CN113215162B

CN113215162B - Reduction of aluminum-induced Abeta 1-42 Expression level of interfering RNA and application thereof

Info

Publication number: CN113215162B
Application number: CN202110614914.7A
Authority: CN
Inventors: 宋静; 郝嘉瑞; 袁春满; 高婷; 李文静
Original assignee: Shanxi Medical University
Current assignee: Shanxi Medical University
Priority date: 2021-06-02
Filing date: 2021-06-02
Publication date: 2023-08-22
Anticipated expiration: 2041-06-02
Also published as: CN113215162A

Abstract

The application discloses a method for reducing aluminum-induced Abeta _1‑42 The interfering RNAs in the expression level are respectively siRNA-1, siRNA-2 and siRNA-3, and the RNA provided by the application can effectively interfere Abeta in cells affected by aluminum _1‑42 Expression, APP protein levels on lipid rafts and changes in palmitoylation levels. The interfering RNA in the application can be applied to in vitro researches on pathogenesis and treatment mechanism of aluminum-induced cognitive function injury diseases, and has important significance on prevention and treatment of the aluminum-induced cognitive function injury diseases, particularly Alzheimer's disease.

Description

Reduction of aluminum-induced Abeta 1-42 Expression level of interfering RNA and application thereof

Technical Field

The application relates to the technical field of biological medicine, in particular to a method for reducing aluminum-induced Abeta _1-42 Interfering RNA with expression level and application thereof.

Background

Alzheimer's Disease (AD), also known as Alzheimer's disease, is a neurodegenerative disease that causes progressive decline in brain function (Neurodegenerative diseases). Epidemiological studies have shown that the incidence of AD is 2-5% in elderly people over 65 years old, rising twice every 5 years later, up to 47% in elderly people over 85 years old, severely affecting the quality of life of the elderly. The influence factors of AD are more, and pathological characteristics are mainly beta-amyloid (Abeta) deposition, tau protein hyperphosphorylation, and finally the sustained shrinkage of cortex and the loss of hippocampal neurons.

Aluminum (Aluminum) is one of the most abundant metal elements on earth, and Aluminum and its compounds are widely present in water, foods, tableware, tea, seasonings, deodorizers, food additives, cosmetics and medicines, and can enter the human body through various ways such as foods, drinking water, medicines, etc., so that it is very easy for people to eat by mistake and accumulate in the human body. The theory that aluminum accumulated in a human body can be accumulated in brain tissues through blood brain barrier to generate neurotoxicity and cause damage to cognitive functions has been proved by a great deal of researches, and a plurality of scholars at home and abroad consider aluminum as one of environmental causative factors of neurodegenerative diseases such as Alzheimer's Disease (AD), parkinson's disease, dialysis encephalopathy and the like, and epidemiological researches show that the incidence rate of AD of people exposed to high-aluminum drinking water for a long time is remarkably improved. Animal studies demonstrate that excessive aluminum intake in rats results in reduced cognitive impairment compared to control, and that similar pathological changes in AD occur. In recent years, with the research on the action mechanism of aluminum adjuvant in vaccines, the interaction mechanism between aluminum and Abeta and the occurrence of neurological diseases such as aluminum and AD are re-caused.

Studies in the prior art have shown that elevated Abeta is an important element in the pathogenesis of AD, and Abeta consists essentially of Abeta _1-40 And Aβ _1-42 Two, produced metabolically by APP (Amyloid precursor protein). There are two metabolic pathways of APP, one is the amyloid pathway that produces aβ by continuous cleavage of β -amyloid precursor protein lyase 1 (BACE 1) and γ -secretase, and the other is the non-amyloid pathway that produces P3 peptide chain by cleavage of α -secretase and γ -secretase. Under normal conditions, aβ production and degradation is in an equilibrium state. However, in the presence of certain causative factors, increased production of aβ may result. In vivo experimental studies have shown that increased aluminum intake results in rat brain hippocampus and cortical areas aβ _1-42 The protein expression level is increased. In vitro experiments show that increased aluminum intake leads to A.beta.in PC12 cells _1-42 The generation of (2) increases.

