CN113862346A

CN113862346A - Use of long non-coding RNA in treating Alzheimer's disease

Info

Publication number: CN113862346A
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: 2021-12-31
Anticipated expiration: 2041-09-26
Also published as: CN113862346B

Abstract

The invention discloses application of long-chain non-coding RNA in treating Alzheimer disease. The long non-coding RNA is any one of the following: (a1) RNA coded by DNA with the sequence shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3; (a2) RNA coded by DNA with the same function and with the nucleotide sequence shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3 substituted and/or deleted and/or added by one or more nucleotide residues; (a3) and (c) RNA having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the nucleotide sequence defined in (a1) or (a2) and having the same function. The long-chain non-coding RNA can be applied to preparation of products for treating and/or preventing Alzheimer's disease or products for improving Alzheimer's disease symptoms.

Description

Use of long non-coding RNA in treating Alzheimer's 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 disorder, which is clinically manifested primarily as cognitive impairment, aphasia, and visuospatial memory impairment. About 4 thousand and 7 million AD patients exist in the world currently, but with the increasing aging of the world population, the prevalence rate of AD is gradually increased, 1 hundred million and 3 million AD patients are expected to exist in the world in 2050, which brings huge economic burden to the world, also seriously affects the social, occupational and living functions of the patients, brings heavy mental and economic burden to families and society, and also brings huge challenges to the medical health systems of countries in the world.

Pathological features of AD mainly include pathological deposition of extracellular β -amyloid (amyloid- β, Α β) to form Senile Plaques (SP), Neuronal Fibrillar Tangles (NFTs) formed by hyperphosphorylation and aggregation of tau protein, and massive neuronal loss. AD is a multifactorial complex disease with a complex pathogenesis and unknown etiology. Several hypotheses are currently prevailing: amyloid cascade hypothesis, tau hyperphosphorylation hypothesis, neuroinflammation hypothesis, cholinergic hypothesis, etc. Among them, the amyloid cascade hypothesis and tau hyperphosphorylation dominate. Clinically, in the study of AD drugs, drugs with Α β, tau protein, etc. as the target of action, although they perform well in vitro and in vivo experiments before clinical treatment, the clinical therapeutic benefits are not significant. More and more studies suggest that a β and tau protein may be the only consequence of AD pathogenesis, not the causative agent.

Mitochondria are the "energy factories" of cells, and have many important functions, including energy metabolism, participation in processes such as membrane electrical excitability, neurotransmitter transmission and synaptic plasticity, which are very important for the survival of neurons, and the "mitochondrial cascade hypothesis" reveals an important role of mitochondria in AD.

With the development of high-throughput sequencing technology and computational methodology, a large amount of non-coding RNA (ncRNA) is found to be capable of being used as a signal molecule, a molecular decoy and a protein complex skeleton, participating in the regulation of epigenetics, and playing a very important role in the occurrence and development of nervous system diseases. In recent years, some studies find that lncRNA (long non-coding RNA) derived from mitochondrial genome can be specifically expressed in nervous system, and is closely related to the occurrence and development of AD diseases.

Disclosure of Invention

The invention aims to provide application of a coding gene of long-chain non-coding RNA or long-chain non-coding RNA.

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

(1) the application in preparing the product for treating and/or preventing the Alzheimer disease;

(2) the application of the product in preparing the product for improving the symptoms of the Alzheimer disease;

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

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

(a2) RNA coded by DNA with the same function and with the nucleotide sequence shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3 substituted and/or deleted and/or added by one or more nucleotide residues;

(a3) and (c) RNA having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the nucleotide sequence defined in (a1) or (a2) and having the same function.

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

Those skilled in the art can self-prepare specific features or primer pairs based on the nucleotide sequence of the long non-coding RNA (lncMtDLoop) and perform subsequent experiments. For example, the primer pair sequences are as follows:

5’-TCTCGATGGTATCGGGTCTA-3’；

5’-CGCAAAACCCAATCACCTAA-3’。

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

the coding gene is any one of the following:

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

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

(b3) and (b) 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 (b1) or (b2) and having the same function.

