CN113862346B

CN113862346B - Application of long-chain non-coding RNA in treatment of Alzheimer disease

Info

Publication number: CN113862346B
Application number: CN202111132412.7A
Authority: CN
Inventors: 李家立; 熊婉迪; 刘斯灵; 徐开宇; 张莹
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2021-09-26
Filing date: 2021-09-26
Publication date: 2023-07-07
Anticipated expiration: 2041-09-26
Also published as: CN113862346A

Abstract

The invention discloses application of long-chain non-coding RNA in treating Alzheimer disease. The long-chain non-coding RNA is any one of the following: (a1) RNA encoded by DNA having the sequence shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3; (a2) RNA encoded by DNA having the same function and having the nucleotide sequence shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3 by substitution and/or deletion and/or addition of one or more nucleotide residues; (a3) RNA having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the nucleotide sequence defined in (a 1) or (a 2) and having the same function. The long non-coding RNA can be used for preparing a product for treating and/or preventing Alzheimer's disease or a product for improving Alzheimer's disease.

Description

Application of long-chain non-coding RNA in treatment of Alzheimer disease

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of long-chain non-coding RNA in treating Alzheimer disease.

Background

Alzheimer's Disease (AD) is a progressive, progressive neurodegenerative Disease that is clinically manifested mainly as cognitive impairment, aphasia, and impairment of visual space memory. Currently, about 4 thousands of 7 million AD patients exist in the world, but as the population of the world is aging and increasing, the prevalence of AD is increasing gradually, and it is expected that 1 hundred million AD patients exist in the world in 2050, which will bring about a huge economic burden to the world, seriously affect social, professional and life functions of the patients, bring about a heavy mental and economic burden to families and society, and bring about a huge challenge to the medical and health system of countries around the world.

The pathological features of AD mainly include pathological deposition of extracellular β -amyloid- β (aβ) to form Senile Plaques (SP), neurofibrillary tangles (neurofibrillary tangles, NFTs) formed by hyperphosphorylation aggregation of tau protein, massive neuronal loss, etc. AD is a multifactorial complex disease with complex pathogenesis and unknown etiology. Several hypotheses of the current mainstream: amyloid cascade hypothesis, tau hyperphosphorylation hypothesis, neuroinflammatory hypothesis, cholinergic hypothesis, and the like. Among them, amyloid cascade hypothesis and tau hyperphosphorylation predominate. Clinically, drugs with Abeta, tau protein and the like as action targets in drug research of AD have good performance in preclinical in-vivo and in-vitro experiments, but clinical curative effects have no obvious benefit. More and more studies suggest that aβ and tau proteins may be the only consequence of AD pathogenesis, rather than causative factors.

Mitochondria are the "energy factories" of cells that take on a number of important functions, including energy metabolism, participation in processes such as membrane electrical excitability, neurotransmitter transmission and synaptic plasticity, and are important for neuronal survival, and the relevant "mitochondrial cascade hypothesis" reveals an important role for mitochondria in AD.

With the development of high-throughput sequencing technology and computational methodologies, a large number of non-coding RNAs (ncrnas) are found to be able to serve as signal molecules, molecular baits and protein complex backbones, to participate in regulating epigenetic inheritance, and to play a very important role in the development and progression of neurological diseases. In recent years, studies have found that lncRNA (long non-coding RNA) derived from mitochondrial genome can be specifically expressed in nervous system, and is closely related to occurrence and development of AD disease.

Disclosure of Invention

It is an object of the present invention to provide a long non-coding RNA or the use of a gene encoding a long non-coding RNA.

The invention provides application of long-chain non-coding RNA in any one of the following:

(1) Use in the preparation of a product for the treatment and/or prophylaxis of alzheimer's disease;

(2) Use in the preparation of a product for ameliorating the symptoms of alzheimer's disease;

the long-chain non-coding RNA is any one of the following:

(a1) RNA encoded by DNA having the sequence shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3;

(a2) RNA encoded by DNA having the same function and having the nucleotide sequence shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3 subjected to substitution and/or deletion and/or addition of one or more nucleotide residues;

(a3) RNA having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the nucleotide sequence defined in (a 1) or (a 2) and having the same function.

