Use of siPRMT3 in HIV infection

Abstract

The application discloses siPRMT3 in HIV infection, and designs a plurality of siRNAs which can target PRMT3, evaluates the effect of the siRNAs on knocking down PRMT3 in MT4 cells, and screens out siRNAs which can knock down PRMT3 in MT4 cells. + MT4 cells infected with HIV-1 virus and primary CD4 T cells of HIV infected patients are used respectively, the influence of the screened siRNAs on HIV-1 virus replication and latent reactivation is evaluated and verified, finally, siRNAs which can inhibit HIV-1 virus replication and latent reactivation by knocking down PRMT3 are found, the evidence chain of treating HIV-1 by targeting PRMT3 is perfected, and a new possibility is provided for the treatment of HIV-1 infection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of siPRMT3 in HIV infection. BACKGROUND

[0002] The main treatment scheme for AIDS at present is that the virus in active propagation can be effectively inhibited through the cocktail therapy [1], and finding a cure for AIDS is a key problem urgently to be solved in the field of public health. Identifying a new HIV-1 transcription regulatory factor and revealing the action mechanism thereof are important bases for exploring a cure for AIDS.

[0003] Although AIDS can be effectively controlled at present, there is no method for removing or long-term inhibiting the replication of the HIV, the highly active antiretroviral therapy (HAART) can inhibit the active propagation of HIV-1, but cannot remove or long-term inhibit the latent HIV-1. When the HARRT is interrupted or the immunity is lowered, the latent virus can be repeatedly activated, leading to the difficulty in curing AIDS. At present, two main directions for curing AIDS are the removal strategy and the functional cure strategy. It is very challenging to comprehensively develop the removal strategy in the clinic due to technical bottlenecks and clinical application obstacles. Therefore, finding a new target and means to long-term control the virus replication is a research hotspot in this field and a major requirement for clinical treatment.

[0004] The patent "PRMT3 protein use and method for regulating HIV-1 transcription" with application number 202110750730.3 discloses that PRMT3 interacts with LTR, down-regulating PRMT3 level by shRNA can inhibit HIV-1 transcription, inhibiting PRMT3 activity can inhibit Tat-activated HIV-1 transcription, inhibiting PRMT3 activity can inhibit JQ-1-activated HIV-1 transcription, and inhibiting PRMT3 activity cannot inhibit Prostratin-activated HIV-1 transcription. In Jurkat 2D10 cells, it is found that inhibiting PRMT3 activity can inhibit JQ-1-activated HIV-1 transcription. The patent uses shRNA gene editing tools to knock down the level of PRMT3 in cells, but the circular delivery efficiency of shRNA plasmid DNA is low (poor targeting in vivo; difficult to cross physiological barriers such as blood-brain barrier, etc.); short half-life; high production cost, and it is difficult to develop drugs [4]; and the evidence that knocking down PRMT3 inhibits HIV-1 replication and transcription activation in the patent is only verified based on cell line models (HeLa and HEK293T), lacking verification of true virus infection models and patient primary immune cells, so whether knocking down PRMT3 can inhibit HIV-1 replication and transcription activation still needs to be further confirmed; in addition, the patent does not cover the broad-spectrum antiviral effect of different HIV-1 subtypes, making it difficult to evaluate its clinical application potential.

[0005] The objective conditions of HIV-1 true virus infection experiments require high, for example: studying HIV-1 replication and transcription activation requires HIV-1 virus strains and biological samples of HIV-infected patients, experiments need to be carried out in BSL-3 laboratories and meet the requirements of WHO and "Regulations on the Management of Biological Safety of Pathogenic Microorganisms", ensuring aerosol containment control and high-efficiency air filtration (HEPA), and full protection is required during operation to avoid HIV-1 exposure. Non-inactivated HIV-1 infected samples should be handled under BSL-3 double supervision, and inactivated virus nucleic acid extraction should be carried out in a secondary biological safety cabinet, etc.

