siRNA and liposome for inhibiting human USP46 gene expression and their application

Abstract

The present invention provides siRNA and liposomes for inhibiting human USP46 gene expression, and their applications. The synthesized siRNA of the present invention has relatively high stability, effectively inhibits USP46 gene expression, and specifically acts on the USP46 gene. The synthesized siRNA is delivered into cells via a nanoliposome encapsulation delivery system, where it binds to the USP46 gene mRNA sequence in the cytoplasm through complementary base pairing, inhibiting USP46 protein expression and, in turn, cell growth. This siRNA can be encapsulated and delivered into breast cancer cells via nanoliposomes, inhibiting their growth and therefore being applicable to the treatment of breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to siRNA and liposome for inhibiting human USP46 gene expression and applications thereof. Background Art

[0002] Small interfering RNA (siRNA), sometimes called short interfering RNA or silencing RNA, is a double-stranded RNA of 20 to 25 nucleotides in length that has many diverse biological applications. siRNA is primarily known to participate in the phenomenon of RNA interference (RNAi), regulating gene expression in a specific manner. It also participates in several RNAi-related pathways, such as antiviral mechanisms and alterations in chromatin structure. However, the pathways underlying these complex mechanisms remain unclear.

[0003] Breast cancer is the uncontrolled proliferation of mammary epithelial cells in response to multiple carcinogens. Early symptoms include breast lumps, nipple discharge, and enlarged axillary lymph nodes. In advanced stages, the disease can metastasize to distant organs, leading to multi-organ lesions and posing a direct threat to the patient's life. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide an siRNA for inhibiting the expression of the human USP46 gene.

[0005] Another object of the present invention is to provide a liposome for inhibiting the expression of human USP46 gene.

[0006] Another object of the present invention is to provide applications of the above-mentioned siRNA and liposome.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] An siRNA for inhibiting human USP46 gene expression, comprising at least one of the following:

[0009] siUSP46#1, the positive strand nucleotide sequence is:

[0010] 5'-CGCUUACCAAUGAAACUCGAUdTdT-3',

[0011] The nucleotide sequence of the antisense strand is:

[0012] 5'-AUCGAGUUUCAUUGGUAAGCGdTdT-3';

[0013] siUSP46#2, the positive strand nucleotide sequence is:

[0014] 5'-AUGGCCUGACGUCAGAUAUAUdTdT-3',

[0015] The nucleotide sequence of the antisense strand is:

[0016] 5'-AUAUAUCUGACGUCAGGCCAUdTdT-3';

[0017] siUSP46#2-Ome, the positive chain nucleotide sequence is:

[0018] 5'-AUGGCCUGACGUCAGAUAUAUdTdT-3', the nucleotides from position 17 to position 20 are 2' methoxy-modified nucleotides,

[0019] The nucleotide sequence of the antisense strand is:

[0020] 5'-AUAUAUCUGACGUCAGGCCAUdTdT-3';

[0021] Or siUSP46#1, siUSP46#2, siUSP46#2-Ome are analogs obtained by base insertion, deletion, or substitution and still have the function of inhibiting human USP46 gene expression.

[0022] The siRNA for inhibiting human USP46 gene expression can promote the siRNA to enter cells through cholesterol modification.

[0023] The siRNA for inhibiting human USP46 gene expression is added with a cy3 fluorescent group so that after the siRNA enters the cell, it not only inhibits the expression function of human USP46 gene but also emits fluorescence to indicate the position of the siRNA in the cell.

[0024] The dT in the nucleotide sequence of the siRNA for inhibiting human USP46 gene expression is deoxythymidine.

[0025] A liposome for inhibiting the expression of human USP46 gene is obtained by encapsulating at least one siRNA for inhibiting the expression of human USP46 gene by nanoliposomes.

[0026] The method for preparing the liposome comprises the following steps:

[0027] (1) Dissolve DLin-MC3-DMA, DMG-PEG2000, DSPC, and cholesterol in anhydrous ethanol and mix to obtain solution A for later use;

[0028] (2) dissolving the siRNA to be encapsulated in citric acid-sodium citrate buffer (pH = 4.0, 100 mM) to obtain solution B;

[0029] (3) Solution A and solution B are mixed to obtain a liposome that inhibits the expression of the human USP46 gene.

[0030] The application of the siRNA or liposome for inhibiting human USP46 gene expression in regulating human USP46 gene expression.

[0031] The application of the siRNA or liposome for inhibiting human USP46 gene expression in the preparation of USP46 protein expression inhibitors.