The long non-coding RNA was designated as lncMtDLoop. The coding gene of the long-chain non-coding RNA can be located in mitochondria, the coding gene of the lncMtDLoop containing 939 nucleotides can be located in 15356-16294 base sequences of the whole length of mitochondrial genome. Specifically, the coding gene sequence of lncMtDLoop can be shown as SEQ ID No.1 (from mice), SEQ ID No.2 (from Chinese macaque) or SEQ ID No.3 (from human).

Those skilled in the art can prepare specific features or primer pairs by themselves based on the nucleotide sequence of long-chain non-coding RNA (lncMtDLoop) to develop subsequent experiments. For example, the primer pair sequences are shown below:

5’-TCTCGATGGTATCGGGTCTA-3’；

5’-CGCAAAACCCAATCACCTAA-3’。

the invention also provides application of the coding gene of the long-chain non-coding RNA in any one of the following:

the coding gene is any one of the following:

(b1) DNA with the sequence shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3;

(b2) DNA with the same function and obtained by substituting and/or deleting and/or adding one or more nucleotide residues of a nucleotide sequence shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3;

(b3) A DNA having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the nucleotide sequence defined in (b 1) or (b 2) and having the same function.

The invention also provides the application of the substance for improving the content of the long-chain non-coding RNA or the substance for improving the expression of the coding gene of the long-chain non-coding RNA in any one of the following:

(2) Use in the preparation of a product for ameliorating the symptoms of alzheimer's disease.

The substance that enhances the coding gene of the long non-coding RNA may specifically be the recombinant adeno-associated virus described in the examples.

The invention also provides the long-chain non-coding RNA related biological material, which is any one of the following A1) to A4):

a1 An expression cassette containing the coding gene of the long non-coding RNA;

a2 A recombinant vector comprising the coding gene of the long non-coding RNA or the expression cassette A1);

a3 A recombinant microorganism prepared from the recombinant vector of A2);

a4 A recombinant microorganism comprising the long non-coding RNA, a gene encoding the long non-coding RNA, the expression cassette of A1) or the recombinant vector of A2).

Alternatively, according to the above biological material, the recombinant microorganism of A3) or A4) is a recombinant adeno-associated virus or a recombinant lentivirus.

The invention also provides application of the biological material in any one of the following:

The invention also provides a product comprising the long-chain non-coding RNA, wherein the product has at least one of the following functions:

In the above, the product may be a medicament.

In the above, the improving the symptoms of alzheimer's disease may be at least one of:

improving spatial cognitive ability; lowering blood pressureLow A beta protein content, e.g. A beta _1-42 A protein; reducing tau protein phosphorylation; improving the morphology of mitochondria; improving autophagy function of mitochondria.

In the above, the improvement of the morphology of mitochondria may be improvement of cristae morphology of mitochondria, increasing the duty cycle of normal cristae structure. In the above, the improvement of autophagy function of mitochondria may be an increase in the content of autophagy-related proteins of mitochondria. The autophagy-related protein may be at least one selected from the group consisting of PINK1, parkin, LC3B and UKL 1.

In the above, the term "homology" refers to sequence similarity to a native sequence. Homology can be assessed visually or by computer software. Using computer software, homology between two or more sequences can be expressed in percent (%), which can be used to evaluate homology between related sequences.

In some specific embodiments, the subject sample is a brain tissue sample of an AD patient subject having significantly lower expression levels of long chain non-coding RNA (lncMtDLoop) than a healthy control sample.

In some specific embodiments, over-expression of long-chain non-coding RNA (lncMtDLoop) can improve mitochondrial morphology.

In some specific embodiments, over-expression of long-chain non-coding RNA (lncMtDLoop) can improve autophagy function of mitochondria.

In some specific embodiments, overexpression of long-chain non-coding RNA (lncMtDLoop) can reduce pathological changes in AD, e.g., decrease AB protein content, decrease tau protein phosphorylation levels.

In some specific embodiments, over-expressing long-chain non-coding RNA (lncMtDLoop) can improve spatial cognitive ability.

Drawings

FIG. 1 shows the results of the expression level detection of long-chain non-coding RNA (lncMtDLoop) in example 1.

Fig. 2 shows the result of electron microscopy observation of mitochondria in example 2.

FIG. 3 shows the autophagy-related factors detected by western blot in example 2.

FIG. 4 shows the test results of example 3.