More and more studies indicate that aluminum intake is responsible for the pathogenesis of AD and Aβ in the pathogenesis pathway of AD _1-42 The generation of (2) has important influence, but the action mechanism is not clear, and no effective prevention and treatment means exist.

Disclosure of Invention

The object of the present application is to provide a method for regulating Aβ in cells affected by aluminum _1-42 Expression ofThe RNA provided by the application can effectively interfere with Abeta in cells affected by aluminum _1-42 Expression, APP protein levels on lipid rafts and changes in palmitoylation levels.

The interfering RNA provided by the application comprises any one antisense nucleotide sequence of the following three:

siRNA-1：GUGCUCUUCACCAUGUACA

siRNA-2：GCAAUGACGAAACUGAGAU

siRNA-3：CACCUAAGAACCAUGCUCA。

preferably, the interfering RNA comprises an antisense nucleotide sequence that is siRNA-2.

Further, the interfering RNA may further comprise any one of the sense nucleotide sequences that is continuously complementary to the three antisense nucleotide sequences.

Further, the interfering RNA comprises siRNA-1 and a sense nucleotide sequence that is continuously complementary to siRNA-1.

Further, the interfering RNA comprises siRNA-2 and a sense nucleotide sequence that is continuously complementary to siRNA-2.

Further, the interfering RNA comprises siRNA-3 and a sense nucleotide sequence that is continuously complementary to siRNA-3.

In a preferred embodiment of the application, the interfering RNA is a double stranded RNA with perfectly complementary sense and antisense strands.

The interfering RNA of the present application may be obtained synthetically, such as by in vitro transcription, siRNA expression vectors, or PCR expression cassettes.

The interfering RNA provided by the application can effectively reduce the influence of aluminum on Abeta in cells _1-42 Is a target expression level.

The interfering RNA provided by the application can effectively reduce the expression level of APP protein on lipid rafts in cells affected by aluminum.

The interference RNA provided by the application can effectively reduce the level of APP palmitoylation in the cell affected by aluminum.

In the use according to the application, the cells are mammalian cells.

Further, the cell is a mammalian neural cell.

In one embodiment of the application, the cells used are mouse PC12 cells.

In the application of the application, the mammal is human, mouse, rat, monkey or pig.

On the other hand, the application also provides application of the interfering RNA in preparing a medicament for treating aluminum-induced Alzheimer disease.

Furthermore, the application also provides a transfection method of the interfering RNA, which comprises the following specific steps:

1) Diluting siRNA, namely diluting 30 mu l of 1x riboFECTTM CP Buffer to 1.25 mu l of 20 mu M siRNA stock solution, and lightly mixing according to the corresponding volume of the hole number.

2) Add 3. Mu. l riboFECTTMCP Reagent (v 4), gently blow mix, incubate at room temperature for 15min to prepare transfection complexes.

3) The 137. Mu.l of the transfection complex thus prepared was added to 1863. Mu.l of the double antibody-free medium, and the mixture was gently mixed, and after 6 hours, the medium containing the double antibody was changed.

4) Selecting cells in good condition and logarithmic phase, and concentrating according to 1×10 ⁵ Each/ml the cell mixture was seeded into six well plates and 1.5ml of cell suspension was added to each well. Culturing the six-hole plate in an incubator for about 24 hours, and observing the cell density under a light microscope to reach 40-50%.

5) siRNA transfection was performed for 24 hours.

In one embodiment of the application, the cells in the transfection method described above are PC12 cells.

The cells in the transfection methods described above may include, but are not limited to, mammalian cells, particularly mammalian neural cells. The mammal may be a human, mouse, rat, monkey, pig.

Compared with the prior art, the application has the following advantages and beneficial effects:

the siRNA in the application can effectively interfere with Abeta in cells affected by aluminum _1-42 The expression and the change of APP protein level and palmitoylation level on lipid raft provide important for mechanism and medicine research of aluminum-induced ADCompared with chemical regulator, the tool has the advantages of strong targeting property, obvious expression regulating effect and far lower dosage than the chemical regulator.