[0006] In addition, HIV-1 true virus infection experiments are difficult, and sample acquisition and preservation are relatively difficult, for example: the change of TCID50 during virus use, the influence of different MOI and different virus subtypes on experimental results need a lot of exploration; the acquisition of biological samples of HIV-infected patients is relatively difficult, peripheral blood mononuclear cells (PBMC) need to be separated within 6 hours after collection, stored in liquid nitrogen (-196℃), transported on dry ice (-78℃), and the survival rate after recovery is usually <70%; the sample is rare and precious, and the primary CD4 + T cells need to be enriched by magnetic bead sorting, and the purity requirement is ≥95%; primary cells cannot be expanded in vitro, and only a limited amount of cells (usually ≤6×10 6(cells / sample), multiple patient samples need to be pooled to increase statistical power; primary cells are fragile, and conditions need to be continuously optimized during electroporation experiments to reduce cell damage and improve transfection efficiency, etc.

[0007] In summary, the existing technologies for shRNA targeting PRMT3 to inhibit HIV-1 have the following problems: low efficiency of shRNA plasmid DNA circular delivery, high production cost, and difficulty in drug development; whether targeting PRMT3 is effective against HIV-1 lacks validation using true viral infection models and primary immune cells from patients, and further in-depth research is needed to confirm its clinical translation 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,AmyJustice,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-infectedindividualsAIDS(London,England)2010 Jan 02;24(1):123-37doi:10.1097 / QAD.0b013e3283324283

[0010] [2] Marie Armani-Tourret, Ce Gao, Ciputra Adijaya Hartana, Wei Wei 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 privileged HIV-1 proviruses during treatment with panobinostat and interferon-a2a Cell 2024 Feb 29; 187(5): 1238-1254.e14 doi: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 Perez, 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; RNAitherapeutics: an update on delivery Current opinion in molecular therapeutics 2008 Apr; 10(2): 158-67 SUMMARY

[0013] In view of the problems of the prior art, the present application designs multiple siRNAs that can target PRMT3. The siRNA drug has the advantages of high targeting, specificity, long-acting effect, reduced dosing frequency, and wide target range. The effect of siRNA in knocking down PRMT3 in MT4 cells is evaluated, and siRNAs that can knock down PRMT3 in MT4 cells are screened. Then, the siRNAs are used in HIV-1 virus-infected MT4 cells and primary CD4 + T cells from HIV-infected patients to evaluate and verify the effect of the screened siRNAs in knocking down PRMT3 on HIV-1 virus replication and latent reactivation. Finally, siRNAs that can knock down PRMT3 to inhibit HIV-1 virus replication and latent reactivation are determined. The specific technical solutions of the present application are as follows:

[0014] First aspect, 7 siRNAs possibly targeting PRMT3 are designed

[0015] 7 siRNAs possibly targeting PRMT3 designed by software and 1 random control siRNA (sicon) are used as control.

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

[0017] 7 siRNAs possibly targeting PRMT3 in the first aspect are respectively transiently electroporated into MT4 cells, and after 48 hours, cells are collected and total RNA is extracted using a kit, and after reverse transcription into cDNA, PRMT3 gene is amplified by Realtime q-PCR to detect the effect of the 7 siRNAs on PRMT3 knockdown.

[0018] Conclusion: siRNA-1, siRNA-2, siRNA-3, and siRNA-4 have good effects on PRMT3 knockdown in MT4 cells, so siRNA-1, siRNA-2, siRNA-3, and siRNA-4 are selected for subsequent evaluation and verification.

[0019] Third aspect, different siRNAs knock down PRMT3 in MT4 cells, and the effect of inhibiting HIV-1 replication is evaluated by examining the copy number of HIV-1 RNA

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

[0021] Results:

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

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

[0024] Conclusion:

[0025] The siRNA-1 and siRNA-2 can significantly inhibit the replication of different subtypes of HIV-1 virus (NL4-3 and CRF01-AE) by knocking down PRMT3 in MT4 cells.