FIG. 5 shows the test results of example 4.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The following examples were run on GraphPad Prism7 software package, and the experimental results were expressed as mean ± standard deviation, P < 0.05 (x) indicated significant differences, P < 0.01 (x) indicated very significant differences, P < 0.001 (x) indicated very significant differences, and P < 0.0001 (x) indicated very significant differences.

The experimental animals were 3xTg (APPSwe/TAUP 301L/PSEN 1) tri-transgenic mice (hereinafter, abbreviated as AD model mice in the examples) and C57/BL6 mice (hereinafter, abbreviated as wild type mice) as control groups, all purchased from Nanjing university model animal institute (introduction from Jackson laboratories, U.S.). Animals were kept in a fixed animal house at temperature (22.+ -. 2 ℃), humidity (50.+ -. 10%) and illumination time (12 hours light/dark alternation, 8:00am off lamp) with free drinking water for feeding.

Recombinant adeno-associated virus AAV virus of SYN-EGFP-lncMtDLoop: and metabiotechnology company, cat No. H15544. The recombinant adeno-associated virus AAV contains a coding gene (the sequence is shown as SEQ ID No. 1) of lncMtDLoop, and expresses long-chain non-coding RNA lncMtDLoop.

Recombinant adeno-associated virus AAV virus of SYN-EGFP: and Meta Biotechnology Inc., cat# H12990.

RNAiso plus TaKaRa company product number 9108

Q RT SuperMix for qPCR goods number R233-01

ChamQ Universal SYBR Green PCR Master goods number Q711-02

100 Xprotease inhibitor Bimake company cat# B14001

ECL luminous liquid Tanon company product number 180-5001

Hippocampal neuron planting Medium Gibco corporation hippocampal neuron maintenance Medium Gibco corporation cat# A35829-01

The level of long-chain non-coding RNA (lncMtDLoop) was significantly lower in example 1, AD pathology than in healthy control.

RNAiso plus extraction of brain tissue RNA:

1) The RNAiso plus method is used for extracting total RNA of the brain tissue of the mice:

A. 100mg of fresh 12-month AD model and wild mouse hippocampus or cortex tissue are placed into a homogenizing tube, 1ml of RNAiso plus is respectively added, the homogenizing tube is placed into an ice bath for homogenizing until the homogenate in the homogenizing tube is particle-free transparent, the homogenate is transferred into a centrifuge tube, and the mixture is kept stand for 5 minutes at room temperature.

B. And C, centrifuging 12000g of the centrifuge tube after standing in the step A for 5 minutes at the temperature of 4 ℃ to obtain supernatant.

C. The centrifuged supernatant obtained in the step B was transferred to a new centrifuge tube, 200. Mu.l of chloroform was added, the centrifuge tube lid was closed, and the mixture was vigorously shaken by hand for 15 seconds and allowed to stand at room temperature for 5 minutes.

D. Centrifuge tube 12000g after standing in step C, centrifuge at 4℃for 15 minutes to obtain a centrifuge supernatant.

E. And D, transferring the centrifugal supernatant in the step to a new centrifuge tube, adding 500 mu l of isopropanol, reversing the centrifuge tube upside down, fully and uniformly mixing, and standing at room temperature for 10 minutes.

F. Centrifuge tube 12000g after standing in step E, centrifuge at 4℃for 10 min.

G. F, discarding the supernatant in the centrifugal tube after the centrifugation in the step F, adding 1ml of 75% ethanol, washing the centrifugal tube upside down,

H. step G the centrifuge tube after reverse washing was centrifuged at 12000G for 5 minutes at 4℃and the ethanol was carefully discarded and the pellet was dried at room temperature for 5 minutes.

I. And (3) adding 30 mu l of DEPC H2O into the centrifuge tube after the drying and precipitation in the step (H) to dissolve and precipitate, thus obtaining the extracted brain tissue RNA sample.

2) Extracting total RNA of human brain tissue by RNAiso plus method:

A. 100mg of the prefrontal cortex brain area and the hippocampus (the brain tissue paraffin sections of patients are supplied by Herrup laboratories of hong Kong university) of AD patients and control patients were placed in a homogenate tube, 1ml of RNAiso plus was added respectively, the homogenate tube was placed in an ice bath for homogenate until the homogenate in the homogenate tube was particle-free transparent, the homogenate was transferred into a centrifuge tube, and left standing for 5 minutes at room temperature.