Drawings

FIG. 1 shows the Aβ of the dry and pre-stained aluminum cells of each experimental group in example 3 _1-42 Expression quantity Condition

FIG. 2 shows the results of the dry and post-infection aluminum cell APP palmitoylation levels of the experimental groups of example 4

FIG. 3 shows the expression of APP protein on lipid rafts of infected aluminum cells after the dry state of each experimental group in example 5

FIG. 4 shows Abeta of different siRNA stem pre-post-staining aluminum cells in example 6 _1-42 Expression quantity Condition

Detailed Description

The present application is further described below by way of specific examples, and the scope of application of the present application is not limited to the following examples. Having described the application in terms of the following preferred embodiments, certain modifications and equivalent changes will be apparent to those of ordinary skill in the art and are included within the scope of the application.

Unless defined otherwise herein, scientific and technical terms used in connection with the present application shall have the meanings commonly understood by one of ordinary skill in the art. In the present application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms is not limiting.

In the application, a mouse PC12 cell (source: university of Shanxi medical university labor and health science textroom) is adopted as a nerve cell model, and is subjected to aluminum staining and interference experiments, and Abeta in the PC12 cell is observed respectively _1-42 Expression, APP protein levels on lipid rafts and palmitoylation levels.

The main experimental instruments adopted in the application are as follows: carbon dioxide constant temperature incubator-Thermo Fisher scientific, U.S. SW-CJ-2F ultra clean bench-Antai air technologies Co., ltd., china, universal Hood II gel imager-Bio-Rad, U.S. SpectraMaxM 2 type microplate reader-Molecular Divices, U.S. decolorizing shaker-six instrument factories, china, film transfer apparatus-Bio-Rad Co., U.S. A.

The main experimental reagents adopted in the application are as follows: maltol-Sigma, U.S. and aluminum chloride-Fengsha chemical reagent, china, DMEM high sugar medium-sea clone biochemistry Co., ltd, china, hydroxylamine (Hydroxylamine, HAM) -Thermo Fisher scientific, U.S. and N-ethyls-succinimide (N-ethyllmaleimide, NEM) -Sigma, U.S. and biological amino-4- [4'- (maleimidomethyl) cyclohexane carboxamide butane ((1-Biotinamido-4-4' -maleimidomethyl cyclohexanecarboxamido) bunane, BMCC-Biotin-Thermo Fisher scientific, U.S. and streptavidin antibody-Thermo Fisher scientific, U.S. and UltraRIPA lipid raft extraction kit-funakoshi, japan and Nib dyeing kit-Solebao technology Co., ltd, china, protein A+G agarose beads-Kakon being century technology Co., china, 2-hexadecanoic acid (2-Bromohexadecanoic acid, BP, U.S. and AβA) _1-42 ELISA kit- -Wudonglin technology Co., ltd., china and RNA extraction kit- -kang is century biotechnology Co., ltd., china.

Example 1 cell culture Process

Cell passage: PC12 cells were cultured in 25cm containing 5ml of complete medium ² In a cell culture flask, the cell culture flask was placed in a constant temperature cell incubator (37 ℃ C., 5% CO) ₂ 100% humidity). And when the density reaches about 90%, carrying out passage treatment. Firstly, sucking and discarding old culture solution in a culture bottle, adding D-hank's for 2-3 times, and discarding waste liquid; in the second step, 1ml of trypsin digestion solution was added, and after incubation in an incubator at 37℃for 1min, 2ml of DMEM medium containing serum was added to terminate digestion. All liquid was aspirated into a 15ml EP tube and centrifuged. The supernatant was discarded, 4ml of culture medium was added to the cell pellet, and after thoroughly mixing, the mixture was uniformly added to four cell culture flasks, and after adding 4ml of complete medium to each flask, the flask was placed in a cell incubator.