[0032] The application of the siRNA or liposome for inhibiting human USP46 gene expression in the preparation of anti-tumor drugs.

[0033] The tumor is breast cancer.

[0034] The present invention has the following advantages and effects compared to the prior art:

[0035] (1) The present invention synthesizes an siRNA sequence that inhibits human USP46 gene expression. The synthesized siRNA is delivered into cells via a nanoliposome encapsulation delivery system, where it binds to the mRNA sequence of the USP46 gene in the cytoplasm through base complementary pairing, inhibiting the expression of the USP46 protein and, in turn, cell growth. This siRNA can be encapsulated and delivered into breast cancer cells by nanoliposomes, inhibiting their growth and having potential application in the treatment of breast cancer.

[0036] (2) The siRNA synthesized by the present invention has relatively high stability, can effectively inhibit the expression of the USP46 gene, can specifically act on the USP46 gene, can be encapsulated by nanoliposomes and delivered into cells, and effectively inhibits the growth of breast cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a graph showing the qPCR detection results after siUSP46#1 and siUSP46#2 were transfected into T47D cells in Example 2.

[0038] Figure 2 This is a graph showing the qPCR detection results after T47D cells were transfected with siUSP46#2-LNP and siNC-LNP in Example 3.

[0039] Figure 3 This is a graph showing the results of agarose gel electrophoresis after nanoliposome encapsulation in Example 4.

[0040] Figure 4This is a graph showing the qPCR detection results after siUSP46-LNP and siNC-LNP transfection into T47D and MDA-MB-231 cells in Example 4.

[0041] Figure 5 This is a graph showing the detection results of Western Blot experiments after siUSP46-LNP and siNC-LNP were transfected into T47D and MDA-MB-231 cells in Example 4.

[0042] Figure 6 This is a diagram showing the results of the clone formation experiment in Example 4. DETAILED DESCRIPTION

[0043] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0044] If specific experimental conditions are not specified in the following embodiments, conventional experimental conditions or those recommended by the reagent company will generally be used. Materials and reagents used were commercially available unless otherwise specified.

[0045] Example 1

[0046] The human USP46 gene, whose full name is ubiquitin specific peptidase 46, encodes a protein that deubiquitinates cellular proteins and is a deubiquitinating enzyme. The gene ID number included in the NCBI (National Center for Biotechnology Information) is 64854. The gene is approximately 68,341 base pairs long and has 11 exons. Currently, there are six mRNA transcript variants, including four confirmed mRNA sequences and two mRNA sequences in the prediction stage. To achieve inhibition of the USP46 gene, the present invention designed two sets of siRNAs to verify their inhibitory effects:

[0047] siUSP46#1:

[0048] sense strand (5′-CGCUUACCAAUGAAACUCGAUdTdT-3′);

[0049] antisense strand (5′-AUCGAGUUUCAUUGGUAAGCGdTdT-3′);

[0050] siUSP46#2:

[0051] sense strand (5′-AUGGCCUGACGUCAGAUAUAUdTdT-3′);

[0052] antisense strand (5′-AUAUAUCUGACGUCAGGCCAUdTdT-3′);

[0053] A random sequence siNC was designed for control, and its nucleotide sequence is:

[0054] Sense strand (5'-UUCUCCGAACGUGUCACGUdTdT-3')

[0055] Antisense strand (5'-ACGUGACACGUUCGGAGAAdTdT-3')

[0056] The above sequence was synthesized by Guangzhou Aiji Biotechnology Co., Ltd.

[0057] Example 2

[0058] 1. Cell culture

[0059] The experiment involved two breast cancer cells, T47D and MDA-MB-231 cells. T47D cells were cultured in 1640 medium containing 10% fetal bovine serum, and MDA-MB-231 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2.

[0060] 2. Transient transfection assay to detect USP46 mRNA expression level

[0061] (1) Inoculate T47D cells in a six-well plate at a density of 300,000 cells / well and culture until adherent;

[0062] (2) siUSP46#1 and siUSP46#2 prepared in Example 1 and Lipo2000 (Thermo) were added separately, with the nucleic acid concentration being 50 nM. Each group was repeated for 3 wells and the cells were cultured for 48 h for transfection.

