SaRNA for promoting expression of human gene HIF-1alpha and application of saRNA

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to saRNA for promoting expression of a human gene HIF-1alpha and application of the saRNA. The invention provides saRNA capable of promoting the expression of a human gene HIF-1alpha, wherein the saRNA comprises saRNA-1, saRNA-2, saRNA-3 or saRNA-4; the invention also provides nucleotide sequences of the positive-sense strand and the antisense strand of each saRNA. The invention also provides an application of the saRNA in improving the expression quantity of the HIF-1alpha protein and promoting cell proliferation, especially in a low-oxygen environment. The result of the embodiment shows that the saRNA can be effectively transfected into the cell and up-regulate expression of mRNA and protein level of the Jurkat cell, and can obviously enhance the proliferation capacity of the cell under the condition of low oxygen (1% O2).

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a saRNA that promotes the expression of the human gene HIF-1α and its application. Background Art

[0002] Small activating RNA (saRNA) is a small RNA targeting gene promoters, which can promote gene expression at the gene transcription and epigenetic levels. This phenomenon is RNA activation (RNAa). This small double-stranded RNA is 21 nucleosides (nt) in length and contains 2 nucleotides of deoxyribonucleic acid (DNA) protrusion at the 3' end. The mechanism of action of saRNA requires the participation of AGO protein. First, saRNA is loaded onto the AGO2 protein to form a saRNA-AGO complex. Afterwards, the saRNA guide strand and the formed saRNA-AGO complex enter the cell nucleus and interact with the promoter region of the target gene. The AGO protein recruits other proteins such as RNA polymerase II, histone modification factors, etc. to form an RNA-induced transcriptional activation complex (RNA-induced transcriptional activation, RITA), which ultimately triggers increased gene transcription and epigenetic activation.

[0003] The unique immunosuppressive microenvironment of solid tumors leads to poor persistence of cell therapy, impaired cell function, and cell exhaustion, resulting in poor therapeutic efficacy. Hypoxia is a key feature of the tumor microenvironment, which can promote tumor cell invasion and metastasis while inhibiting the function of immune cells.

[0004] Studies have shown that in a hypoxic microenvironment, hypoxia-inducible factor 1 (HIF-1) is a key transcriptional regulator that participates in angiogenesis, erythropoiesis, cell proliferation and survival by regulating protein expression, and plays an important role in a variety of life activities such as vascular remodeling and vasoconstriction control. Hypoxia-inducible factor is a dimeric protein complex composed of an α subunit (HIF-1α, HIF-2α or HIF-3α) and a β subunit (HIF-1β). Among the HIF-α protein family, HIF-1α is the most widely expressed and oxygen-sensitive. In the presence of oxygen, the proline residues of HIF-1α are degraded by the proteasome after being hydroxylated by prolyl hydroxylases (PHDs). Under hypoxic conditions, the activity of oxygen-sensitive prolyl hydroxylases is reduced, and the hydroxylation process of HIF-1α cannot proceed. Due to the inhibition of degradation, HIF-1α accumulates and then transfers from the cytoplasm into the nucleus, forming HIF dimers with HIF-1β. The transcription factor HIF can recognize and bind to hypoxia response elements (HREs), thereby initiating the transcription process of related target genes. These include regulatory genes that affect angiogenesis, lipid metabolism, cell growth, apoptosis, metastasis, etc. There is evidence that HIF-1α participates in the formation of the vascular system through synergistic effects with other angiogenic factors (such as VEGF), and may also contribute to the formation of an immunosuppressive tumor microenvironment, highlighting the complexity of its role in tumor biology.

[0005] Therefore, providing a substance that regulates HIF-1α is a technical problem that urgently needs to be solved in the art. Summary of the invention

[0006] The object of the present invention is to provide a saRNA that promotes the expression of the human gene HIF-1α. The saRNA provided by the present invention can regulate the expression level of HIF-1α and can also be applied to the field of solid tumor treatment through gene-assisted therapy.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a saRNA for promoting the expression of human gene HIF-1α, wherein the saRNA comprises a sense chain and an antisense chain; the saRNA is saRNA-1, saRNA-2, saRNA-3 or saRNA-4; the sense chains of saRNA-1, saRNA-2, saRNA-3 and saRNA-4 are as shown in SEQ ID NOs: 1, 3, 5 and 7; 80% of the sequences in the antisense chain are complementary to the sense sequence; and the sense sequence and the antisense sequence have a vertical tail at the 3' end for enhancing stability.

[0009] Preferably, the vertical tail is 2 deoxythymidine nucleotides, which are represented by dTdT in the specification.