Cell cryopreservation: when PC12 cells were in the logarithmic growth phase and in good condition, cell pellets were collected and added to a freezing tube containing 700. Mu.l of complete medium, 100. Mu.l of DMSO and 200. Mu.l of fetal bovine serum, and after being blown and mixed uniformly, they were sealed with a sealing film and labeled. Placing in refrigerator at-20deg.C for 30min and-80deg.C for 1 hr, and long-term storage.

Cell resuscitation: rapidly dissolving the frozen cells at-80 ℃ in a preheated 37 ℃ water bath kettle (the process is completed within 5min as much as possible), centrifuging, discarding the supernatant, adding 2mL of complete culture medium to resuspend the cells, blowing and uniformly mixing, transferring to a culture bottle containing 3mL of culture solution, and placing in a culture box for culture (instant dissolving).

Wherein, the cell complete culture medium is: DMEM high sugar medium (89%) + fetal bovine serum (10%) + green streptomycin mixture (1%), was thoroughly mixed and stored at 4 ℃.

Example 2 interference test grouping and procedure

The experimental PC12 cells were divided into 7 groups, each group being a blank group, a solvent control group (solvent), an NC group, and 200. Mu. Mol/L Al (mal) ₃ siRNA group, 50nMsiRNA (cell density 30-50%) +200. Mu. Mol/L Al (mal) ₃ Group, chemical interferent group, wherein the siRNA is siRNA-2 and the chemical interferent is 2-bromopalmitate (2-BP).

The steps of siRNA group interference RNA transfection in this experiment are:

3) The 137. Mu.l of the transfection complex was added to 1863. Mu.l of the double antibody-free medium, gently mixed, and after six hours the medium containing the double antibody was changed.

4) PC12 cells in good condition and in logarithmic growth phase were selected at a concentration of 1X 10 ⁵ Each/ml the cell mixture was seeded into six well plates and 1.5ml of cell suspension was added to each well. Culturing the six-hole plate in an incubator for about 24 hours, and observing the cell density under a light microscope to reach 40-50%.

5) siRNA transfection was performed for 24 hours.

The interference steps of the chemical interference agent are as follows:

the palmitoylation inhibitor 2-bromopalmitate (2-BP) (Sigma) was dissolved in DMSO to prepare a stock solution, which was then diluted according to the desired concentration. The concentrations were (0, 25. Mu.g/ml), respectively, and PC12 cells were treated for 9 hours.

EXAMPLE 3Aβ in each experimental group _1-42 Content detection

(1) 200. Mu. Mol/L aluminum maltol (Al (mal)) was added to the cells in each experimental group in example 2 ₃ ) After that, the culture was continued, and after 36 hours of staining with aluminum, the culture solution of the cells was collected and quantified by BCA method protein.

(2) Preparing a standard product: 1ml of diluent was added to each bottle of standard, covered, left to stand at room temperature for about 10 minutes, and inverted repeatedly to aid dissolution at a concentration of 1000pg/ml. EP tubes of 7 dilution standards were prepared, and 250. Mu.l of diluent was added to each EP tube, and diluted in turn at a multiple ratio of 1000pg/ml,500pg/ml,250pg/ml,125pg/ml,62.5pg/ml,31.2pg/ml,15.6pg/ml,0pg/ml.

(3) Sample adding: and respectively arranging a standard hole, a sample hole to be tested and a blank hole. A standard hole 7 is arranged, and 50ml of standard substances with different concentrations are sequentially added. Then 50ml of the working solution of the detection solution A was added to each well, mixed with gentle vibration, and the ELISA plate was covered with a film and incubated at 37℃for 1 hour.

(4) The liquid in the wells was discarded, and each well was washed with 300ml of washing liquid and immersed for 1 minute. And (3) throwing away the liquid in the ELISA plate, beating the ELISA plate downwards for several times, and repeatedly washing the plate for 3 times. After the last washing, the washing liquid in the holes is completely dried.

(5) 100ml of working solution (prepared just before use) of detection solution B was added to each well, and the mixture was incubated at 37℃for 1 hour.

(6) The liquid in the holes is discarded, the plate is dried by spin-drying and washed 5 times.

(7) 90ml of primer solution is added to each hole, a coating film is added to the ELISA plate, and color development is carried out at 37 ℃ in a dark place.

