, and providing a new possibility for treatment of HIV-1 infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of siPRMT3 in HIV infection. Background Art

[0002] AIDS is a major global infectious disease. At present, the main treatment plan for AIDS is that the highly active antiretroviral therapy (HAART) can effectively inhibit the virus in active reproduction [1]. Finding a cure for AIDS is a key issue that urgently needs to be solved in the field of public health. Identifying new HIV-1 transcriptional regulators and revealing their mechanisms of action are important bases for exploring the cure for AIDS. There is still no effective cure for AIDS [2].

[0003] Although AIDS can be effectively controlled at present, there is no method to clear or long-term inhibit the replication of the HIV virus. HAART can inhibit the active reproduction of HIV-1, but it cannot clear or long-term inhibit the latent HIV-1. When HARRT is interrupted or the immune system is weakened, the latent virus can be repeatedly activated, resulting in the difficulty of curing AIDS. The main reason for the difficulty in curing AIDS is that it is impossible to eradicate the latent HIV-1 or silence it for a long time [3]. Since patients need to take medicine for life, it brings a great economic burden to the country and society. At present, the two main directions for curing AIDS are the clearance strategy and the functional cure strategy. It is very challenging to comprehensively carry out the clearance strategy clinically due to technical bottlenecks and clinical application obstacles. Therefore, finding new targets and means to long-term control virus replication is a research hotspot in this field and also a major clinical need.

[0004] The patent "Use of PRMT3 Protein and Method for Regulating HIV-1 Transcription" with the application number 202110750730.3 discloses that PRMT3 interacts with LTR. Downregulating the level of PRMT3 by shRNA can inhibit HIV-1 transcription. Inhibiting the activity of PRMT3 can inhibit Tat-activated HIV-1 transcription and JQ-1-activated HIV-1 transcription. Inhibiting the activity of PRMT3 cannot inhibit Prostratin-activated HIV-1 transcription. In Jurkat 2D10 cells, treatment with a PRMT3 inhibitor was found to inhibit JQ-1-activated HIV-1 transcription. This patent uses the shRNA gene editing tool to knockdown the intracellular level of PRMT3. However, the circular delivery efficiency of the shRNA plasmid DNA is low (poor in vivo targeting; difficult to cross physiological barriers such as the blood-brain barrier, etc.); it has a short half-life; and the production cost is high, making it difficult to develop into a drug [4]. Moreover, the evidence for knockdown of PRMT3 inhibiting HIV-1 replication and transcriptional activation in this patent is only verified based on cell line models (HeLa and HEK293T), lacking verification in a true virus infection model and primary immune cells of patients. Therefore, whether knockdown of PRMT3 inhibits HIV-1 replication and transcriptional activation still needs to be further confirmed. Additionally, this patent does not cover the broad-spectrum antiviral effects of different HIV-1 subtypes, making it difficult to evaluate its clinical application potential.

[0005] The objective conditions for experiments such as true HIV-1 virus infection are demanding. For example, studying HIV-1 replication and transcriptional activation requires HIV-1 virus strains and biological samples from HIV-infected patients. The experiment needs to be carried out in a BSL-3 laboratory and meet the requirements of WHO and the "Regulations on the Biosafety Management of Pathogenic Microorganisms Laboratories", ensuring aerosol tight control and high-efficiency air filtration (HEPA). Adequate protection is required during operation to avoid HIV-1 exposure. Uninactivated HIV-1 infection samples should be processed under the supervision of two people in a BSL-3 laboratory. Nucleic acid extraction of inactivated virus needs to be carried out in a secondary biological safety cabinet, etc.

[0006] In addition, experiments such as true HIV-1 virus infection are difficult, and sample acquisition and preservation are relatively difficult. For example, a large amount of exploration is required for the changes in TCID50 during the use of the virus, the effects of different MOIs and different virus subtypes on the experimental results. Biological samples from HIV-infected patients are relatively difficult to obtain. Peripheral blood mononuclear cells (PBMCs) need to be separated within 6 hours after collection, cryopreserved in liquid nitrogen (-196 °C), and transported with dry ice (-78 °C). The survival rate after resuscitation is usually <70%. Samples are scarce and precious. Primary CD4 + T cells need to be enriched by magnetic bead sorting, and the purity requirement is ≥95%. Primary cells cannot be amplified in vitro, and only a limited amount of cells can be obtained in a single batch experiment (usually ≤6×10 6For (cells / samples), it is necessary to combine multiple patient samples to increase statistical power; primary cells are in a fragile state, and conditions need to be continuously optimized during the electroporation experiment to reduce trauma to the cells and improve transfection efficiency, etc.