[0026] The siRNA-3 cannot inhibit the replication of different subtypes of HIV-1 virus (NL4-3 and CRF01-AE) by knocking down PRMT3 in MT4 cells.

[0027] The fourth aspect is to knock down PRMT3 in MT4 cells by different siRNAs, and to investigate the effect of inhibiting HIV-1 replication by evaluating the content of HIV-1 P24 protein.

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

[0029] Results: Compared with the control group, the siPRMT3-1 group and the siPRMT3-2 group can significantly reduce the expression of HIV-1 P24 protein in MT4 cells, and the P value is less than 0.01.

[0030] Conclusion: The siRNA-1 and siRNA-2 can significantly inhibit the replication of HIV-1 virus by knocking down PRMT3 in MT4 cells.

[0031] The fifth aspect is to knock down PRMT3 in the primary CD4 + T cells of HIV-infected patients by different siRNAs, and to verify whether it effectively inhibits the transcriptional reactivation of HIV.

[0032] The siRNA-1, siRNA-2 and siRNA-4 sequences are used to knock down PRMT3 in the primary CD4 + T cells of HIV-infected patients, and to investigate the copy number of HIV RNA in CD4 + T cells, and to verify the impact on the latent reactivation of HIV virus.

[0033] Results:

[0034] The siRNA-1 and siRNA-2 are used to knock down PRMT3 in the primary CD4 + T cells of HIV-infected patients, and compared with the control group, it can significantly inhibit the copy number of HIV RNA in CD4 + T cells, and the P value is less than 0.01 and 0.05, respectively.

[0035] The siRNA-4 is used to knock down PRMT3 in the primary CD4 + T cells of HIV-infected patients, and compared with the control, it can significantly inhibit the copy number of HIV RNA in CD4 +The copy number of HIV RNA in T cells did not decrease, but even increased.

[0036] Conclusion:

[0037] The sequence of siRNA-1 and siRNA-2 was used to knock down the PRMT3 in the primary CD4 + T cells of HIV infected patients, which can significantly inhibit the transcription reactivation of HIV;

[0038] The sequence of siRNA-4 was used to knock down the PRMT3 in the primary CD4 + T cells of HIV infected patients, which cannot inhibit the transcription reactivation of HIV.

[0039] Compared with the prior art, the beneficial effects of the present application are:

[0040] The present application first found that the sequence of siRNA-1 or siRNA-2 was used to knock down PRMT3, which can significantly inhibit the replication and transcription reactivation of HIV-1 virus; and the sequence of siRNA-3 or siRNA-4 was used to knock down PRMT3, which cannot inhibit the replication and transcription reactivation of HIV-1 virus, which provides a new possibility for the treatment of HIV-1 infection. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 , 7 siRNA knockdown PRMT3 in MT4 cells, the expression level of PRMT3 gene is reduced by multiple, wherein, sicon is random sequence siRNA 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 (the expression amount of pol) and the expression amount of gag in MT4 cells infected with NL4-3 and knocked down PRMT3 gene, wherein,

[0043] siPRMT3-1 is that siRNA-1 is used to knock down PRMT3 in MT4 cells, and then NL4-3 is infected,

[0044] siPRMT3-2 is that siRNA-2 is used to knock down PRMT3 in MT4 cells, and then NL4-3 is infected, siPRMT3-3 is that siRNA-3 is used to knock down PRMT3 in MT4 cells, and then NL4-3 is infected, sicon is that sicon is used to electrotransfer MT4 cells, and then NL4-3 is infected, as a control;

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

[0046] siPRMT3-1 is to knock down PRMT3 in MT4 cells by siRNA-1, then infect CRF01-AE again,