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

(2) application in preparing a product for improving Alzheimer disease symptoms.

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

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

A1) an expression cassette containing a gene encoding the long non-coding RNA;

A2) a recombinant vector comprising a gene encoding the long non-coding RNA or the expression cassette described in A1);

A3) a recombinant microorganism prepared from the recombinant vector of a 2);

A4) a recombinant microorganism comprising the long non-coding RNA, a gene encoding the long non-coding RNA, A1) or A2) the recombinant vector.

Optionally, according to the above-mentioned biological material, A3) or a4) the recombinant microorganism is a recombinant adeno-associated virus or a recombinant lentivirus.

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

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

Hereinbefore, the product may be a medicament.

As above, the ameliorating alzheimer's disease symptoms may be at least one of:

improving spatial cognitive ability; reducing A beta protein content, e.g. A beta_1-42A protein; reducing phosphorylation levels of tau protein; improving the morphology of mitochondria; improving the autophagy function of mitochondria.

In the above, the improvement of the morphology of mitochondria may be an improvement of the cristae morphology of mitochondria, increasing the proportion of normal cristae structure. As described above, the improvement of the autophagy function of mitochondria can be the increase of the content of a protein involved in mitochondrial autophagy. The autophagy-related protein may be selected from at least one 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 as a percentage (%), which can be used to assess homology between related sequences.

In some specific embodiments, the subject sample is a brain tissue sample from an AD patient subject with significantly lower expression levels of long non-coding rna (lncmtdlop) than a sample from a healthy control.

In some specific embodiments, overexpression of long non-coding rna (lncmtdlop) can improve mitochondrial morphology.

In some specific embodiments, overexpression of long non-coding rna (lncmtdlop) can improve autophagy function of mitochondria.

In some specific embodiments, overexpression of long non-coding rna (lncmtdlop) can reduce pathological changes in AD, such as reducing AB protein content, reducing tau protein phosphorylation levels.

In some specific embodiments, overexpression of long non-coding rna (lncmtdlop) may improve spatial cognitive ability.

Drawings

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

FIG. 2 shows the results of the electron microscopic observation of mitochondria in example 2.

FIG. 3 is a graph of the detection of autophagy-related factors by western blot in example 2.

FIG. 4 shows the results of the test in example 3.

FIG. 5 shows the results of the test in example 4.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The following examples were processed using the GraphPad Prism7 software package and the results of the experiments were expressed as mean ± standard deviation, P < 0.05 (x) for significant differences, P < 0.01 (x) for very significant differences, P < 0.001 (x) for very significant differences and P < 0.0001 (x) for very significant differences.

The experimental animals were 3xTg (APPWE/TAUP 301L/PSEN1) triple transgenic mice (hereinafter referred to as AD model mice) and C57/BL6 mice (hereinafter referred to as wild type mice) as a control group, all purchased from the university of Nanjing institute of model animals (introduced from Jackson laboratories, USA). Animals were kept in a room with constant temperature (22. + -. 2 ℃), humidity (50. + -.10%) and illumination time (12 hours light/dark alternating, 8: 00am light off) and were fed with free water.

Recombinant adeno-associated virus AAV virus of SYN-EGFP-lncMtDLoop: and Yuanzhi corporation, cat # H15544. The recombinant adeno-associated virus AAV contains a coding gene (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 YuanBiotech, Inc., cat number H12990.

Goods number 9108 of RNAioso plus TaKaRa

Q RT Supermix for qPCR product number R233-01

ChamQ Universal SYBR Green PCR Master Cat number Q711-02

100 Xprotease inhibitor Bimake K.K. No. B14001

ECL luminescence liquid Tanon company's goods number 180-

Culture Medium for hippocampal neuron culture Medium Gibco Inc. Hippocampus neuron maintenance Medium Gibco Inc. Cat No. A35829-01

Example 1, long-chain non-coding rna (lncmtdlop) levels were significantly lower in AD pathological conditions than in healthy controls.