[0007] In summary, the problems existing in the prior art of shRNA targeting PRMT3 to inhibit HIV-1 virus are as follows: the circular delivery efficiency of shRNA plasmid DNA is low, the production cost is high, and it is relatively difficult to become a drug; the effect of targeting PRMT3 on HIV-1 lacks verification in a true virus infection model and primary immune cells of patients, and it needs to be further studied in depth to be confirmed before it has clinical transformation and application value.

[0008] References:

[0009] [1]HIV-CAUSAL Collaboration, Maile Ray, Roger Logan, Jonathan A CSterne, Sonia James M Robins, Caroline Sabin, Loveleen Bansi, Ardvan Sighem, Frank de Wolf, Dominique Costagliola, Emilie Lanoy, Heiner C Bucher, Viktor von Wyl, Anna Esteve, Jordi Casbona, Julia del Amo, Santiago Moreno, Amy Justice, Joseph Goulet, Sara Lodi, Andrew Phillips, Rémonie Seng, Laurence Meyer, Santiago Pérez-Hoyos, Patricia de Olalla, Miguel A The effect of combined antiretroviral therapy on the overall mortality of HIV-infected individuals AIDS (London, England) 2010 Jan 02; 24(1): 123-37 doi:10.1097 / QAD.0b013e3283324283

[0010] [2]Marie Armani-Tourret,Ce Gao,Ciputra Adijaya Hartana,WeiWei Sun,Leah Carrere,Liliana Vela,Alexander Hochroth,Maxime Bellefroid,Amy Sbrolla,Katrina Shea,Theresa Flynn,Isabelle Roseto,Yelizaveta Rassadkina,Carole Lee,Francoise Giguel,Rajeev Malhotra,Frederic D Bushman,Rajesh T Gandhi,Xu G Yu,Daniel R Kuritzkes,Mathias Lichterfeld;Selection of epigenetically privilegedHIV-1 proviruses during treatment with panobinostat and interferon-α2aCell2024 Feb 29;187(5):1238-1254.e14doi:10.1016 / j.cell.2024.01.037

[0011] [3]Iain C Clark, Prakriti Mudvari, Shravan Thaploo, Samuel Smith, Mohammad Abu-Laban, Mehdi Hamouda, Marc Theberge, Sakshi Shah, Sung Hee Ko, Liliana Pérez, Daniel G Bunis, James S Lee, Divya Kilam, Saami Zakaria, Sally Choi, Samuel Darko, Amy R Henry, Michael A Wheeler, Rebecca Hoh, Salwan Butrus, Steven G Deeks, Francisco J Quintana, Daniel C Douek, Adam R Abate, Eli A Boritz; HIV silencing and cell survival signatures in infected T cell reservoirs Nature 2023 02; 614(7947):318-325 doi:10.1038 / s41586-022-05556-6

[0012] [4]Thu Nguyen, Ellen M Menocal, Jens Harborth, Johannes H Fruehauf; RNA therapeutics: an update on delivery Current opinion in molecular therapeutics 2008 Apr; 10(2):158-67 Summary of the Invention

[0013] In view of the problems existing in the prior art, this application designed multiple siRNAs that may target PRMT3. The siRNA drug has the advantages of high targeting, specificity, long-term effect, reduced dosing frequency, and a wide target range. The effect of siRNA on knocking down PRMT3 in MT4 cells was evaluated, and the siRNAs that can knock down PRMT3 in MT4 cells were screened out; then MT4 cells infected with HIV-1 virus and primary CD4 + T cells of HIV-infected patients were used to evaluate and verify the effect of the screened siRNAs on knocking down PRMT3 on HIV-1 virus replication and latent reactivation. Finally, it was found and determined that the siRNAs that can inhibit HIV-1 virus replication and latent reactivation by knocking down PRMT3. The specific technical solutions of the present invention are as follows:

[0014] First aspect: Designing 7 siRNAs that may target PRMT3

[0015] 7 siRNAs that may target PRMT3 designed by software and 1 random control siRNA were used as a control (sicon).

[0016] Second aspect: Knockdown of PRMT3 in MT4 cells using the siRNA sequences of the first aspect and evaluation of the effect

[0017] The 7 siRNAs that may target PRMT3 in the first aspect were respectively transiently electroporated into MT4 cells. After 48 hours, the cells were collected and total RNA was extracted using a kit. After reverse transcription into cDNA, the effect of these 7 siRNAs on knocking down PRMT3 was detected by amplifying the PRMT3 gene through Realtime q-PCR.