[0010] Preferably, the antisense strands of saRNA-1, saRNA-2, saRNA-3, and saRNA-4 are as shown in SEQ ID NOs: 2, 4, 6, and 8;

[0011] The present invention also provides the use of the above-mentioned saRNA in transfecting tumor cells.

[0012] The present invention also provides the use of the above-mentioned saRNA in increasing the expression of HIF-1α protein.

[0013] The present invention also provides the use of the above-mentioned saRNA-2 and saRNA-3 in promoting cell proliferation.

[0014] Preferably, the environment used to increase the expression of HIF-1α protein and promote cell proliferation is a hypoxic environment.

[0015] The present invention also provides an anti-tumor product, which comprises the above-mentioned saRNA.

[0016] The present invention also provides a method for screening saRNA that promotes HIF-1α gene expression, comprising the following steps:

[0017] (1) Using the database to query the coding sequence (CDS) of the human HIF-1α gene and obtain the promoter sequence information of the HIF-1α gene;

[0018] (2) analyzing the gene sequences that need to be removed in the promoter region, wherein the gene sequences that need to be removed include CG content-rich regions and low-complexity sequences, wherein the low-complexity sequences include CpG islands and Alu repeat elements;

[0019] (3) According to the prior art saRNA design method, the TATA box, the start site of gene transcription, was determined and a saRNA sequence with a length of 19-21 bp was designed upstream of the transcription start site (TSS);

[0020] (4) performing Blast homology analysis on the saRNA obtained in step (3) to confirm that it has no homology with known gene sequences other than the human HIF-1α gene;

[0021] (5) Add two dTdT tails to the 3' end of the sequence.

[0022] Preferably, the database in step (1) is the Ensemble website.

[0023] Beneficial effects:

[0024] The saRNA provided by the present invention can promote the expression of human gene HIF-1α and improve the expression level of HIF-1α protein. The saRNA specifically targets the promoter region of gene HIF-1α, effectively improving the activation efficiency and expression level of HIF-1α.

[0025] The saRNA provided by the present invention can effectively enhance cell proliferation ability under different oxygen concentration conditions, especially under hypoxic (1% O2) culture conditions, laying a foundation for transforming new immune cells for use in solid tumor treatment. The saRNA can show great potential and application prospects in the field of solid tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in 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 paying creative labor.

[0027] Figure 1 This is the cell fluorescence intensity result after the saRNA sequence was transfected into Jurkat cells with Cy3 fluorescence labeling, indicating that saRNA was successfully transfected into Jurkat cells.

[0028] Figure 2 The results of RT-qPCR experiments after saRNA sequences were transfected into Jurkat cells, indicating that saRNA sequences can promote the expression of HIF-1α mRNA.

[0029] Figure 3 The results of WB experiments after saRNA sequences were transfected into Jurkat cells, indicating that saRNA sequences can promote the expression of HIF-1α protein.

[0030] Figure 4 This is a diagram of a cell proliferation experiment, which shows that the screened saRNA sequence can effectively promote cell proliferation. DETAILED DESCRIPTION

[0031] The present invention provides a saRNA that promotes the expression of the human gene HIF-1α; by designing a saRNA targeting the promoter sequence of the HIF-1α gene, the natural processing process of endogenous double-stranded small RNA is imitated to specifically promote the transcription and expression of the HIF-1α gene.

[0032] The present invention provides saRNAs that promote the expression of the human gene HIF-1α: saRNA-1, saRNA-2, saRNA-3, and saRNA-4.

[0033] The saRNA-1 sequence is:

[0034] Sense strand: 5′-AGAUAUUGUUCUAAGUAUA-3′ (SEQ ID NO: 1)

[0035] Antisense strand: 5′-UAUACUUAGAACAAUAUCU-3′ (SEQ ID NO: 2);

[0036] The saRNA-2 sequence is:

[0037] Sense strand: 5′-GGUUUAUAUGCUUAUUAAA-3′ (SEQ ID NO: 3)

[0038] Antisense strand: 5′-UUUAAUAAGCAUAUAAACC-3′ (SEQ ID NO: 4);

[0039] The saRNA-3 sequence is:

[0040] Sense strand: 5′-CGCUAAGGUCAAUGUUUAA-3′ (SEQ ID NO: 5)

[0041] Antisense strand: 5′-UUAAACAUUGACCUUAGCG-3′ (SEQ ID NO: 6);

[0042] The saRNA-4 sequence is:

[0043] Sense strand: 5′-AGAUAUAUGUGCAAUGCUA-3′ (SEQ ID NO: 7)

[0044] Antisense strand: 5′-UAGCAUUGCACAUAUAUCU-3′ (SEQ ID NO: 8).