Extraction of brain tissue RNA by RNAiso plus:

1) extraction of total RNA from mouse brain tissue by the RNAlso plus method:

A. 100mg of fresh model AD in 12 months and wild mouse hippocampus or cortical tissue are put into a homogenate tube, 1ml of RNAiSo plus is added into the homogenate tube, the homogenate tube is placed into an ice bath for homogenate until homogenate liquid in the homogenate tube is transparent without particles, the homogenate liquid is transferred into a centrifuge tube, and the centrifuge tube is kept stand for 5 minutes at room temperature.

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

C. Transferring the centrifugal supernatant obtained in the step B into a new centrifugal tube, adding 200 mu l of chloroform, tightly covering the centrifugal tube, violently shaking for 15 seconds by hands, and standing for 5 minutes at room temperature.

D. And C, centrifuging 12000g of the centrifuge tube after standing in the step C for 15 minutes at 4 ℃ to obtain a centrifugal supernatant.

E. Transferring the centrifugal supernatant obtained in the step D into a new centrifugal tube, adding 500 mu l of isopropanol, turning the centrifugal tube upside down, fully mixing the mixture, and standing the mixture at room temperature for 10 minutes.

F. And E, centrifuging 12000g of the centrifuge tube after standing in the step E for 10 minutes at 4 ℃.

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

H. step G the washed tubes were inverted 12000G, centrifuged at 4 ℃ for 5 minutes and carefully discarded after ethanol, and the pellet was dried at room temperature for 5 minutes.

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

2) The RNAlso plus method is used for extracting the total RNA of the brain tissue of the human:

A. the brain area of the prefrontal cortex and the hippocampus (paraffin sections of the brain tissue of the patient provided by Herrup laboratories, university of hong kong science and technology) of 100mg of AD patients and control patient groups were taken and placed in a homogenization tube, 1ml of rnaasso plus was added, and the homogenization tube was placed in an ice bath for homogenization until the homogenate in the homogenization tube was transparent without particles, and the homogenate was transferred to a centrifuge tube and allowed to stand at room temperature for 5 minutes.

3) NanoDrop determination of Total RNA concentration (1. mu.l loading)

Taking 1. mu.l of RNA sample extracted from the above 1) or 2), using NanoDrop2000, measuring the RNA concentration, and recording the data.

Reverse transcription of lncRNA:

the reverse transcription reaction system is shown in Table 1. Carrying out reverse transcription reaction on the RNA sample extracted in the step 1) or 2) to obtain a 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

The 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 the difference is significant when P is less than 0.05.

The results are shown in FIG. 1, in which PFC is prefrontal cortex, HP is hippocampus, left-side graph is expression level of human brain tissue lncMtDLoop, AD is AD patient, control is control patient group, right-side graph is expression level of mouse brain tissue lncMtDLoop, WT is wild type mouse, and 3 × Tg is AD model mouse. By collecting brain tissues of AD patients and control patients, brain tissues of AD model mice at 12 months of age and wild type mice, including hippocampal and cortical tissues, it was found that the expression level of long-chain non-coding rna (lncmtdlop) appears significantly down-regulated in the brains of AD patients or AD model mice at 12 months of age compared to control patients or wild type mice.

Example 2 Long non-coding RNA (lncMtDLoop) significantly improved mitochondrial morphology and mitochondrial autophagy function under AD pathological conditions

1) Nuclear mass stereotactic surgery and adeno-associated virus microinjection:

after anesthetizing the mouse with amobarbital, the mouse was placed on a brain stereotaxic apparatus in the prone position, and an ear stick was inserted to fix the mouse. The cranial hair of the mice was shaved and the mice were wiped of the operative field with iodophors and medical alcohol. The skull is exposed and bregma are adjusted to the same level. Two shallow wells were drilled with a cranial drill at the hippocampal coordinate position (AP, -2.0 mm; ML, ± 1.5 mm; DV, -2.0 mm). Taking 1 μ l of recombinant adeno-associated virus AAV loaded with SYN-EGFP-lncMtDLoop or SYN-EGFP, the virus titer is 1x10¹³Placed on ice. Injecting mouse hippocampal brain region with 2.5 μ l Hamilton microinjector with a needle insertion depth of 2mm and an injection speedAt 1. mu.l/min, 0.5. mu.l per hippocampus was injected, and the needle was left for 10 minutes after injection. After the injection is finished, the scalp is sutured by operation, erythromycin ointment is smeared, and the mixture is marked and placed into a cage for continuous breeding for 6 weeks.