[0047] siPRMT3-2 is to knock down PRMT3 in MT4 cells by siRNA-2, then infect CRF01-AE again, siPRMT3-3 is to knock down PRMT3 in MT4 cells by siRNA-3, then infect CRF01-AE again, sicon is to infect MT4 cells by sicon electroporation, then infect CRF01-AE again, as a control;

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

[0049] siPRMT3-1 is to knock down PRMT3 in MT4 cells by siRNA-1, then infect CRF01-AE again, siPRMT3-2 is to knock down PRMT3 in MT4 cells by siRNA-2, then infect CRF01-AE again, sicon is to infect MT4 cells by sicon electroporation, then infect CRF01-AE again, as a control;

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

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

[0052] Figures 1-6

[0053] *, ** and *** respectively represent: P<0.05, P<0.01, P<0.001, indicating that the difference between the two groups has statistical significance, ns represents no statistical difference between the two groups. DETAILED DESCRIPTION

[0054] ​The following examples are intended to illustrate the present application and are not intended to limit the scope of the present application. Unless otherwise indicated, the techniques utilized in the examples are routine techniques commonly used in the art.

[0055] The experimental methods used in the following examples are routine methods unless otherwise specified.

[0056] All materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0057] Biological materials

[0058]

[0059]

[0060] Reagents

[0061]

[0062]

[0063] Instrument

[0064]

[0065] Example 1, siRNA design

[0066] Seven siRNA sequences possibly targeting PRMT3 and one random control siRNA sequence were designed by software, see Table 1.

[0067] Table 1, siRNA sequences possibly targeting PRMT3 designed by software

[0068]

[0069]

[0070] In Table 1,

[0071] siRNA-1: the first siRNA possibly targeting PRMT3;

[0072] siRNA-2: the second siRNA possibly targeting PRMT3;

[0073] ...

[0074] siRNA-7: the seventh siRNA possibly targeting PRMT3;

[0075] sicon: siRNA of random sequence, as control.

[0076] According to the sequence of siRNA-1, siRNA-2, …, siRNA-7 in Table 1, siRNA was synthesized respectively;

[0077] The freeze-dried powder of siRNA-1, siRNA-2, …, siRNA-7 was fully dissolved with RNase-free water, with a concentration of 20 μM, and was divided into 15 μL / tube and stored at -20°C.

[0078] Example 2, PRMT3 knockdown in MT4 cells using siRNA sequences in Table 1 and effect evaluation

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

[0080] 1. Method

[0081] After the MT4 cells were cultured in a 37°C carbon dioxide incubator for 24 hours, 800,000 cells per well (12-well plate) were counted, and siRNA (control), siRNA-1, siRNA-2, …, siRNA-7 in Table 1 were respectively transiently electroporated into MT4 cells using a Lonza electroporator. After the 1640 medium was cultured in a 37°C carbon dioxide incubator for 48 hours, the cells were collected by centrifugation at 500 x g for 3 minutes.

[0082] The total RNA in the above cell precipitate was extracted using a total RNA rapid extraction kit, and after reverse transcription into cDNA by a reverse transcription kit, the PRMT3 gene was amplified by Realtime q-PCR to detect the effect of the seven siRNAs on knocking down the PRMT3 gene expression in MT4 cells. GAPDH was used as an internal reference gene.

[0083] 2. Results

[0084] As shown in Figure 1 , taking the PRMT3 expression level in the control group (sicon) as the reference, GAPDH was used as the internal reference gene, and the qPCR data were normalized by relative quantification by ΔΔCt method, and the relative expression level of PRMT3 mRNA in the experimental group was calculated.

[0085] The PRMT3 gene expression level in MT4 cells knocked down by siRNA-1, siRNA-2, siRNA-3, and siRNA-4 was reduced by more than 0.5 times compared with the control group (sicon).

[0086] The PRMT3 gene expression level of MT4 cells knocked down by siRNA-5, siRNA-6 and siRNA-7 was less than 0.5 times compared with the control group (sicon).