[0018] Conclusion: siRNA-1, siRNA-2, siRNA-3, and siRNA-4 have good effects on knocking down PRMT3 in MT4 cells. Therefore, siRNA-1, siRNA-2, siRNA-3, and siRNA-4 were selected for subsequent evaluation and verification.

[0019] Third aspect: Knocking down PRMT3 in MT4 cells with different siRNAs and investigating the effect of inhibiting HIV-1 replication by evaluating the copy number of HIV-1 RNA

[0020] By transiently electroporating the control (sicon), siRNA-1, siRNA-2, and siRNA-3 into MT4 cells respectively, and infecting the electroporated MT4 cells with HIV-1 CRF01-AE (Chinese strain) and NL4-3 virus strain (European and American prevalent strain) 16 hours after electroporation, the effect of knocking down PRMT3 in MT4 cells on inhibiting HIV-1 virus replication was investigated by measuring the copy number of HIV-1 RNA (expression level of pol) and the expression level of gag in the MT4 cells infected with HIV-1 virus.

[0021] Results:

[0022] Compared with the control group, the siPRMT3-1 group and the siPRMT3-2 group could significantly reduce the copy number of HIV-1 RNA (expression level of pol) and the expression level of gag in MT4 cells;

[0023] Compared with the control group, there was no significant difference in the copy number of HIV-1 RNA (expression level of pol) and the expression level of gag in MT4 cells in the siPRMT3-3 group.

[0024] Conclusion:

[0025] Knockdown of PRMT3 in MT4 cells using siRNA-1 and siRNA-2 respectively can significantly inhibit the replication of different subtypes of HIV-1 viruses (NL4-3 and CRF01-AE);

[0026] Knockdown of PRMT3 in MT4 cells using siRNA-3 cannot inhibit the replication of different subtypes of HIV-1 viruses (NL4-3 and CRF01-AE).

[0027] Fourth aspect, different siRNAs were used to knockdown PRMT3 in MT4 cells, and the inhibitory effect on HIV-1 replication was evaluated by examining the content of HIV-1 P24 protein.

[0028] The content of HIV-1 P24 protein in the cell culture supernatant after infection with HIV-1 CRF01-AE (Chinese strain) was detected.

[0029] Results: Compared with the control group, the siPRMT3-1 group and the siPRMT3-2 group could significantly reduce the expression level of HIV-1 P24 protein in MT4 cells, and the P values were all less than 0.01.

[0030] Conclusion: Knockdown of PRMT3 in MT4 cells using the sequences of siRNA-1 and siRNA-2 respectively can significantly inhibit the replication of HIV-1 virus.

[0031] Fifth aspect, different siRNAs were used to knockdown PRMT3 in primary CD4 + T cells of HIV-infected patients to verify whether it can effectively inhibit the transcriptional reactivation of HIV

[0032] The sequences of siRNA-1, siRNA-2, and siRNA-4 were used to knockdown PRMT3 in primary CD4 + T cells in the peripheral blood of HIV-infected patients, and the copy number of HIV RNA in CD4 + T cells was examined to verify the effect on the latent reactivation of HIV virus.

[0033] Results:

[0034] Using siRNA-1 and siRNA-2 to knockdown PRMT3 in primary CD4 + T cells of HIV-infected patients, compared with the control group, could significantly inhibit the copy number of HIV RNA in CD4 + T cells, and the P values were less than 0.01 and 0.05 respectively;

[0035] Using siRNA-4 to knockdown PRMT3 in primary CD4 + T cells of HIV-infected patients, compared with the control, CD4 +The copy number of HIV RNA in T cells did not decrease and even increased.

[0036] Conclusion:

[0037] Using the sequences of siRNA-1 and siRNA-2 to knockdown PRMT3 in primary CD4 + T cells of HIV-infected patients can significantly inhibit the transcriptional reactivation of HIV;

[0038] Using the sequence of siRNA-4 to knockdown PRMT3 in primary CD4 + T cells of HIV-infected patients cannot inhibit the transcriptional reactivation of HIV.