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

2) Electron microscope treatment:

fixing: hippocampal tissue was taken and fixed with 2.5% glutaraldehyde buffer overnight at 4 ℃. Rinsing with 0.1M phosphoric acid rinse solution for 15 minutes × 3 times; fixation with 1% osmate solution at 4 ℃ for 2 hours and rinsing with 0.1M phosphoric acid rinse 15 min X3 times;

and (3) dehydrating: after gradient dehydration with ethanol, the mixture was dehydrated in acetone solution for 2 times, each time for 20 minutes.

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

Slicing: microtome sections were taken to a thickness of 60 nm.

3)Western blot：

Protein sample preparation: brain tissue protein was weighed, RIPA protein lysate (10 mM PMSF, 100 x protease inhibitor added) was added to the brain tissue at a weight of 100mg/1mL, homogenized on ice, and allowed to stand for 30 minutes. The homogenate was centrifuged at 12000rpm for 15 minutes at 4 ℃ to extract the supernatant, 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 protein to obtain a protein sample, subpackaging, and freezing and storing in a refrigerator at-80 ℃ for later use.

Sample adding: the protein sample prepared above and the marker were dissolved at room temperature in advance. And after the concentrated gel is solidified, putting the gel into an electrophoresis tank, adding an electrophoresis buffer solution, pulling out a comb, and sequentially adding samples, wherein the sample loading amount of each hole is 20 mu g.

Electrophoresis: switching on the positive electrode and the negative electrode of a power supply, performing constant-voltage electrophoresis for 100V and 30 minutes, and performing constant-voltage electrophoresis for 100V and 90 minutes after the bromophenol blue indicator crosses a boundary between the separation gel and the concentration gel.

Film transfer: cutting a PVDF membrane and filter paper with the same size as the gel, and precooling the membrane transferring liquid at 4 ℃ for standby. And taking out the gel after the dye reaches the bottom of the gel, and placing the gel in a film transfer clip according to the following sequence: black surfaces, a spongy cushion, filter paper, gel, a PVDF film, filter paper and white surfaces are correspondingly placed in a film rotating groove according to the positive and negative electrodes. Set to constant current 250mA for 120 minutes.

And (3) sealing: after the completion of the membrane transfer, the PVDF membrane was taken out and placed in a 5% BSA blocking solution, and blocked at room temperature for 1 hour.

Primary antibody incubation: the primary antibody was diluted with blocking solution according to the antibody specification, the area of the PVDF membrane where the target protein was located was carefully excised, and the membrane was placed in the primary antibody dilution and incubated overnight at 4 ℃. The primary antibodies are Rabbit polyclonal anti-PINK1, Rabbit polyclonal anti-Parkin, Rabbit polyclonal anti-ULK1 and Rabbit polyclonal anti-LC 3B.

And (3) secondary antibody incubation: the next day, primary anti-diluent was recovered and the membrane washed on a TBST shaker for 5 minutes × 5 times. Add secondary antibody and incubate on shaker at room temperature for 1.5 hours. The secondary antibody was a goat anti-mouse antibody to Alexa Flour 555.

Strip color development: the next day, the secondary antibody diluent was recovered and the membrane was washed on a TBST shaker for 5 minutes × 5 times. Clear bands were obtained by applying ECL luminescence (1: 1 solution a: B) to PVDF membranes.

Quantitative analysis: the bands were grey value analyzed by Image J software.

4) Statistical analysis

Experimental data one-way ANOVA was performed using GraphPad Prism7 software package, with differences of significance when P < 0.05.