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

[0088] Example 3, different siRNAs knock down PRMT3 in MT4 cells, and the copy number of HIV-1 RNA is investigated to evaluate the effect of inhibiting HIV-1 replication

[0089] 1. Method

[0090] After the MT4 cells were cultured in a 37℃ carbon dioxide incubator for 24 hours, 800,000 cells per well (twelve-well plate) were counted, and sicon (control), siRNA-1, siRNA-2 and siRNA-3 were respectively instantaneously electroporated into the 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 16 hours, the cells were collected by centrifugation to obtain MT4 cell precipitate;

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

[0093] After the MT4 cell precipitate collected by centrifugation was mixed with 200 μL of diluted virus liquid at a MOI of 0.005, it was infected for 8 hours; after 8 hours, the cells were transferred to a 12-well plate and supplemented with 1.5 mL of liquid, and then cultured for 40 hours;

[0094] After the MT4 cell precipitate infected with the virus was collected by centrifugation, the cells were lysed with cell lysis solution in the virus RNA extraction kit for 10 minutes to inactivate the virus, and then HIV-1 virus RNA was extracted using the virus RNA extraction kit;

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

[0096] 2. Results

[0097] Table 2 shows the copy number of HIV-1 RNA (pol expression level) and gag expression level in NL4-3-infected PRMT3-knockdown MT4 cells.

[0098] Table 2. HIV-1 RNA copy number (pol expression level) and gag expression level in NL4-3-infected PRMT3-knockdown MT4 cells.

[0099]

[0100] In Table 2,

[0101] siPRMT3-1: PRMT3 in MT4 cells was knocked down using siRNA-1 as shown in Table 1, and then NL4-3 cells were infected.

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

[0103] siPRMT3-3: PRMT3 in MT4 cells was knocked down using siRNA-3 as shown in Table 1, and then NL4-3 cells were infected.

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

[0105] Depend on Figure 2 It can be seen that,

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

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

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

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

[0110] Table 3, the copy number of HIV-1 RNA (the expression level of pol) and the expression level of gag in the PRMT3-knocked-down MT4 cells infected with CRF01-AE

[0111]

[0112] In Table 3,

[0113] siPRMT3-1: PRMT3 in MT4 cells was knocked down by siRNA-1 in Table 1, and then the cells were infected with CRF01-AE;

[0114] siPRMT3-2: PRMT3 in MT4 cells was knocked down by siRNA-2 in Table 1, and then the cells were infected with CRF01-AE;

[0115] siPRMT3-3: PRMT3 in MT4 cells was knocked down by siRNA-3 in Table 1, and then the cells were infected with CRF01-AE;

[0116] Control sicon: MT4 cells were electroporated with sicon in Table 1, and then the cells were infected with CRF01-AE.

[0117] It can be seen from Figure 3 that

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

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

[0120] The siPRMT3-3 group has 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 compared with the control group;

[0121] 3、Conclusion

[0122] PRMT3 in MT4 cells was knocked down by siRNA-1 and siRNA-2 respectively, which can significantly inhibit the replication of different subtypes of HIV-1 virus (NL4-3 and CRF01-AE);

[0123] Knocking down PRMT3 in MT4 cells by siRNA-3 could not inhibit the replication of different subtypes of HIV-1 virus (NL4-3 and CRF01-AE).

[0124] Example 4, Knocking down PRMT3 in MT4 cells by different siRNAs, and investigating the effect of HIV-1 P24 protein content on the inhibition of HIV-1 replication

[0125] 1. Method

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

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

[0128] After the cell precipitate collected by centrifugation was mixed with 200 μL of the diluted virus liquid with a MOI of 0.005, it was infected for 8 hours. After 8 hours, the cells were transferred to a 12-well plate and supplemented with 1.5 mL of liquid for further culture for 40 hours;

[0129] The cells were centrifuged, and the cell culture supernatant was retained for detection of the expression of HIV-1 virus P24 protein in the supernatant;