3) NanoDrop assay total RNA concentration (1. Mu.l loading)

1. Mu.l of the RNA sample extracted in 1) or 2) above was used to measure RNA concentration using NanoDrop2000, and data were recorded.

Reverse transcription of lncRNA:

the reverse transcription reaction system is shown in Table 1. And (3) carrying out reverse transcription reaction on the RNA sample extracted in the step (1) or the step (2) to obtain template DNA.

TABLE 1 reverse transcription reaction system

Lncrna real-time PCR reaction:

qRT-PCR was performed using the DNA template obtained in 1.3) above, lncRNA sense primer and lncRNA antisense primer as primers.

lncRNA sense primer: 5'-TCTCGATGGTATCGGGTCTA-3'

lncRNA antisense primer: 5'-CGCAAAACCCAATCACCTAA-3'

The qRT-PCR reaction system is shown in Table 2.

TABLE 2 qRT-PCR reaction System

qRT-PCR procedure is shown in Table 3.

TABLE 3 qRT-PCR procedure

3. Statistical analysis

The qRT-PCR experimental data adopts GraphPad Prism7 software package to carry out unpaired t-test, and P is less than 0.05, which is significant.

The results are shown in FIG. 1, wherein PFC is the prefrontal cortex, HP is the hippocampus, the left graph is a graph of the expression level of human brain tissue lncMtDLoop, AD is an AD patient, control is a control patient group, the right graph is a graph of the expression level of mouse brain tissue lncMtDLoop, WT is a wild type mouse, and 3xTg is an AD model mouse. By collecting brain tissue from AD patients and control patients, brain tissue from 12 month old AD model mice and wild type mice, including hippocampal and cortical tissue, long chain non-coding RNA (lncMtDLoop) expression levels were found to be significantly down-regulated in AD patients or 12 month old AD model mice brains compared to control patients or wild type mice.

Example 2 Long chain non-coding RNA (lncMtDLoop) significantly improves mitochondrial morphology and mitochondrial autophagy function in AD pathological conditions

1) Nucleolus stereotactic surgery and adeno-associated virus microinjection:

after anesthetizing the mice with isopentobarbitude, the mice were placed on a brain stereotactic apparatus in the prone position, and the ear stick was inserted to fix the mice. The cranium top hair of the mice is shaved, and the operation field of the mice is wiped by iodophor and medical alcohol. The skull was exposed and the bregma were adjusted to the same level. Two shallow holes were drilled in the hippocampal coordinate locations (AP, -2.0mm; ML, + -1.5 mm; DV, -2.0 mm) with a cranial drill. 1 μl of recombinant adeno-associated virus AAV virus loaded with SYN-EGFP-lncMtDLoop or SYN-EGFP was taken at a viral titer of 1x10 ¹³ Placed on ice. The hippocampus was injected with 2.5 μl Hamilton microinjector at a needle depth of 2mm at a rate of 1 μl/min and 0.5 μl per side hippocampus for 10 minutes after injection. After the injection is finished, the scalp is sutured by operation, erythromycin ointment is smeared, and the scalp is placed in a cage for continuous feeding for 6 weeks after marking.

The method is divided into 3 treatment groups, wherein a normal control group is a wild-type mice injected with the recombinant adeno-associated virus AAV virus of SYN-EGFP, a negative control group is an AD model mice injected with the recombinant adeno-associated virus AAV virus of SYN-EGFP, and an experimental group is an AD model mice injected with the recombinant adeno-associated virus AAV virus of SYN-EGFP-lncMtDLoop. Each group had 10 mice. Wherein, wild mice and AD model mice are male and female in half, 12 months old.

2) Electron microscope processing:

fixing: hippocampal tissue was harvested and fixed overnight at 4℃with 2.5% glutaraldehyde buffer. Rinse with 0.1M phosphoric acid rinse for 15 min x 3 times; fixing with 1% osmium acid fixing solution at 4deg.C for 2 hours, rinsing with 0.1M phosphoric acid rinsing solution for 15 min×3 times;

dehydrating: after gradient dehydration of ethanol, the mixture was placed in an acetone solution for dehydration for 2 times, each for 20 minutes.

Embedding and solidifying: in acetone: embedding in an embedding agent solution, and standing overnight at room temperature; embedded in Epon 812 resin and polymerized at 60 ℃ for 48 hours.

Slicing: the microtome was sectioned to 60nm thickness.