[0045] The 3' end of the above sequences SEQ ID NO: 1-8 also has dTdT as a tail.

[0046] The present invention does not limit the vertical tail, as long as it plays the role of enhancing stability.

[0047] According to the ST.26 standard, U is represented by T in the sequence listing file.

[0048] The present invention provides a method for screening saRNA that promotes efficient expression of HIF-1α gene, comprising the following steps:

[0049] (1) The coding sequence (CDS) of the human HIF-1α gene was queried using the Ensemble website, and the promoter sequence information of the HIF-1α gene was obtained.

[0050] (2) Analyze the gene sequences that need to be removed in the promoter region: including CG content-rich regions and low-complexity sequences such as CpG islands, Alu repeat elements, etc.

[0051] (3) According to the prior art saRNA design method, the TATA box, the start site of gene transcription, was determined and a saRNA sequence with a length of 19-21 bp was designed upstream of the transcription start site (TSS).

[0052] (4) Perform Blast homology analysis to confirm that there is no homology with known gene sequences other than saRNA.

[0053] (5) Two dTdT tails were added to the 3' end of the sequence to enhance the stability of the saRNA sequence.

[0054] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0055] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0056] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0057] Example 1 Design and synthesis of HIF-1α gene saRNA

[0058] The coding sequence (CDS) of the human HIF-1α gene was queried using the Ensemble website, and the promoter sequence information of the HIF-1α gene was obtained; the gene sequences that needed to be removed in the promoter region were analyzed: including CG content-rich regions and low-complexity sequences such as CpG islands, Alu repeat elements, etc.; according to the prior art saRNA design method, the TATA box at the gene transcription start site was determined and a saRNA sequence with a length of 19-21bp was designed upstream of the transcription start site (TSS); Blast homology analysis was performed to confirm that saRNA had no homology with known gene sequences other than sRNA; and two dTdT tails were added to the 3' end of the sequence to enhance the stability of the saRNA sequence. In order to observe the transfection efficiency of the cells, the saRNA sequence was fluorescently labeled with Cy3. Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize the saRNA sequence and negative control sequence. The saRNA sequence was designed as follows:

[0059] Table 1. saRNA sequences

[0060]

[0061] Example 2 Screening of saRNA targeting human HIF-1α gene

[0062] Jurkat cells, a human acute T-cell leukemia cell line, were used as a cell tool for saRNA screening. First, the cell morphology and fluorescence intensity were observed under an inverted fluorescence microscope, and the observations were recorded and photographed. The expression of HIF-1α mRNA and protein in the cells was detected by RT-qPCR and Western blotting experiments.

[0063] Cell transfection and fluorescence imaging

[0064] (1) Jurkat cells, a human acute T-cell leukemia cell line, were cultured in RPMI 1640 medium containing 10% FBS at 37°C and 5% CO2. When the cell confluence reached 80%-90%, the cells were passaged. After the second passage, the cells were cultured at 10 6 The number of cells / well was inoculated into a 6-well cell culture plate and cultured for 24 h for cell transfection;

[0065] (2) Add 12.5 μL (0.25 nmol) of the human saRNA working solution in Example 1 to a centrifuge tube and dilute to 50 μL with OPTI-MEM reduced serum medium; add 5 μL Entranster TM-R4000 transfection reagent to another centrifuge tube and dilute to 50 μL with OPTI-MEM reduced serum medium; after standing at room temperature for 5 minutes, mix the solutions in the two centrifuge tubes thoroughly, and after standing at room temperature for 15 minutes, the transfection complex is configured;

[0066] (3) The blank wells were not treated, and 2500 μL RPMI 1640 complete medium (containing 10% FBS) was added; 2400 μL RPMI 1640 complete medium (containing 10% FBS) and 100 μL transfection complex were added to the experimental wells. The transfection concentration of saRNA was 100 nmol / L; 6 hours after transfection, the cell status was observed and the medium was changed as appropriate; if the cell status was good, the culture was continued for 24 hours;

[0067] (4) After 24 hours, the cell culture plate was removed and the cell morphology and fluorescence expression intensity were observed under an inverted fluorescence microscope. Figure 1 As shown, saRNA was successfully transfected into Jurkat cells.

[0068] RT-qPCR steps

[0069] (1) Cells were collected, lysed with Trizol, RNA was extracted with isopropanol, and then precipitated with ethanol; according to TaRaKaPrimeScript TM II 1st Strand cDNA Synthesis Kit Instructions: Reverse transcribe the obtained total RNA into cDNA; Table 2 shows the cDNA synthesis system and PCR instrument expansion program.