[0039] Compared with the prior art, the beneficial effects of this application are as follows:

[0040] This application first discovers that using the sequences of siRNA-1 or siRNA-2 to knockdown PRMT3 can significantly inhibit the replication and transcriptional reactivation of HIV-1 virus; while using the sequences of siRNA-3 or siRNA-4 to knockdown PRMT3 cannot inhibit the replication and transcriptional reactivation of HIV-1 virus, providing a new possibility for the treatment of HIV-1 infection. Description of the Drawings

[0041] Figure 1 The reduction multiples of the PRMT3 gene expression level by knocking down PRMT3 in MT4 cells with 7 siRNAs, where sicon is a siRNA with a random sequence as a control, si-1 is siRNA-1, si-2 is siRNA-2,..., si-7 is siRNA-7;

[0042] Figure 2 The copy number of HIV-1 RNA (expression level of pol) and the expression level of gag in MT4 cells infected with NL4-3 and with PRMT3 gene knocked down, where

[0043] siPRMT3-1 is to knockdown PRMT3 in MT4 cells with siRNA-1 and then infect with NL4-3,

[0044] siPRMT3-2 is to knockdown PRMT3 in MT4 cells with siRNA-2 and then infect with NL4-3,, siPRMT3-3 is to knockdown PRMT3 in MT4 cells with siRNA-3 and then infect with NL4-3, sicon is to electrotransfer MT4 cells with sicon and then infect with NL4-3 as a control;

[0045] Figure 3, the copy number of HIV-1 RNA (expression level of pol) and the expression level of gag in MT4 cells infected with CRF01-AE and with PRMT3 gene knockdown, where

[0046] siPRMT3-1 means knocking down PRMT3 in MT4 cells using siRNA-1 and then infecting with CRF01-AE,

[0047] siPRMT3-2 means knocking down PRMT3 in MT4 cells using siRNA-2 and then infecting with CRF01-AE, siPRMT3-3 means knocking down PRMT3 in MT4 cells using siRNA-3 and then infecting with CRF01-AE, sicon means electrotransfecting MT4 cells with sicon and then infecting with CRF01-AE, as a control;

[0048] Figure 4 , the expression level of HIV-1 P24 protein in the culture supernatant of MT4 cells infected with CRF01-AE and with PRMT3 knockdown, where

[0049] siPRMT3-1 means knocking down PRMT3 in MT4 cells using siRNA-1 and then infecting with CRF01-AE, siPRMT3-2 means knocking down PRMT3 in MT4 cells using siRNA-2 and then infecting with CRF01-AE, sicon means electrotransfecting MT4 cells with sicon and then infecting with CRF01-AE, as a control;

[0050] Figure 5 , knocking down PRMT3 in primary CD4 + T cells of HIV-infected patients (No. 1, 2, 3) using siRNA-1 and siRNA-2 respectively, and the copy number of HIV RNA in CD4 + T cells;

[0051] Figure 6 , knocking down PRMT3 in primary CD4 + T cells of HIV-infected patients (No. 4, 5, 6) using siRNA-4, and the copy number of HIV RNA in CD4 + T cells.

[0052] Figure 1-6 Among them,

[0053] *, **, and *** respectively represent: P < 0.05, P < 0.01, P < 0.001, indicating the statistical significance of the difference between two groups, and ns indicates no statistical difference between two groups. Specific implementation manners

[0054] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0055] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0056] Unless otherwise specified, all materials, reagents, etc. in the following examples can be obtained from commercial sources.

[0057] Biological materials

[0058]

[0059]

[0060] Reagents

[0061]

[0062]

[0063] Instruments

[0064]

[0065] Example 1. siRNA Design

[0066] Seven siRNA sequences that may target PRMT3 and one random control siRNA sequence designed by software are shown in Table 1.

[0067] Table 1. siRNA Sequences That May Target PRMT3 Designed by Software

[0068]

[0069]

[0070] In Table 1,

[0071] siRNA-1: The first siRNA that may target PRMT3;

[0072] siRNA-2: The second siRNA that may target PRMT3;

[0073] ……;

[0074] siRNA-7: The seventh siRNA that may target PRMT3;

[0075] sicon: siRNA with a random sequence, used as a control.

[0076] Synthesize siRNAs according to the sequences of siRNA-1, siRNA-2, ……, siRNA-7 in Table 1 respectively.

[0077] Dissolve the lyophilized powders of siRNA-1, siRNA-2, ……, siRNA-7 in RNase-free water sufficiently, with a concentration of 20 μM. Aliquot 15 μL per tube and store at -20 °C.

[0078] Example 2: Knockdown of PRMT3 in MT4 cells using the siRNA sequences in Table 1 and evaluation of the effect

[0079] The MT4 cell line is derived from human T lymphocytes in the peripheral blood of an adult T cell leukemia patient, highly expresses the CD4 receptor and the CXCR4 co-receptor, and is widely used to evaluate the HIV-1 virus infection and replication mechanism.

[0080] 1. Method

[0081] After culturing MT4 cells in 1640 medium in a 37 °C carbon dioxide incubator for 24 hours, count 800,000 cells per well (12-well plate). Use the Lonza electroporator to transiently electroporate sicon (control), siRNA-1, siRNA-2, ……, siRNA-7 in Table 1 into MT4 cells respectively. After culturing in 1640 medium in a 37 °C carbon dioxide incubator for 48 hours, centrifuge at 500 × g for 3 minutes to collect cell pellets.