(8) The reaction was terminated by adding 50ml of the termination solution to each well, and the blue color turned yellow immediately.

(9) After ensuring that there are no water drops at the bottom of the microplate and no air bubbles in the wells, the optical density (OD value) of each well was measured immediately with a microplate reader at a wavelength of 450 nm.

And (3) making a standard curve according to the OD value of the standard hole, and calculating the concentration of each group according to the OD value of each group.

Wherein, maltol aluminum (Al (mal) ₃ ) The preparation method of the solution comprises the following steps: dissolving maltol in PBS (0.1%) to obtain maltol solution, alCl ₃ ·6H ₂ O is dissolved in physiological saline to prepare aluminum chloride solution. And AlCl is added again ₃ The solution and the maltol solution are mixed in equal volume to prepare the maltol aluminum solution with different concentrations. The pH was adjusted to 7.4 with HCl and 10% NaOH solution and suction filtered through a 0.22 μm filter.

As a result, FIG. 1 shows that A.beta.of the control group, the solvent control group, and the NC group _1-42 The difference in content is not statistically significant. 200 mu mol/L Al (mal) ₃ Aβ compared to the control, solvent control, and NC groups _1-42 Has a significant increase in content (P<0.05). siRNA group and control group, solvent control group, NC and 200. Mu. Mol/L Al (mal) ₃ In comparison, Aβ _1-42 The content is obviously reduced (P)<0.05)。siRNA+200μmol/L Al(mal) ₃ Aβ compared to the remaining five groups _1-42 The content is statistically significant (P)<0.05 Indicated that the siRNA in the application can significantly improve the Abeta caused by aluminum in cells _1-42 The content was increased (fig. 1A). The chemical agent (2-BP) used in the present application also has the same concentration of aluminum as that of aluminum-induced Abeta _1-42 The increase in content has a certain reduction effect (FIG. 1B), but from the experimental results, it can be seen that the chemical disruptor, Aβ, in the group of 25. Mu.g/ml 2-BP _1-42 The content is reduced by 18.7%, while the Aβ in the siRNA group _1-42 The content is reduced to 60.2%, and the effect is far higher than that of the chemical interference agent group.

Example 4 determination of APP palmitoylation level in each experimental group

(1) Protein extraction and quantification: centrifuging the cell sample, removing the supernatant, adding 100 μl of lysate, blowing and mixing with gun tip, and standing on ice for 30min; centrifuging at 12,000 rpm for 15min; aspirate supernatant solution to a new EP tube; and the total protein concentration of each group was determined by BCA protein assay and adjusted to a uniform concentration.

(2) Each sample was incubated overnight at 4℃with 3-5. Mu.l of primary APP protein. The supernatant was pipetted off and an equal volume of protein (a+g) agarose beads was added to each antibody-added complex and incubated at 4 ℃ for 4 hours to immunoprecipitate APP. A blank sample is set as negative group mark "-" in advance; the remainder were HAM positive groups, labeled "+".

(3) The sample was resuspended vertically in 0.5ml of a mixture of Lysate (LB) +10mM NEM for 1min and centrifuged (4 ℃,100 rpm/min), the supernatant was discarded and placed on ice for 10 min.

(4) The beads were washed rapidly with 0.5ml of string buffer and the supernatant was discarded by centrifugation. All samples were gently washed 3 times by adding 0.5ml of pH 7.2 lysate.

(5) 0.5ml of HAM buffer was added to the positive samples and the negative group was added with 0.5ml of lysate at pH 7.2 as a control. All samples were mixed for 50 minutes at room temperature (on shaker).

(6) Agarose beads were gently washed once in lysis solution at pH 6.2 to remove residual HAM buffer. To each sample 0.5ml Biotin-BMCC buffer was added to label the reactive cysteines. Mix at low speed for 50 minutes at 4 ℃.