FIG. 2 shows the results of mitochondrial observation by electron microscopy. In FIG. 2, A is an electron microscope image of mitochondria, 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, the number of observed mitochondria in each group is 71-89, and the morphology of mitochondria can be improved by over-expressing long non-coding RNA (lncMtDLoop). FIG. 2B is an electron microscope observation of mitochondrial morphology, wherein the left panels ClassI, ClassII, and ClassIII are respectively the number of the mitochondrial cristae exceeding 3, 2-3, 1 and below, the right panels ClassA and ClassB are respectively the mitochondrial cristae arrangement order and the mitochondrial stroma porosity, the cristae arrangement disorder or no cristae structure, Wildtype is the normal control group, 3 × Tg-AAV-control is the negative control group, and 3 × Tg-AAV-lncMtDLoop is the experimental group. ClassI, ClassII, ClassIII, ClassA and ClassB of the normal control groups account for 57.00% + -3.90%, 37.00% + -4.54%, 6.00% + -3.54% and 87.27% + -3.53, 12.73% + -3.53%, respectively, ClassI, ClassII, ClassIII, ClassA and ClassB of the negative control groups account for 16.33% + -3.60%, 52.25% + -2.75%, 31.42% + -4.47% and 73% + -4.50%, 39.27% + -4.50%, respectively, ClassI, ClassII, ClassIII and ClassA of the experimental groups, 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 ratios of the experimental group to the normal control group are closer to those of ClassI, ClassII, ClassIII, ClassA and ClassB, and the over-expression of the 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, and the over-expression of the long-chain non-coding RNA (lncMtDLoop) can obviously improve the number and the structure of mitochondrial cristae in the hippocampal brain region of the AD model mouse (negative control group) and restore to the level of a wild type mouse (normal control group).

FIG. 3 shows the results of western blot detection of the changes in the expression levels of autophagy-related factors PINK1, Parkin, LC3B-II, and ULK 1. A in FIG. 3 is the result of electrophoresis, Wildtype-AAV-control is the normal control group, 3 XTG-AAV-control is the negative control group, and 3 XTG-AAV-lncMtDLoop is the experimental group. In FIG. 3, B is the statistical result of the grayscale values of the electrophoretic bands, Wildtype-AAV-control is a normal control group, 3 × Tg-AAV-control is a negative control group, 3 × Tg-AAV-IncMtDLoop is an experimental group, and 4 individuals are present in each group. Wherein, the relative contents of PINK1 in three groups are respectively 1.188 +/-0.089, 0.688 +/-0.040 and 1.183 +/-0.196, the relative contents of Parkin in three groups are respectively 1.087 +/-0.091, 0.749 +/-0.038 and 0.901 +/-0.107, the relative contents of LC3B-II in three groups are respectively 0.967 +/-0.084, 0.614 +/-0.078 and 0.937 +/-0.065, and the relative contents of ULK1 in three groups are respectively 0.962 +/-0.043, 0.572 +/-0.092 and 1.060 +/-0.13. Compared with a negative control group, the method has the advantages that long-chain non-coding RNA (lncMtDLoop) is overexpressed in a hippocampal brain region of an AD model mouse, the level of self-expression of PINK1, Parkin, LC3B-II and ULK1 can be improved, and the fact that the autophagy function of mitochondria is improved by overexpression of the long-chain non-coding RNA (lncMtDLoop) is demonstrated.

Example 3 Long non-coding RNA (lncMtDLoop) significantly reduced pathological changes in AD

Mice were prepared according to the method of example 21) and divided into 3 treatment groups, wherein the normal control group was wild-type mice injected with recombinant adeno-associated virus AAV virus using SYN-EGFP, the negative control group was AD model mice injected with recombinant adeno-associated virus AAV virus using SYN-EGFP, and the experimental group was AD model mice injected with recombinant adeno-associated virus AAV virus using SYN-EGFP-lncmtdoop. Each group had 4-5 mice.

1.Aβ_1-42ELISA: (kit purchased from R)&D Biotech company, Cat number DAB142, the following reagents were provided in the kit)

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

2) Preparing a standard substance: firstly, A beta is_1-42And diluting the standard sample to 5000pg/mL by using the sample diluent, and standing and uniformly mixing for 15 minutes for later use. 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 to the last tube. Zero hole: the sample diluent was added directly.