[0130] The expression of p24 in the virus supernatant was detected by a reverse transcription virus titer p24 detection kit (EasyQuarter). The standard curve of HIV-1 p24 was prepared using the standard provided in the kit;

[0131] 25 μL of lysis buffer was added to each well, and then 75 μL of sample or standard was added to the corresponding well. 75 μL of enzyme conjugate was added to the reaction well, shaken for 60 seconds, and then placed at 37°C for 50 minutes. The well was washed with 300 μL of buffer for 5 times. Then, 100 μL of TMB developing solution was added to each well, and incubated at 37°C for 10 minutes. 50 μL of stop solution was added to each well, shaken for 60 seconds, and the OD 450 nm was measured.

[0132] 2. Results

[0133] The expression of HIV-1 P24 protein in the culture supernatant of PRMT3-knocked-down MT4 cells infected by CRF01-AE is shown in Table 4.

[0134] Table 4. HIV-1p24 protein expression levels in the supernatant of MT4 cell culture with CRF01-AE-infected PRMT3 knockdown.

[0135]

[0136] In Table 4,

[0137] siPRMT3-1: PRMT3 in MT4 cells was knocked down using siRNA-1 as shown in Table 1, and then CRF01-AE was infected.

[0138] siPRMT3-2: PRMT3 in MT4 cells was knocked down using siRNA-2 as shown in Table 1, and then CRF01-AE was infected.

[0139] siPRMT3-3: PRMT3 in MT4 cells was knocked down using siRNA-3 as shown in Table 1, and then CRF01-AE was infected.

[0140] Control: MT4 cells were electroporated using the method shown in Table 1 and then infected with CRF01-AE.

[0141] Depend on Figure 4 It can be seen that,

[0142] Compared with the control group, the siPRMT3-1 group significantly reduced the expression level of HIV-1P24 protein in MT4 cells (P < 0.01).

[0143] Compared with the control group, the siPRMT3-2 group significantly reduced the expression level of HIV-1P24 protein in MT4 cells (P < 0.01).

[0144] Compared with the control group, the expression level of HIV-1P24 protein in MT4 cells decreased significantly less in the siPRMT3-3 group than in the siPRMT3-1 and siPRMT3-2 groups.

[0145] 3. Conclusion

[0146] Knocking down PRMT3 in MT4 cells using siRNA-1 and siRNA-2 sequences significantly inhibited HIV-1 viral replication.

[0147] Knocking down PRMT3 in MT4 cells using the siRNA-3 siRNA sequence did not significantly inhibit HIV-1 viral replication.

[0148] Example 5: Different siRNAs knock down primary CD4 in HIV-infected patients + PRMT3 in T cells was used to verify whether it effectively inhibited HIV transcriptional reactivation.

[0149] The siRNA-1, siRNA-2 and siRNA-4 were used to knock down the PRMT3 in the primary CD4 + T cells of the HIV infected patients, and the copy number of HIV RNA in the CD4 + T cells was detected to verify the effect on the reactivation of HIV virus latency.

[0150] 1. Method

[0151] The PBMC cells of the HIV infected patients were recovered, and after the cells were stabilized in the 37℃ carbon dioxide incubator for 3 hours, the CD4 + T cells in the PBMC were sorted by using the CD4 + T cell sorting kit, and after the cell counting, at least 1 million cells were taken from each group, and the cells of each patient were divided into three groups: sicon (control), siPRMT3-1 and siPRMT3-2 groups, and the cells were collected by centrifugation.

[0152] Phorbol ester (PMA) 200nM was prepared, 400μL for each group, and the prepared phorbol ester (PMA) solution was used to resuspend the CD4 + T cell precipitate, and the virus reservoir in the patient cells was activated by stimulation in the 37℃ carbon dioxide incubator for 12 hours.