3)Western blot：

Protein sample preparation: brain tissue protein was weighed, RIPA protein lysate (10 mM PMSF,100 Xprotease inhibitor added) was added according to 100mg/1mL brain tissue weight, homogenized on ice, and allowed to stand for 30 minutes. The homogenate was centrifuged at 12000rpm at 4℃for 15 minutes, and the supernatant was aspirated, and 200. Mu.l of 5 XSDS was added to the supernatant to obtain a lysate. Boiling the lysate in a water bath kettle at 100 ℃ for 10 minutes to denature the protein to obtain a protein sample, subpackaging, and freezing in a refrigerator at-80 ℃ for standby.

Sample adding: the protein samples prepared above and markers were dissolved at room temperature in advance. After the concentrated gel is solidified, placing the gel into an electrophoresis tank, adding electrophoresis buffer solution, pulling out a comb, sequentially adding samples according to the sequence, and adding 20 mug of sample amount in each hole.

Electrophoresis: the positive and negative poles of the power supply are connected, the constant voltage electrophoresis is carried out for 100V and 30 minutes, and when the bromophenol blue indicator passes through the separation gel and the concentrated gel, the constant voltage electrophoresis is carried out for 100V and 90 minutes.

Transferring: cutting a PVDF film and filter paper with the same size as the gel, and pre-cooling the film transferring liquid at 4 ℃ for later use. After the dye reached the bottom of the gel, the gel was removed and placed in the transfer clips in the following order: black surface, foam-rubber cushion, filter paper, gel, PVDF film, filter paper, white surface, and the like are placed in a film transfer groove according to the positive and negative correspondence. Constant current 250mA was set for 120 minutes.

Closing: after the transfer, the PVDF membrane was taken out and put into a 5% BSA blocking solution, and blocked at room temperature for 1 hour.

Incubation resistance: the primary antibody was diluted with blocking solution according to the antibody instructions, the region of the PVDF membrane where the target protein was located was carefully cut off, the membrane was placed in the primary antibody dilution and incubated overnight at 4 ℃. The primary antibodies were Rabbit polyclonal anti-PINK1, rabbit polyclonal anti-Parkin, rabbit polyclonal anti-ULK1 and Rabbit polyclonal anti-LC3B.

Secondary antibody incubation: the next day the primary anti-dilution was recovered, and the membranes were washed on a TBST shaker for 5 min. Times.5. Secondary antibody was added and incubated on a shaker at room temperature for 1.5 hours. The secondary antibody was a goat anti-mouse antibody to Alexa flow 555.

Band color development: the next day the secondary antibody dilutions were recovered, and the membranes were washed on a TBST shaker for 5 min X5 times. ECL luminescence (A: B solution=1:1) was applied to PVDF film to obtain clear bands.

Quantitative analysis: the grey value analysis was performed on the strips by Image J software.

4) Statistical analysis

The experimental data were significantly different using the GraphPad Prism7 software package for one-way ANOVA with P < 0.05.

Fig. 2 shows the result of electron microscopy. A in FIG. 2 is an electron microscope image of mitochondria, wildtype-AAV-control is a normal control group, 3 XTg-AAV-control is a negative control group, 3 XTg-AAV-lncMtDLoop is an experimental group, the number of mitochondria is 71-89 in each group, and the morphology of mitochondria can be improved by over-expression of long-chain non-coding RNA (lncMtDLoop). In fig. 2, B is an electron microscope observation of mitochondrial morphology, wherein the left graph ClassI, classII, classIII is a graph showing the number of mitochondrial cristae exceeding 3,2-3,1 and below, the right graph ClassA, classB is a graph showing the ordered arrangement of mitochondrial cristae and the loose mitochondrial matrix, the cristae arrangement is disordered or no cristae structure, the wild type is a normal control group, the 3×tg-AAV-control is a negative control group, and the 3×tg-AAV-lncmtdoop is an experimental group. Normal control groups ClassI, classII, classIII and ClassA, classB account for 57.00% ± 3.90%, 37.00% ± 4.54%, 6.00% ± 3.54% and 87.27% ± 3.53, 12.73% ± 3.53%, respectively, negative control groups ClassI, classII, classIII and ClassA, classB account for 16.33% ± 3.60%, 52.25% ± 2.75%, 31.42% ± 4.47% and 73% ± 4.50%, 39.27% ± 4.50%, respectively, and experimental groups ClassI, classII, classIII and ClassA, classB account for 60.19% ± 3.54%, 26.30% ± 4.71%, 13.51% ± 4.40% and 84.90% ± 3.30%, 16.00% ± 3.30%, respectively. Compared with a negative control group, the ratio of ClassI, classII, classIII and ClassA, classB of the experimental group to that of the normal control group is closer, which indicates that the over-expression of long-chain non-coding RNA (lncMtDLoop) can improve the mitochondrial cristae morphology and increase the ratio of the normal cristae structure.