3) Sample detection:

a. and (3) adding 100 mu L of the standard substance prepared in 1.2) into each standard substance hole of the standard plate in turn according to the concentration sequence, and directly adding 100 mu L of the sample diluent into each zero hole. Gently shake, cover the sealing plate membrane, incubate at 2-8 ℃ for two hours.

b. 100ul of the sample lysate supernatant obtained in 1.1) was added to each well of the standard. Gently shake, cover the sealing plate membrane, incubate at 2-8 ℃ for two hours.

c. Diluting 25 times of washing concentrated solution with distilled water for later use.

d. Slightly uncovering the unsealing plate film, discarding liquid, spin-drying, adding 400ul of cleaning solution into each hole, standing for 30 seconds, discarding, repeating for four times, and patting dry.

e. Add 200. mu. l A. beta. to each well_1-42A binding agent. Gently shake, cover the sealing plate membrane, incubate at 2-8 ℃ for two hours.

f. Add 50. mu.l stop buffer to each well. Each well changed in color from blue to yellow.

g. And adjusting to zero by using a zero hole, and sequentially measuring the OD value of each hole by using the wavelength of 450 nm.

h. Preparing a standard curve according to the concentration and OD value of the standard substance, and then calculating the Abeta of the sample according to a standard curve equation_1-42And (4) concentration.

2.Western blot：

The specific method is the same as that of Western blot 3) in example 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 Flour 555.

3. Statistical analysis

The results are shown in FIG. 4. In FIG. 4, A is Abeta_1-42The content detection result shows that Wildtype-AAV-control is a normal control group, 3 × Tg-AAV-control 1 is a negative control group, 3 × Tg-AAV-lncMtDLoop is an experimental group, and Abeta is_1-42The contents were 329.60 + -47.36, 1667.00 + -290.50 and 509.20 + -73.64 (pg/ml), respectively. Over-expressing long-chain non-coding RNA (lncMtDLoop) in the brain area of the hippocampi of an AD model mouse, and comparing a negative control group injected with unloaded recombinant adeno-associated virus to obtain the over-expressing long-chain non-coding RNA (lncMtDLoop) which can obviously reduce Abeta of the brain area of the hippocampi of the mouse_1-42The content of (a). B in FIG. 4 is the result of Western blot electrophoresis, Wildtype-AAV-control is the normal control group, 3 XTG-AAV-control is the negative control group, 3 XTG-AAV-lncMtDLoop is the experimental group, C in FIG. 4 is the statistical result of the electrophoretic band gray scale, Wildtype-AAV-control is the normal control group, 3 XTG-AAV-control is the negative control group, 3 XTG-AAV-lncMtDLoopp is an experimental group, wherein the relative content of Tau-Ps202+ T205 phosphorylated protein (antibody AT8) in the three groups is 0.745 +/-0.208, 1.158 +/-0.311 and 0.848 +/-0.071 respectively. The relative content of Tau-pT231 phosphorylated protein (antibody AT180) in the three groups is 0.645 + -0.075, 1.391 + -0.171 and 0.549 + -0.121 respectively. The relative content of Human Tau protein (Human Tau, antibody HT7) in three groups was 0.388 + -0.048, 1.033 + -0.0488 and 0.753 + -0.085, respectively. Detecting the change of phosphorylation level of Tau protein, and finding 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 levels of Tau-Ps202+ T205 phosphorylated protein, Tau-pT231 phosphorylated protein and human Tau protein, reduce the phosphorylation level of Tau protein and restore the phosphorylation level of Tau protein to the level of a normal control group mouse. Indicating that overexpression of long non-coding rna (lncmtdlop) significantly reduced pathological changes in AD.

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

Mice were prepared according to the method of example 21) and divided into 3 treatment groups, wherein the normal control group was wild-type mice injected with recombinant adeno-associated virus AAV virus using SYN-EGFP, the negative control group was AD model mice injected with recombinant adeno-associated virus AAV virus using SYN-EGFP, and the experimental group was AD model mice injected with recombinant adeno-associated virus AAV virus using SYN-EGFP-lncmtdoop. Each group had 7 mice.