[0082] Use a cell total RNA rapid extraction kit to extract the total cell RNA from the above cell pellets. After reverse transcribing it into cDNA with a reverse transcription kit, detect the effect of these 7 siRNAs on knocking down the PRMT3 gene expression in MT4 cells by amplifying the PRMT3 gene through Realtime q-PCR, with GAPDH as the internal reference gene for detection.

[0083] 2. Results

[0084] As Figure 1 shown, taking the PRMT3 expression level in the control group (sicon) as the benchmark, using GAPDH as the internal reference gene, perform relative quantitative normalization processing on the qPCR data by the ΔΔCt method, and calculate the relative expression level of PRMT3 mRNA in the experimental group.

[0085] Using siRNA-1, siRNA-2, siRNA-3, siRNA-4 to knock down PRMT3 in MT4 cells, compared with the control group (sicon), the reduction multiples of their PRMT3 gene expression levels are all greater than 0.5 times.

[0086] PRMT3 was knocked down in MT4 cells using siRNA-5, siRNA-6, and siRNA-7. Compared with the control group (sicon), the reduction multiples of the PRMT3 gene expression level were all less than 0.5-fold.

[0087] The above shows that siRNA-1, siRNA-2, siRNA-3, and siRNA-4 have good effects on knocking down PRMT3 in MT4 cells. Therefore, siRNA-1, siRNA-2, siRNA-3, and siRNA-4 were selected for the evaluation and verification of Examples 3-5.

[0088] Example 3: Different siRNAs were used to knock down PRMT3 in MT4 cells, and the copy number of HIV-1 RNA was examined to evaluate the effect of inhibiting HIV-1 replication.

[0089] 1. Method

[0090] After MT4 cells were cultured in 1640 medium in a 37°C carbon dioxide incubator for 24 hours, 800,000 cells per well (12-well plate) were counted. Sicon (control), siRNA-1, siRNA-2, and siRNA-3 were transiently electroporated into MT4 cells using a Lonza electroporator, and the electroporation system was 100 μL.

[0091] After the electroporated MT4 cells were cultured in 1640 medium for another 16 hours, the cell precipitate was collected by centrifugation to obtain the MT4 cell precipitate.

[0092] The virus strain to be infected: HIV-1 virus NL4-3 virus strain (European and American epidemic strain) or CRF01-AE (Chinese strain) was diluted with 1640 medium at an MOI of 0.005.

[0093] The centrifuged MT4 cell precipitate was thoroughly mixed with 200 μL of the diluted virus solution with an MOI of 0.005, and then left standing for 8 hours for infection; after 8 hours, the cells were transferred to a 12-well plate and the volume was made up to 1.5 mL, and then cultured for another 40 hours.

[0094] The MT4 cell precipitate infected with the virus was collected by centrifugation, and the cell lysate in the virus RNA extraction kit was used to fully lyse the cells for 10 minutes to inactivate the virus completely, and then the HIV-1 virus RNA was extracted using the virus RNA extraction kit.

[0095] After the RNA was reverse transcribed into cDNA using a reverse transcription kit, the viral genes pol and gag were amplified by Realtime q-PCR, and the intracellular GAPDH was used as an internal reference gene to measure the cell quantity. By measuring the copy number of HIV-1 RNA (the expression level of pol) and the expression level of gag in MT4 cells infected with the HIV-1 virus, the effect of knocking down PRMT3 in MT4 cells on inhibiting the replication of the HIV-1 virus was investigated.

[0096] 2. Results

[0097] The copy number of HIV-1 RNA (the expression level of pol) and the expression level of gag in NL4-3-infected MT4 cells with knocked-down PRMT3 are shown in Table 2.

[0098] Table 2. The copy number of HIV-1 RNA (the expression level of pol) and the expression level of gag in NL4-3-infected MT4 cells with knocked-down PRMT3

[0099]

[0100] In Table 2,

[0101] siPRMT3-1: PRMT3 in MT4 cells was knocked down using siRNA-1 in Table 1, and then infected with NL4-3;

[0102] siPRMT3-2: PRMT3 in MT4 cells was knocked down using siRNA-2 in Table 1, and then infected with NL4-3;

[0103] siPRMT3-3: PRMT3 in MT4 cells was knocked down using siRNA-3 in Table 1, and then infected with NL4-3;

[0104] Control sicon: MT4 cells were electroporated with sicon in Table 1 and then infected with NL4-3.