(7) The samples were gently washed 3 times in LB buffer pH 7.5. To each sample, 15-20 μl (5×) of sample buffer was added, the beads were vortexed to thoroughly mix with the sample buffer, and then all samples were boiled at 100deg.C for 5 minutes. The sample was allowed to cool to room temperature and then (12000 g,3 min) agarose beads were completely precipitated for use in subsequent experiments of Western blotting.

(8) The level of APP palmitoylation is expressed as the ratio of palmitoylated APP (palAPP) to APP total protein (APP) (palAPP/APP).

The results are shown in fig. 2, where there was no statistical significance in differences in APP palmitoylation levels in the blank, solvent, and NC groups. 200 mu mol/L Al (mal) ₃ APP palmitoylation levels were significantly elevated in the group compared to the control group, solvent control group, and NC group (P<0.05). siRNA group and control group, solvent control group, NC group and 200. Mu. Mol/L Al (mal) ₃ In comparison, APP palmitoylation level was significantly reduced (P<0.05)。siRNA+200μmol/L Al(mal) ₃ APP compared to the remaining five groupsThe difference in palmitoylation level was statistically significant (P<0.05 (fig. 2A). At a certain aluminum concentration, the chemical agent (2-BP) used in the application also has a certain reduction effect on the increase of the aluminum-caused palmitoylation APP level (figure 2B), but from the experimental result, the palmitoylation APP level in the chemical interfering agent 25 mug/ml 2-BP group is reduced by 34.7%, and the palmitoylation APP level in the siRNA group is reduced by 50.4%, so that the effect is obviously higher than that of the chemical interfering agent group.

Example 5 detection of APP protein expression on lipid rafts from each experimental group

(1) Lipid raft extraction of cells

Cells were collected into EP tubes, and 200. Mu.l of UltraRIPAKITAbuffer (this procedure was performed on ice) in UltraRIPA lipid raft extraction kit was added to each EP tube. The supernatant was removed by crushing using an ultrasonic crusher and centrifuging at 14,000rpm for 5 minutes. After 0.5ml of ice-cold A buffer was added to the pellet (RIPA insoluble fraction), centrifuged at 14,000rpm for 5 minutes, the supernatant was removed, 50-200. Mu.l of UltraRIPAkit B buffer was added to the pellet, and the pellet was resuspended for about 5 minutes, centrifuged at 14,000rpm for 5 minutes, and the supernatant was collected in a new tube and assayed for each group of total protein concentration by BCA protein assay (total protein final concentration was set to 1-10 mg/ml).

(2) Determination of APP protein expression

The corresponding proteins were isolated by Western blotting. Preparing separating gel with corresponding concentration according to protein molecular weight, electrophoresis voltage and transfer condition, and sealing with 5% skimmed milk powder at room temperature after transfer. Antibodies to the corresponding proteins (dilution ratio: β -Tubulin,1:3000; app,1:6500; bace1,1:5000; flotillin,1:3000; zdhc7, 1:3000), were added, incubated overnight at 4 ℃, followed by 5 washes with TBST for 8min each; the secondary antibodies (dilution ratio 1:4000) were incubated at 37℃for 2h, then washed 5 times with TBST for 8min each, and after ECL development, the grey values of the proteins were analyzed by image J software and the relative expression amounts were calculated.

The results are shown in FIG. 3, where there was no statistical significance in differences in APP protein expression on lipid rafts in the blank, solvent, and NC groups. 200 mu mol/L Al (mal) ₃ Group and control group, solvent control group,APP protein expression on lipid rafts was elevated compared to NC group. siRNA group and control group, solvent control group, NC group and 200. Mu. Mol/L Al (mal) ₃ In contrast, APP protein expression on lipid rafts was significantly reduced (P<0.05)。siRNA+200μmol/L Al(mal) ₃ The difference in APP protein expression on lipid rafts was statistically significant compared to the remaining five groups (P<0.05 (fig. 3A). Under the condition of the same aluminum concentration, the chemical agent (2-BP) used in the application has a certain reduction effect on the increase of the palmitoylation APP level content caused by aluminum (figure 3B), but from the experimental result, the APP expression level on lipid rafts in the chemical interference agent 25 mug/ml 2-BP group is reduced by 9.5%, and the APP expression level on lipid rafts in the siRNA group reaches 57.0%, so that the effect is obviously higher than that of the chemical interference agent group.