[0063] 3. qPCR assay to detect USP46 mRNA expression level

[0064] (1) Collect cells, extract total RNA from cells, and reverse transcribe total RNA into cDNA using a Novozymes reverse transcription kit (Cat. No. R323-01);

[0065] (2) The mRNA expression level of USP46 in cells was detected using the qPCR kit (Cat. No.: Q711-02) of Novozymes. The primers used were:

[0066] USP46 amplification upstream primer: 5'-AGAAGAAGGTTGGCGTCATCC-3',

[0067] USP46 amplification downstream primer: 5′-TGTCCGCAATAGTGTTTAGCAAA-3′;

[0068] Upstream primer for internal control β-actin amplification: 5′-GAGCTACGAGCTGCCTGACG-3′;

[0069] Downstream primer for internal control β-actin amplification: 5′-GTAGTTTCGTGGATGCCACAG-3′;

[0070] The experimental results are as follows Figure 1 As shown, the data uses 2^ -ΔΔCt The experimental results showed that after siUSP46#2 transfection, the mRNA expression level of the USP46 gene in T47D cells was low, so this siRNA sequence was selected for subsequent experiments.

[0071] Example 3

[0072] 1. Nanoliposome encapsulation

[0073] (1) Prepare the various lipid materials required for the siUSP46-LNP alcohol phase at a molar mass ratio of DLin-MC3-DMA:DMG-PEG2000:DSPC:cholesterol for injection = 50:1.5:10:38.5, and mix them to obtain solution A for later use;

[0074] (2) Preparation of nanoliposome aqueous solution: 22 nmol of siUSP46#2 or siNC nucleotides to be encapsulated were dissolved in 900 μl of citric acid-sodium citrate buffer (pH = 4.0, 100 mM) to obtain solution B;

[0075] (3) Using a nanolipid particle packaging device (Chengqi Bio), solution A and solution B were mixed at a volume ratio of 1:3 by microfluidics to obtain nanoliposome-encapsulated siUSP46#2 (hereinafter referred to as siUSP46#2-LNP) and nanoliposome-encapsulated siNC (hereinafter referred to as siNC-LNP), respectively;

[0076] (4) siUSP46#2-LNP and siNC-LNP were concentrated and replaced with 10 kD ultrafiltration tubes and RNase-free PBS, respectively, by centrifugation at 4000 g for 90 min at 4°C, and then stored in a refrigerator at 4°C until use;

[0077] 2. Nanoliposome transient transfection experiment

[0078] Referring to step 2 in Example 2, the siUSP46#2-LNP and siNC-LNP prepared above were transfected into T47D cells.

[0079] 3. qPCR assay to detect USP46 mRNA expression level

[0080] After 48 h of culture, the expression level of mRNA in T47D cells transfected with siUSP46#2-LNP and siNC-LNP was detected with reference to step 3 of Example 2. The experimental results are as follows: Figure 2 As shown in the figure, after LNP delivered siUSP46#2 into T47D cells, the mRNA expression level of the USP46 gene did not change significantly. This may be because after LNP encapsulation, the properties of siUSP46#2-LNP became unstable and the expected technical effect could not be achieved.

[0081] Example 4

[0082] 1. Methylation of siRNA

[0083] In Example 3, siUSP46#2 did not achieve the expected effect after being encapsulated and delivered into cells via LNP. Therefore, siUSP46#2 was methoxy-modified to obtain siUSP46#2-Ome:

[0084] Sense strand (5'-ATGGCCTGACGTCAGA TATA TdTdT-3');

[0085] antisense strand (5′-ATATATCTGACGTCAGGCCATdTdT-3′);

[0086] The underlined portion is the 2'-methoxy-modified base sequence, which was synthesized by Guangzhou Aiji Biotechnology Co., Ltd.

[0087] 2. Nanoliposome encapsulation and detection

[0088] (1) Referring to step 1 in Example 3, siUSP46#2-Ome-LNP (referred to as siUSP46-LNP) was prepared and used together with the previously prepared siNC-LNP for subsequent experiments;

[0089] (2) Take a small amount of siUSP46-LNP and siNC-LNP encapsulated in step (1) and break the seal with TritonX-100. TM The siRNA concentrations of the encapsulated and unencapsulated samples were detected using the RNA (BR) Quantification Kit (Cat. No. Q10211A), and the encapsulation efficiency was calculated (formula: encapsulation efficiency = (unencapsulated sample siRNA concentration - encapsulated sample siRNA concentration) / unencapsulated sample siRNA concentration * 100%).

[0090] (3) The encapsulated and unencapsulated samples were detected by 2% agarose gel electrophoresis. The results are shown in Table 1. Both siUSP46-LNP and siNC-LNP have good encapsulation efficiency, both exceeding 90%. Figure 3 As shown, when the encapsulated sample is not broken, there is no band in the electrophoresis lane corresponding to the electrophoresis position of pure siRNA, while the broken sample shows the presence of electrophoresis bands. Combined with Table 1, the results show that siRNA can be well encapsulated using nanoliposomes.