[0105] From Figure 2 it can be seen that

[0106] Compared with the control group, the siPRMT3-1 group could significantly reduce the copy number of HIV-1 RNA (the expression level of pol) and the expression level of gag in MT4 cells, and the P values were all less than 0.01;

[0107] Compared with the control group, the siPRMT3-2 group could significantly reduce the copy number of HIV-1 RNA (the expression level of pol) and the expression level of gag in MT4 cells, and the P values were all less than 0.05;

[0108] Compared with the control group, there was no significant difference in the copy number of HIV-1 RNA (the expression level of pol) and the expression level of gag in MT4 cells in the siPRMT3-3 group.

[0109] The copy number of HIV-1 RNA (expression level of pol) and the expression level of gag in MT4 cells with PRMT3 knockdown infected with CRF01-AE are shown in Table 3.

[0110] Table 3. Copy number of HIV-1 RNA (expression level of pol) and expression level of gag in MT4 cells with PRMT3 knockdown infected with CRF01-AE

[0111]

[0112] In Table 3,

[0113] siPRMT3-1: Knockdown of PRMT3 in MT4 cells using siRNA-1 in Table 1, and then infect with CRF01-AE;

[0114] siPRMT3-2: Knockdown of PRMT3 in MT4 cells using siRNA-2 in Table 1, and then infect with CRF01-AE;

[0115] siPRMT3-3: Knockdown of PRMT3 in MT4 cells using siRNA-3 in Table 1, and then infect with CRF01-AE;

[0116] Control sicon: Transfect MT4 cells with sicon in Table 1, and then infect with CRF01-AE.

[0117] From Figure 3 it can be seen that

[0118] Compared with the control group, the siPRMT3-1 group can significantly reduce the copy number of HIV-1 RNA (expression level of pol) and the expression level of gag in MT4 cells, and the P values are all less than 0.01;

[0119] Compared with the control group, the siPRMT3-2 group can significantly reduce the copy number of HIV-1 RNA (expression level of pol) and the expression level of gag in MT4 cells, and the P values are all less than 0.01;

[0120] Compared with the control group, there is no significant difference in the copy number of HIV-1 RNA (expression level of pol) and the expression level of gag in MT4 cells in the siPRMT3-3 group;

[0121] 3. Conclusion

[0122] Knockdown of PRMT3 in MT4 cells using siRNA-1 and siRNA-2 respectively can significantly inhibit the replication of different subtypes of HIV-1 virus (NL4-3 and CRF01-AE);

[0123] Knockdown of PRMT3 in MT4 cells using siRNA-3 did not inhibit the replication of different subtypes of HIV-1 virus (NL4-3 and CRF01-AE).

[0124] Example 4: Knockdown of PRMT3 in MT4 cells using different siRNAs, and evaluation of the effect of inhibiting HIV-1 replication by examining the content of HIV-1 P24 protein

[0125] 1. Method

[0126] After culturing MT4 cells in 1640 medium in a 37°C carbon dioxide incubator for 24 hours, 800,000 cells per well (12-well plate) were counted. Sicon (control), siRNA-1, siRNA-2, and siRNA-3 were transiently electroporated into MT4 cells using a Lonza electroporator, and the electroporation system was 100 μL. The electroporated MT4 cells were continuously cultured in 1640 medium for 16 hours, and then the cell precipitate was collected by centrifugation;

[0127] Dilute HIV-1 CRF01-AE (Chinese strain) with 1640 medium at an MOI of 0.005;

[0128] The centrifuged MT4 cell precipitate was thoroughly mixed with 200 μL of the diluted virus solution with an MOI of 0.005, and then left to stand for 8 hours for infection; after 8 hours, the cells were transferred to a 12-well plate and the volume was made up to 1.5 mL with additional medium, and the cells were continuously cultured for 40 hours;

[0129] Centrifuge the cells, retain the cell culture supernatant, and detect the expression level of HIV-1 virus P24 protein in the supernatant;

[0130] The expression of p24 in the virus supernatant was detected using a retrovirus titer p24 detection kit (EasyQuarter); using the standard product provided in the kit, an HIV-1 p24 standard curve was prepared;

[0131] Add 25 μL of lysis buffer to each well, and then add 75 μL of sample or standard product to the corresponding wells; add 75 μL of enzyme conjugate to the reaction wells, shake for 60 seconds, and then place at 37°C for 50 minutes; wash the wells 5 times with 300 μL of buffer; then, add 100 μL of TMB developing solution to each well and incubate at 37°C for 10 minutes; add 50 μL of stop solution to each well, shake for 60 seconds, and measure the OD at 450 nm.