[0070] (2) Fluorescence quantitative detection was performed according to the instructions of the QuantiNovaSYBR Green PCR Kit. The primer sequences are shown in Table 3, and the reaction system and PCR amplification program are shown in Table 4. Three replicate wells were set for each sample, and GAPDH was used as the internal reference.

[0071] Table 2. cDNA synthesis system and procedures

[0072]

[0073] Table 3. RT-qPCR primer sequences

[0074]

[0075]

[0076] Table 4. Reverse transcription system and procedures

[0077]

[0078] Total protein extraction steps:

[0079] (1) preparing total protein extraction solution, including RIPA lysis buffer, PMSF, and protease inhibitors;

[0080] (2) Collect the cells by centrifugation at 1000 rpm / min for 10 min, and flick the cells with your fingers. Add 200 μL of total protein extract to each well of cells. Flick the cells with your fingers to fully lyse them and transfer them to a 1.5 mL centrifuge tube;

[0081] (3) Place on ice for 30 min, invert twice to mix; centrifuge at 12000 rpm / min for 20 min, carefully aspirate the supernatant (to avoid aspirating the precipitate) and transfer to a new 1.5 mL EP tube;

[0082] (4) The total protein concentration in the supernatant was determined by the BCA method.

[0083] The Western blotting experimental steps are as follows:

[0084] (1) Prepare 8% SDS-PAGE separation gel;

[0085] (2) add an appropriate amount of RIPA cell lysis buffer to adjust the total protein concentration and 5× protein loading buffer so that the total protein concentration of different samples is close to the same;

[0086] (3) Place the protein solution in boiling water at 100°C and boil for 10 minutes to denature the protein;

[0087] (4) Electrophoresis: 20 μg of total protein from each sample was added to the SDS-PAGE gel well. The voltage was kept constant at 80V for about 30 min. After the protein entered the separation gel, the voltage was adjusted to 120V. The electrophoresis was terminated when the protein sample reached the bottom of the glass plate.

[0088] (5) Transfer: Take a PVDF membrane of appropriate size and soak it in methanol for about 20 min. Assemble the gel, PVDF membrane, filter paper, and sponge into a sandwich structure and transfer it at a constant current of 300 mA for 2 h in an ice water bath.

[0089] (6) Blocking: After electroporation, soak the PVDF membrane in 5% skimmed milk powder at 4°C and block on a shaker for 2 h at room temperature;

[0090] (7) Incubation with primary antibody: After blocking, wash the membrane three times with TBST solution, 5 min each time, and then incubate with HIF-1α primary antibody (1:1000) at 4°C overnight; for the internal reference, incubate with GAPDH primary antibody (1:1000) at 4°C overnight;

[0091] (8) Incubation with secondary antibody: Take the membrane incubated with primary antibody overnight from the 4°C refrigerator, wash the membrane three times with TBST, 5 min each time; add secondary antibody (1:5000) and incubate on a shaker at room temperature for 2 h;

[0092] (9) Development: After the incubation with the secondary antibody is completed, wash the membrane three times with TBST, each time for 5 min; place the membrane in the prepared ECL chemiluminescent developer for 1-2 min, place the membrane in a gel imaging system for chemiluminescence, and adjust the exposure time according to the brightness of the image.

[0093] (10) Use ImagJ software to perform density analysis on the WB bands obtained by exposure.

[0094] All experiments were performed in triplicate, and the results are expressed as the mean ± SD of three independent experiments, with *p < 0.05; **p < 0.01; ***p < 0.001.

[0095] RT-qPCR experimental results are as follows Figure 2 As shown, it can be seen that compared with the blank control Blank group, the expression level of HIF-1α mRNA in saRNA-2 group (P<0.001), saRNA-3 group (P<0.01) and saRNA-4 group (P<0.01) was significantly increased.

[0096] Western blotting results Figure 3 As shown, it can be seen that compared with the blank control Blank group, the expression of HIF-1α protein in the saRNA-2 group (P<0.01) and the saRNA-3 group (P<0.05) was significantly upregulated, so saRNA-2 and saRNA-3 were selected for subsequent experiments.

[0097] Example 3 Evaluation of the effect of saRNA transfection on cell proliferation

[0098] Jurkat cells, a human acute T-cell leukemia cell line, were used as tool cells, and the CCK-8 assay was used to detect cell proliferation under culture conditions of different oxygen concentrations.