The long-chain non-coding RNA (lncMtDLoop) is over-expressed in the hippocampal brain region of the AD model mouse, compared with the injection of the empty recombinant adeno-associated virus (negative control group), the over-expression of the long-chain non-coding RNA (lncMtDLoop) can obviously improve the quantity and the structure of mitochondrial cristae in the hippocampal brain region of the AD model mouse, and the level of the wild type mouse (normal control group) can be restored.

FIG. 3 shows the results of western blot detection of changes in the expression levels of the autophagy-related factors PINK1, parkin, LC3B-II, ULK 1. A in FIG. 3 is the result of electrophoresis, wildtype-AAV-control is a normal control group, 3 XTg-AAV-control is a negative control group, and 3 XTg-AAV-lncMtDLoop is an experimental group. B in FIG. 3 is the result of statistics of gray values of electrophoresis bands, wildtype-AAV-control is a normal control group, 3 XTg-AAV-control is a negative control group, and 3 XTg-AAV-IncMTDLop is an experimental group, each group comprising 4. Wherein, the relative content of PINK1 in three groups is 1.188+ -0.089, 0.688+ -0.040 and 1.183+ -0.196 respectively, the relative content of Parkin in three groups is 1.087+ -0.091, 0.749+ -0.038 and 0.901+ -0.107 respectively, the relative content of LC3B-II in three groups is 0.967+ -0.084, 0.614+ -0.078 and 0.937+ -0.065 respectively, and the relative content of ULK1 in three groups is 0.962+ -0.043, 0.572+ -0.092 and 1.060+ -0.13 respectively. Compared with a negative control group, the over-expression of the long-chain non-coding RNA (lncMtDLoop) in the hippocampal brain region of the AD model mouse can improve the egg self-expression level of PINK1, parkin, LC3B-II and ULK1, which indicates that the over-expression of the long-chain non-coding RNA (lncMtDLoop) improves the autophagy function of mitochondria.

Example 3 Long chain non-coding RNA (lncMtDLoop) significantly reduces the pathological changes of AD

Mice were prepared according to example 21) and divided into 3 treatment groups, normal control group was a wild-type mice injected with recombinant adeno-associated virus AAV virus using SYN-EGFP, negative control group was an AD model mice injected with recombinant adeno-associated virus AAV virus using SYN-EGFP, and experimental group was an AD model mice injected with recombinant adeno-associated virus AAV virus using SYN-EGFP-lncMtDLop. 4-5 mice per group.

1.Aβ _1-42 ELISA: (kit purchased from R)&D Biotechnology Co., ltd., product number DAB142, the following reagents were supplied from the kit

1) Sample preparation: brain tissue was added with an appropriate amount of PBS, homogenized, added with an equal amount of RIPA lysate, and lysed on ice for 30 min. The supernatant was collected by centrifugation at 3000 rpm for 10 minutes.

2) And (3) preparation of a standard substance: aβ is first added _1-42 The standard substance is diluted to 5000pg/mL by a sample diluent, and is kept stand and mixed for 15 minutes for standby. 7 clean EP tubes were prepared and diluted in proportion to 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.3pg/mL, 15.6pg/mL, 7.8pg/mL, and so on, in turn, until the last tube. Zero hole: the sample dilutions were added directly.