[0132] 2. Results

[0133] The expression levels of HIV-1 P24 protein in the culture supernatants of MT4 cells with knockdown of PRMT3 infected with CRF01-AE are shown in Table 4.

[0134] Table 4. Expression level of HIV-1 p24 protein in the culture supernatant of MT4 cells with knocked-down PRMT3 infected with CRF01-AE

[0135]

[0136] In Table 4,

[0137] siPRMT3-1: Knock down PRMT3 in MT4 cells using siRNA-1 in Table 1, and then infect with CRF01-AE;

[0138] siPRMT3-2: Knock down PRMT3 in MT4 cells using siRNA-2 in Table 1, and then infect with CRF01-AE;

[0139] siPRMT3-3: Knock down PRMT3 in MT4 cells using siRNA-3 in Table 1, and then infect with CRF01-AE;

[0140] Control sicon: Transfect MT4 cells with sicon in Table 1, and then infect with CRF01-AE.

[0141] It can be seen from Figure 4 that

[0142] Compared with the control group, the siPRMT3-1 group can significantly reduce the expression level of HIV-1 p24 protein in MT4 cells, P < 0.01;

[0143] Compared with the control group, the siPRMT3-2 group can significantly reduce the expression level of HIV-1 p24 protein in MT4 cells, P < 0.01.

[0144] Compared with the control group, the degree of decrease in the expression level of HIV-1 p24 protein in MT4 cells in the siPRMT3-3 group is significantly lower than that in siPRMT3-1 and siPRMT3-2.

[0145] 3. Conclusion

[0146] Knock down PRMT3 in MT4 cells using the sequences of siRNA-1 and siRNA-2 can significantly inhibit the replication of HIV-1 virus;

[0147] Knock down PRMT3 in MT4 cells using the siRNA sequence of siRNA-3 cannot significantly inhibit the replication of HIV-1 virus.

[0148] Example 5: Knock down PRMT3 in primary CD4 + T cells of HIV-infected patients to verify whether it can effectively inhibit the transcriptional reactivation of HIV

[0149] Using siRNA-1, siRNA-2, and siRNA-4 to knockdown PRMT3 in primary CD4 + T cells in the peripheral blood of HIV-infected patients, and examining the copy number of HIV RNA in CD4 + T cells to verify the effect on the latent reactivation of HIV virus.

[0150] 1. Method

[0151] Resuscitate PBMC cells from HIV-infected patients. After stabilizing the cell state in 1640 medium in a 37°C carbon dioxide incubator for 3 hours, use a human CD4 + T cell sorting kit to sort CD4 + T cells from PBMC. After cell counting, take at least 1 million cells per group. The cells of each patient are divided into 3 groups: sicon (control), siPRMT3-1, and siPRMT3-2 groups. Centrifuge to collect cell pellets;

[0152] Prepare phorbol myristate acetate (PMA) at 200 nM, 400 μL per group. Resuspend the CD4 + T cell pellets with the prepared PMA solution and stimulate them in a 37°C carbon dioxide incubator for 12 hours to activate the latent virus reservoir in the patient cells;

[0153] After 12 hours, use a Lonza electroporator to transiently electroporate sicon (control), siRNA-1, siRNA-2, and siRNA-4 into primary CD4 + T cells of HIV-infected patients. The electroporation system is 100 μL. After electroporation, the primary cells are cultured in 1640 medium for another 40 hours, and then centrifuged to collect cell pellets; Add 300 μL of Trizol reagent to the cell pellets to protect RNA and perform HIV virus copy number detection.

[0154] 2. Results

[0155] Using siRNA-1 and siRNA-2 to knockdown PRMT3 in primary CD4 + T cells in HIV-infected patients, the copy number of HIVRNA in CD4 + T cells is shown in Table 5;

[0156] Table 5. Using siRNA-1 and siRNA-2 to knockdown PRMT3 in primary CD4 + T cells in HIV-infected patients, the copy number of HIV RNA in CD4 + T cells

[0157]

[0158] In Table 5,

[0159] siPRMT3-1: Knockdown of PRMT3 in primary CD4 + T cells of HIV-infected patients using siRNA-1 in Table 1;

[0160] siPRMT3-2: Knockdown of PRMT3 in primary CD4 + T cells of HIV-infected patients using siRNA-2 in Table 1;

[0161] sicon: Transfecting primary CD4 + T cells with siRNA of sicon in Table 1 as a control;

[0162] Undet.: Indicates that the CA RNA / cells per million (copies / 1×E 6 cells) in the representative samples is less than 20.