[0099] CCK-8 Assay Procedure

[0100] (1) Cultivating cells using the method of Example 2, the transfection method is the same as the transfection method of Example 2, and only the culture time is different. Add 100 μL of transfection complex to the cells, mix well, and continue to culture in a 37° C., 5% CO2 cell culture incubator for 24 hours;

[0101] (2) Adjust the cell number to 4×10 4 / mL, and then inoculated into 96-well cell culture plates at a volume of 100uL / well, with 6 replicates per group. Incubate in a 37°C, 5% CO2, 21% O2 cell culture incubator and a 37°C, 5% CO2, 1% O2 cell culture incubator, respectively. CCK-8 kit was used to detect cell viability at 0, 12, 24, 48, 72, and 96h;

[0102] (3) When the detection time point is reached, the cell plate is removed and 10 μL of CCK-8 detection reagent is added to each well. After incubation in a cell culture incubator at 37°C and 5% CO2 for 1 h, the absorbance (OD) at 450 nm is measured using a continuous wavelength multifunctional microplate reader. The blank culture medium reading is subtracted from the measured value as a background signal and normalized with the absorbance value of NC cells on the first day to calculate the growth changes of Jurkat cells.

[0103] The experiment was repeated three times, and the results are expressed as the mean ± SD of three independent experiments, with *p < 0.05; **p < 0.01; ***p < 0.001.

[0104] The results are as follows Figure 4 As shown: After transfection with saRNA, the proliferation ability of Jurkat cells transfected with saRNA sequences was significantly enhanced compared with the blank control Blank group, regardless of whether it was cultured under normoxic (21% O2) or hypoxic (1% O2) conditions, indicating that saRNA transfection can upregulate HIF-1α gene expression and promote Jurkat cell proliferation; and under hypoxic (1% O2) culture conditions, compared with the blank control Blank group, the ability of saRNA-2 group and saRNA-3 group to promote cell proliferation was more obvious.

[0105] In summary, the saRNA designed and synthesized by the present invention to promote the expression of the gene HIF-1α can be effectively transfected into cells and upregulate the expression of Jurkat cell mRNA and protein levels. The cell proliferation ability can be significantly enhanced under hypoxic conditions (1% O2). Therefore, the present invention designs and screens out gene-targeted saRNA-2 and saRNA-3 sequences that can promote the expression of the gene HIF-1α, and can effectively enhance cell proliferation in a hypoxic environment. As an emerging gene-assisted therapy, saRNA shows great potential and application prospects in the field of solid tumor treatment.

[0106] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A saRNA that promotes the expression of human gene HIF-1α, characterized in that The saRNA includes a sense strand and an antisense strand; the saRNA is saRNA-1, saRNA-2, saRNA-3 or saRNA-4; the sense strands of the saRNA-1, saRNA-2, saRNA-3 and saRNA-4 are shown in SEQ ID NOs: 1, 3, 5 and 7; 80% of the sequences in the antisense strand are complementary to the sense sequence; the sense sequence and the antisense sequence have a vertical tail at the 3' end for enhancing stability.

2. The saRNA according to claim 1, characterized in that The vertical tail is 2 deoxythymidine nucleotides.

3. The saRNA according to claim 1, characterized in that The antisense strands of the saRNA-1, saRNA-2, saRNA-3, and saRNA-4 are shown in SEQ ID NOs: 2, 4, 6, and 8.

4. Use of the saRNA according to any one of claims 1 to 3 in transfecting tumor cells.

5. Use of the saRNA according to any one of claims 1 to 3 in increasing the expression of HIF-1α protein.

6. Use of saRNA-2 and saRNA-3 according to any one of claims 1 to 3 in promoting cell proliferation.

7. The use according to claim 6, characterized in that: The environment used to promote cell proliferation is a hypoxic environment.

8. An anti-tumor product, characterized in that: The product comprises the saRNA according to any one of claims 1 to 3.

9. A method for screening saRNA that promotes HIF-1α gene expression, characterized in that: The following steps are involved: (1) Using the database to query the coding sequence (CDS) of the human HIF-1α gene and obtain the promoter sequence information of the HIF-1α gene; (2) analyzing the gene sequences that need to be removed in the promoter region, wherein the gene sequences that need to be removed include CG content-rich regions and low-complexity sequences, wherein the low-complexity sequences include CpG islands and Alu repeat elements; (3) According to the prior art saRNA design method, the TATA box, the start site of gene transcription, was determined and a saRNA sequence with a length of 19-21 bp was designed upstream of the transcription start site (TSS); (4) performing Blast homology analysis on the saRNA obtained in step (3) to confirm that it has no homology with known gene sequences other than the human HIF-1α gene; (5) Add two dTdT tails to the 3' end of the sequence.

10. The method according to claim 9, characterized in that The database in step (1) is the Ensemble website.

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