[0091] Table 1 Calculation of sample encapsulation efficiency

[0092]

[0093] 3. Nanoliposome transient transfection experiment

[0094] Referring to step 2 in Example 2, siUSP46-LNP and siNC-LNP were transfected into T47D cells; in addition, referring to Example 2, MDA-MB-231 cells were cultured and siUSP46-LNP and siNC-LNP were transfected into MDA-MB-231 cells.

[0095] 4. qPCR assay to detect USP46 mRNA expression level

[0096] After culturing for 48 h, the expression levels of mRNA in T47D cells transfected with siUSP46-LNP and siNC-LNP were detected with reference to step 3 in Example 2. The results are as follows: Figure 4 As shown, after T47D and MDA-MB-231 cells were treated with siUSP46-LNP for 48 h, the expression of USP46 mRNA was downregulated, while siNC-LNP had no significant change.

[0097] 5. Western Blot assay to detect USP46 protein expression level

[0098] After 72 h of culture, cells were collected and total protein was extracted. Proteins were electrophoresed on gradient SDS-PAGE gels and transferred to PVDF membranes by wet transfer. The membranes were blocked with 5% milk powder and incubated with USP46 antibody (Proteintech, Catalog No.: 13502-1-AP) or β-actin (Proteintech, Catalog No.: 66009-1-Ig) antibody overnight. The membranes were then incubated with secondary antibodies for 2 h before development (n = 3 replicates). Grayscale values ​​were analyzed using Image J software, and the data were statistically analyzed using the Student's paired t-test. P < 0.05: *, P < 0.01: **, P < 0.001: ***, P < 0.0001: ****

[0099] The experimental results are as follows Figure 5 As shown, after T47D and MDA-MB-231 cells were treated with siUSP46-LNP for 72 h, the expression of USP46 protein was downregulated.

[0100] 6. Clone formation assay to detect cell growth

[0101] (1) T47D and MDA-MB-231 cells were seeded in six-well plates at 500 cells / well and siUSP46-LNP or siNC-LNP was added to the cells at a concentration of 50 nM (the concentration was calculated based on the molar amount of siRNA encapsulated in the nanoliposomes);

[0102] (2) On the 7th day of culture, 2 ml of culture medium was added per well;

[0103] (3) On the 14th day of culture, the cells were fixed with anhydrous methanol and then stained with 0.2% crystal violet for observation.

[0104] The experimental results are as follows Figure 6 As shown, there are only sporadic cell growth in the cell wells treated with siUSP46-LNP, and the number is very small, while there are more cell growth in the cell wells treated with siNC-LNP. It can be seen that siUSP46-LNP can significantly inhibit the growth of breast cancer cells.

[0105] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An siRNA for inhibiting human USP46 gene expression, characterized in that The positive chain nucleotide sequence is: siUSP46#2-Ome. 5'-AUGGCCUGACGUCAGAUAUAUdTdT-3', the nucleotides from position 17 to position 20 are 2' methoxy-modified nucleotides, The nucleotide sequence of the antisense strand is: 5'-AUAUAUCUGACGUCAGGCCAUdTdT-3'.

2. The siRNA for inhibiting human USP46 gene expression according to claim 1, characterized in that: The siRNA for inhibiting human USP46 gene expression is added with a cy3 fluorescent group so that after the siRNA enters the cell, it not only inhibits the expression function of human USP46 gene but also emits fluorescence to indicate the position of the siRNA in the cell.

3. The siRNA for inhibiting human USP46 gene expression according to claim 1, characterized in that: The dT in the nucleotide sequence of the siRNA for inhibiting human USP46 gene expression is deoxythymidine.

4. A liposome for inhibiting human USP46 gene expression, characterized in that: The siRNA for inhibiting human USP46 gene expression according to any one of claims 1 to 3 is encapsulated by nanoliposomes.

5. The method for preparing the liposome for inhibiting human USP46 gene expression according to claim 4, comprising the following steps: (1) Dissolve DLin-MC3-DMA, DMG-PEG2000, DSPC, and cholesterol in anhydrous ethanol and mix to obtain solution A for later use; (2) dissolving the siRNA to be encapsulated in a citric acid-sodium citrate buffer solution with a pH of 4.0 and a concentration of 100 mM to obtain solution B; (3) Solution A and solution B are mixed to obtain a liposome that inhibits the expression of the human USP46 gene.

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