[0163] As Figure 5 can be seen,

[0164] Knockdown of PRMT3 in primary CD4 + T cells of HIV-infected patients using siRNA-1 and siRNA-2 can significantly inhibit the copy number of HIV RNA in CD4 + T cells, with P values less than 0.01 and 0.05 respectively.

[0165] Knockdown of PRMT3 in primary CD4 + T cells of HIV-infected patients using siRNA-4, the copy number of HIV RNA in CD4 + T cells is shown in Table 6;

[0166] Table 6. Copy number of HIV RNA in primary CD4 + T cells of HIV-infected patients after knockdown of PRMT3 using siRNA-4 + in CD4

[0167]

[0168] In Table 6,

[0169] siPRMT3-4: Knockdown of PRMT3 in primary CD4 + T cells of HIV-infected patients using siRNA-4 in Table 1;

[0170] sicon: Transfecting primary CD4 + T cells with siRNA of sicon in Table 1 as a control.

[0171] As Figure 6As shown, primary CD4 T cells from HIV-infected patients were knocked down for PRMT3 using siRNA-4. Compared with the control, the copy number of HIV RNA in CD4 T cells did not decrease. + Knocking down PRMT3 in primary CD4 T cells from HIV-infected patients using siRNA-1 and siRNA-2 respectively could significantly inhibit the transcriptional reactivation of HIV. + The copy number of HIV RNA in CD4 T cells did not decrease.

[0172] 3. Conclusion

[0173] Knocking down PRMT3 in primary CD4 T cells from HIV-infected patients using siRNA-1 and siRNA-2 respectively could significantly inhibit the transcriptional reactivation of HIV. + Knocking down PRMT3 in primary CD4 T cells from HIV-infected patients using siRNA-1 and siRNA-2 respectively could significantly inhibit the transcriptional reactivation of HIV.

[0174] Knocking down PRMT3 in primary CD4 T cells from HIV-infected patients using siRNA-4 could not inhibit the transcriptional reactivation of HIV. + Knocking down PRMT3 in primary CD4 T cells from HIV-infected patients using siRNA-4 could not inhibit the transcriptional reactivation of HIV.

[0175] In summary, knocking down PRMT3 using the sequences of siRNA-1 and siRNA-2 could significantly inhibit the replication and transcriptional reactivation of HIV-1 virus; while knocking down PRMT3 using the sequences of siRNA-3 and siRNA-4 could not inhibit the replication and transcriptional reactivation of HIV-1 virus.

[0176] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. Use of siRNA-1 for knocking down the expression level of PRMT3 in the preparation of a product for treating HIV-1, characterized in that, The antisense strand sequence of the siRNA-1 is shown as SEQ ID NO.1, and the sense strand sequence is shown as SEQ ID NO.

2.

2. The application according to claim 1, characterized in that Using the described siRNA-1 to knockdown PRMT3 in MT4 cells and then infecting with HIV-1 virus NL4-3, compared with the control group, it can significantly reduce the copy number of HIV-1 RNA, the expression levels of pol and gag in MT4 cells.

3. The application according to claim 1, characterized in that Using the described siRNA-1 to knockdown PRMT3 in MT4 cells and then infecting with HIV-1 virus CRF01-AE, compared with the control group, it can significantly reduce the expression level of HIV-1 P24 protein in MT4 cells.

4. The application according to claim 1, characterized in that, Using the siRNA-1 to knockdown PRMT3 in primary CD4 + T cells of HIV-infected patients can significantly inhibit the copy number of HIV RNA in CD4 + T cells compared with the control group.

5. Use of siRNA-2 for knocking down the expression level of PRMT3 in the preparation of a product for treating HIV-1, characterized in that, The antisense strand sequence of the siRNA-2 is shown as SEQ ID NO.3, and the sense strand sequence is shown as SEQ ID NO.

4.

6. The application according to claim 5, wherein Using the described siRNA-2 to knockdown PRMT3 in MT4 cells and then infecting with HIV-1 virus NL4-3, compared with the control group, it can significantly reduce the copy number of HIV-1 RNA, the expression levels of pol and gag in MT4 cells.

7. The application according to claim 5, wherein Using the described siRNA-2 to knockdown PRMT3 in MT4 cells and then infecting with HIV-1 virus CRF01-AE, compared with the control group, it can significantly reduce the expression level of HIV-1 P24 protein in MT4 cells.

8. The application according to claim 5, characterized in that, Using the described siRNA-2 to knockdown PRMT3 in primary CD4 + T cells of HIV-infected patients can significantly inhibit the CD4 + copy number of HIV RNA in T cells compared with the control group.

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