The experimental results prove that: aluminum maltol (Al (mal) ₃ ) The solution is capable of inducing Aβ in cells _1-42 Increased levels of APP palmitoylation modification, increased levels of APP protein expression on cellular lipid rafts, and close correlation with the development and progression of Alzheimer's Disease (AD), wherein the siRNA of the application interferes with Abeta in aluminum-infected cells after the cells are infected with the siRNA _1-42 The content of APP palmitoylation modification level and the APP protein expression amount on the cell lipid raft are obviously reduced.

Example 6 different siRNA interference Effect

The cell culture method of this example is the same as that of example 1.

The interference test grouping conditions are as follows: blank, solvent control (solvent), NC, 200. Mu. Mol/L Al (mal) ₃ Group, siRNA-1 group, siRNA-2 group, siRNA-3 group, siRNA-1 (50 nM) +200. Mu. Mol/L Al (mal) ₃ Group, siRNA-2+200. Mu. Mol/L Al (mal) ₃ Group, siRNA-3+200. Mu. Mol/L Al (mal) ₃ A group.

The interference procedure for each experimental group was the same as in example 2.

Aβ _1-42 The content measurement method was the same as in example 3.

The experimental results are shown in FIG. 4.

Wherein, the antisense nucleotide sequence of the siRNA-1 is shown as SEQ ID 1 in the sequence table, the antisense nucleotide sequence of the siRNA-2 is shown as SEQ ID2 in the sequence table, and the antisense nucleotide sequence of the siRNA-3 is shown as SEQ ID 3 in the sequence table.

As can be seen from the figures: siRNA-1 group, siRNA-2 group and Abeta of siRNA-3 group _1-42 Content and 200. Mu. Mol/L Al (mal) ₃ The differences in group phase are statistically significant (P<0.05)。siRNA(1、2、3)+200μmol/L Al(mal) ₃ Aβ compared to the remaining five groups _1-42 The content is statistically significant (P)<0.05 (fig. 4). While three different siRNA groups (1, 2, 3) +200. Mu. Mol/L Al (mal) ₃ Group reduction of aluminum-induced Abeta _1-42 The rising degree is respectively 50.9%, 64.8% and 41.5%, and good effects are obtained.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.

SEQUENCE LISTING

<110> university of Shanxi medical science

<120> interfering RNA for reducing expression level of aluminum-induced Abeta 1-42 and use thereof

<130> 2021.06

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 19

<212> RNA

<213> artificial sequence

<400> 1

gugcucuuca ccauguaca 19

<210> 2

<211> 19

<212> RNA

<213> artificial sequence

<400> 2

gcaaugacga aacugagau 19

<210> 3

<211> 19

<212> RNA

<213> artificial sequence

<400> 3

caccuaagaa ccaugcuca 19

Claims

1. Use of an interfering RNA that reduces the expression level of aβ1-42 in an aluminum-affected cell, said interfering RNA comprising an antisense nucleotide sequence siRNA-2 and a sense nucleotide sequence complementary thereto, said siRNA-2 having a sequence GCAAUGACGAAACUGAGAU, for the manufacture of a medicament for reducing the expression level of aβ1-42 in an aluminum-affected cell.

2. Use of an interfering RNA as claimed in claim 1 for the manufacture of a medicament for reducing the level of APP protein expression on lipid rafts in an aluminium-affected cell.

3. Use of an interfering RNA as described in claim 1 or 2 for the manufacture of a medicament for reducing the level of APP palmitoylation in an aluminum-affected cell.

4. The use according to any one of claims 1 to 3, wherein the cells are mammalian cells.

5. The use according to claim 4, wherein the cells are mammalian neural cells.

6. The use according to claim 5, wherein the mammal is a human, a mouse, a rat, a monkey, a pig.

7. The use of the interfering RNA of claim 1 for the preparation of a medicament for the treatment of alzheimer's disease.