3) Sample detection:

a. standard substance holes of the standard plate, 100 mu L of the standard substance 1.2) prepared by each hole is added in sequence according to the concentration sequence, and 100 mu L of sample diluent is directly added into the zero holes. Gently shake, cover the sealing plate membrane and incubate for two hours at 2-8deg.C.

b. Sample wells of the standard plate, 100ul 1.1) of the supernatant of the obtained sample lysate was added per well. Gently shake, cover the sealing plate membrane and incubate for two hours at 2-8deg.C.

c. The 25-fold washing concentrate was diluted with distilled water for use.

d. Gently uncovering the sealing plate membrane, discarding the liquid, spin-drying, adding 400ul of washing liquid into each hole, standing for 30 seconds, discarding, repeating four times, and beating.

e. 200 mu l A beta is added to each hole _1-42 And (3) a binding agent. Gently shake, cover the sealing plate membrane and incubate for two hours at 2-8deg.C.

f. Mu.l of stop solution was added to each well. The color of each hole changes from blue to yellow.

g. The OD of each well was measured sequentially at a wavelength of zero Kong Diaoling, 450 nm.

h. Preparing a standard curve according to the concentration and OD value of the standard substance, and calculating the Aβ of the sample according to a standard curve equation _1-42 Concentration.

2.Western blot：

The specific method is the same as the Western blot of the 3) of the embodiment 2, the primary antibodies adopted are anti-AT8, anti-AT180, anti-HT7 and anti-TAU-5, and the secondary antibodies adopted are goat anti-mouse antibodies of Alexa flow 555.

3. Statistical analysis

The results are shown in FIG. 4. A in FIG. 4 is Abeta _1-42 As a result of the content detection, wildtype-AAV-control was used as a normal control group, 3 XTg-AAV-control 1 was used as a negative control group, and 3 XTg-AAV-lncMtDLoop was used as an experimental group, Aβ _1-42 The contents are 329.60 + -47.36, 1667.00 + -290.50 and 509.20 + -73.64 (pg/ml), respectively. The over-expression of long-chain non-coding RNA (lncMtDLoop) in the hippocampal brain region of the AD model mouse can obviously reduce the Abeta in the hippocampal brain region of the mouse compared with the negative control group injected with the empty recombinant adeno-associated virus _1-42 Is contained in the composition. B in FIG. 4 is a Western blot electrophoresis result, wildtype-AAV-control is a normal control group, 3×Tg-AAV-control is a negative control group, 3×Tg-AAV-lncMtDLoop is an experimental group, C in FIG. 4 is an electrophoresis band gray level statistics result, wildtype-AAV-control is a normal control group, 3×Tg-AAV-control is a negative control group, 3×Tg-AAV-lncMtDLoop is an experimental group, wherein the relative contents of Tau-Ps202+T205 phosphorylated proteins (antibodies AT 8) in three groups are 0.745+ -0.208, 1.158 + -0.311 and 0.848+ -0.071, respectively. The relative amounts of Tau-pT231 phosphorylated protein (antibody AT 180) in the three groups were 0.645±0.075, 1.391±0.171, and 0.549±0.121, respectively. The relative amounts of Human Tau protein (Human Tau, antibody HT 7) in the three groups were 0.388±0.048, 1.033± 0.0488 and 0.753±0.085, respectively. The change of Tau protein phosphorylation level is detected, and the result shows that the overexpression of long-chain non-coding RNA (lncMtDLoop) in the hippocampal brain region of an AD model mouse can obviously reduce the expression level of Tau-Ps202+T205 phosphorylated protein, tau-pT231 phosphorylated protein and human Tau protein, reduce the Tau protein phosphorylation level and restore the Tau protein phosphorylation level to the level of a normal control group mouse. It is demonstrated that over-expression of long-chain non-coding RNA (lncMtDLoop) significantly reduces the pathological changes of AD.

Example 4 Long chain non-coding RNA (lncMtDLoop) significantly improves spatial cognitive function in AD pathological conditions

Mice were prepared according to example 21) and divided into 3 treatment groups, normal control group was a wild-type mice injected with recombinant adeno-associated virus AAV virus using SYN-EGFP, negative control group was an AD model mice injected with recombinant adeno-associated virus AAV virus using SYN-EGFP, and experimental group was an AD model mice injected with recombinant adeno-associated virus AAV virus using SYN-EGFP-lncMtDLop. 7 mice per group.

1) Water maze test: the water maze device comprises a black stainless steel pool (diameter 100cm, height 70 cm), a circular hiding platform (diameter 9cm, height about 27 cm) and an automatic recording system. The pool is divided into 4 quadrants, and the circular hiding platform is placed at a fixed position of the first quadrant. White floating beads are added into the water pool to cover the water surface, and the water temperature is kept at about 25 ℃. A camera is placed at the position 2 meters above the pool, swimming images of the mice are automatically collected, and the system automatically analyzes and processes the movement track, escape latency and swimming time of each platform quadrant of the mice.