[0153] After 12 hours, the sicon (control), siRNA-1, siRNA-2 and siRNA-4 were transiently electroporated into the primary CD4 + T cells of the HIV infected patients by using the Lonza electroporator, and the electroporation system was 100μL; after the electroporated primary cells were cultured in the 1640 medium for 40 hours, the cell precipitate was collected by centrifugation; and 300μL Trizol reagent was added to the cell precipitate to protect the RNA, and the HIV virus copy number detection was performed.

[0154] 2. Results

[0155] The siRNA-1 and siRNA-2 were used to knock down the PRMT3 in the primary CD4 + T cells of the HIV infected patients, and the copy number of HIV RNA in the CD4 + T cells was detected to verify the effect on the reactivation of HIV virus latency.

[0156] Table 5, the siRNA-1 and siRNA-2 were used to knock down the PRMT3 in the primary CD4 + T cells of the HIV infected patients, and the copy number of HIV RNA in the CD4 + T cells

[0157]

[0158] In Table 5,

[0159] siPRMT3-1: siRNA-1 in Table 1 was used to knock down PRMT3 in HIV infected patient primary CD4 + T cells;

[0160] siPRMT3-2: siRNA-2 in Table 1 was used to knock down PRMT3 in HIV infected patient primary CD4 + T cells;

[0161] sicon: siRNA of sicon in Table 1 was used to electroporate primary CD4 + T cells as control;

[0162] Undet.: represents less than 20 copies / 1 x 106 6 cells in the sample.

[0163] As Figure 5 can be seen,

[0164] siRNA-1 and siRNA-2 were used to knock down PRMT3 in HIV infected patient primary CD4 + T cells, the copy number of HIV RNA in CD4 + T cells was significantly inhibited, and the P values were less than 0.01 and 0.05, respectively, compared with the control group.

[0165] siRNA-4 was used to knock down PRMT3 in HIV infected patient primary CD4 + T cells; + The copy number of HIV RNA in CD4

[0166] Table 6, siRNA-4 was used to knock down PRMT3 in HIV infected patient primary CD4 + T cells; + The copy number of HIV RNA in CD4

[0167]

[0168] In Table 6,

[0169] siPRMT3-4: siRNA-4 in Table 1 was used to knock down PRMT3 in HIV infected patient primary CD4 + T cells;

[0170] sicon: siRNA of sicon in Table 1 was used to electroporate primary CD4 + T cells as control.

[0171] As Figure 6As shown, using siRNA-4 knockdown HIV infected patients primary CD4 + PRMT3 in CD4 + The copy number of HIV RNA in T cells did not decrease.

[0172] 3、Conclusion

[0173] Using siRNA-1, siRNA-2 knockdown HIV infected patients primary CD4 + PRMT3 in T cells, can significantly inhibit the transcriptional reactivation of HIV;

[0174] Using siRNA-4 knockdown HIV infected patients primary CD4 + PRMT3 in T cells, can not inhibit the transcriptional reactivation of HIV.

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

[0176] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.

Claims

1. knockdown PRMT3 The use of siRNA to reduce the expression level in the preparation of HIV-1 treatment products, characterized in that, The siRNA is siRNA-1, the antisense sequence of which is shown as SEQ ID NO. 1, and the sense sequence is shown as SEQ ID NO.

2.

2. Use according to claim 1, wherein Adopting the siRNA, the expression of MT4 cell in siRNA is knocked down PRMT3 Infecting HIV-1 virus NL4-3 again, compared with the control group, the copy number of HIV-1 RNA in MT4 cell pol The expression of and gag The expression of is reduced.

3. The use according to claim 1, wherein The siRNA is used for knocking down MT4 cells PRMT3 Infection of HIV-1 virus CRF01-AE again, compared with the control group, the expression of HIV-1 P24 protein in MT4 cells is reduced.

4. The use according to claim 1, wherein The siRNA is used to knock down primary CD4 + T cells of HIV infected patients PRMT3 The copy number of HIV RNA in CD4 + T cells is inhibited compared with the control group.

Images