Mice were placed into the pool from the second and third quadrants, respectively, at the same time each day, for 60 seconds each, 2 times per day. The automatic recording system starts to record the time required by the mice to climb the circular hiding platform from the water entering to the limbs, and records the swimming track and escape latency of the mice after the mice enter water for 5 continuous days. If the mouse can find a round hidden platform and stay for more than 2s, the mouse is regarded as finding the platform; if the mouse fails to find the platform within 60s after entering water, the mouse can be artificially guided to stay on the platform for 10s, and the incubation period is calculated according to 60 s. Escape latency refers to the time required for a mouse to successfully find a circular hidden platform for the first time after each water entry.

The round hidden platform was removed on day 6, the mice were placed in water from a random quadrant, and the mice' swim track was started to record the time to first reach the original round hidden platform position (i.e., escape latency time) and the total time in the target quadrant (i.e., first quadrant) at 60 s.

2) Statistical analysis

As a result, as shown in FIG. 5, wildtype-AAV-control was used as a normal control group, 3 XTg-AAV-control was used as a negative control group, and 3 XTg-AAV-lncMtDLoop was used as an experimental group. Fig. 5 a shows the swim trace of the mouse at day 6 at 60 s. The day B escape latency time statistics in fig. 5 were 26.71±5.53S, 45.57 ±5.69S and 23.86±4.23S for the normal control group, the negative control group and the experimental group, respectively. The potential target quadrant dwell time statistics on day C in fig. 5 are 22.72±3.30S, 11.42±0.94S, and 23.10±1.11S, respectively, for each group in order. Compared with a negative control group injected with empty recombinant adeno-associated virus, the over-expression long-chain non-coding RNA (lncMtDLoop) obviously shortens the escape latency time of the AD model mice and prolongs the target quadrant crossing time, which proves that the over-expression long-chain non-coding RNA (lncMtDLoop) obviously improves the space cognitive function under the pathological condition of AD.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<110> university of Beijing

<120> use of long non-coding RNA in the treatment of Alzheimer's disease

<130> 212160

<160> 3

<170> SIPOSequenceListing 1.0

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tcccaaaata tgacttatat tttagtactt gtaaaaattt tacaaaatca tgttccgtga 900

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Claims

1. Use of long non-coding RNA in any of the following:

(1) The application in preparing products for treating Alzheimer disease;

the long-chain non-coding RNA is RNA coded by DNA with a sequence shown as SEQ ID No. 1.

2. Use of the coding gene for long non-coding RNA as claimed in claim 1 in any of the following:

(1) The application in preparing products for treating Alzheimer disease;

the coding gene is DNA with a sequence shown as SEQ ID No. 1.

3. Use of a substance that increases the content of long non-coding RNAs as defined in claim 1 or a substance that increases the expression of genes encoding long non-coding RNAs as defined in claim 2 in any of the following:

(1) The application in preparing products for treating Alzheimer disease;

4. Use of a biological material associated with long non-coding RNA as claimed in claim 1 in any of the following:

(1) The application in preparing products for treating Alzheimer disease;

the biological material is any one of the following A1) -A4):

a1 A cassette comprising a gene encoding a long non-coding RNA as defined in claim 2;

a2 A recombinant vector comprising the coding gene for long non-coding RNA of claim 2 or the expression cassette of A1);

a3 A recombinant microorganism prepared from the recombinant vector of A2);

a4 A recombinant microorganism comprising the long non-coding RNA of claim 1, the gene encoding the long non-coding RNA of claim 2, the expression cassette of A1), or the recombinant vector of A2).

5. The use according to claim 4, characterized in that: a3 Or A4) the recombinant microorganism is a recombinant adeno-associated virus or a recombinant lentivirus.

6. Use according to any one of claims 1-5, characterized in that: the improvement of Alzheimer's disease symptoms is at least one of the following:

improving spatial cognitive ability;

reducing the content of Abeta protein;

reducing tau protein phosphorylation;

improving the morphology of mitochondria;

improving autophagy function of mitochondria.

7. The use according to claim 6, characterized in that: the improvement of the autophagy function of mitochondria is to increase the content of autophagy-related proteins of mitochondria.