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

Mice were placed in the pool from the second and third quadrants, respectively, at the same time each day for 60s each time, 2 trains each day. The automatic recording system starts to record the time required by the mouse to climb up the circular hidden platform from the water inlet to the four limbs, and records the swimming track and the escape latency of the mouse after the mouse enters the water continuously for 5 days. If the mouse can find the circular hidden platform and stays for more than 2s, the mouse is regarded as a found platform; if the mouse can not find the platform within 60s after entering water, the mouse can be artificially guided to the platform to stay for 10s, and the latency 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 entering water each time.

On day 6 the circular hidden platform was removed, the mouse was placed into the water from one of the random quadrants, and the swimming trajectory of the mouse at 60s, the time to first reach the original circular hidden platform position (i.e. the escape latency time) and the total time in the target quadrant (i.e. the first quadrant) were recorded.

2) Statistical analysis

The results are shown in FIG. 5, in which Wildtype-AAV-control is a normal control group, 3 × Tg-AAV-control is a negative control group, and 3 × Tg-AAV-lncMtDLoop is an experimental group. In FIG. 5, A is the swimming trajectory of the mouse at day 6 in 60 s. Day 6 evasion latency statistics for B in fig. 5, normal control, negative control and experimental groups were 26.71 ± 5.53S, 45.57 ± 5.69S and 23.86 ± 4.23S, respectively. The residence time statistics for the potential target quadrant on day 6 of C in FIG. 5 are 22.72 + -3.30S, 11.42 + -0.94S and 23.10 + -1.11S for each group. Compared with a negative control group injected with the no-load recombinant adeno-associated virus, the over-expression long-chain non-coding RNA (lncMtDLoop) remarkably shortens the escape latency time of the AD model mouse and prolongs the target quadrant crossing time, which shows that the over-expression long-chain non-coding RNA (lncMtDLoop) remarkably improves the space cognitive function under the AD pathological condition.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the 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 reference to specific embodiments, it will be appreciated that the invention can 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 use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> Beijing university

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

<130> 212160

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 939

<212> DNA

<213> mouse (Mus musculus)

<400> 1

tcaagaagaa ggagctactc cccaccacca gcacccaaag ctggtattct aattaaacta 60

cttcttgagt acataaattt acatagtaca acagtacatt tatgtatatc gtacattaaa 120

ctattttccc caagcatata agctagtaca ttaaatcaat ggttcaggtc ataaaataat 180

catcaacata aatcaatata tataccatga atattatctt aaacacatta aactaatgtt 240

ataaggacat atctgtgtta tctgacatac accatacagt cataaactct tctcttccat 300

atgactatcc ccttccccat ttggtctatt aatctaccat cctccgtgaa accaacaacc 360

cgcccaccaa tgcccctctt ctcgctccgg gcccattaaa cttgggggta gctaaactga 420

aactttatca gacatctggt tcttacttca gggccatcaa atgcgttatc gcccatacgt 480

tccccttaaa taagacatct cgatggtatc gggtctaatc agcccatgac caacataact 540

gtggtgtcat gcatttggta tctttttatt ttggcctact ttcatcaaca tagccgtcaa 600

ggcatgaaag gacagcacac agtctagacg cacctacggt gaagaatcat tagtccgcaa 660

aacccaatca cctaaggcta attattcatg cttgttagac ataaatgcta ctcaatacca 720

aattttaact ctccaaaccc cccaccccct cctcttaatg ccaaacccca aaaacactaa 780

gaacttgaaa gacatataat attaactatc aaaccctatg tcctgatcaa ttctagtagt 840

tcccaaaata tgacttatat tttagtactt gtaaaaattt tacaaaatca tgttccgtga 900

accaaaactc taatcatact ctattacgca ataaacatt 969

<210> 2

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<212> DNA

<213> Chinese Kiwi berry (Macaca mulatta)

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ggaatccctt gttcaccatc ctccgtgaaa tcaatatccc gcacaagagt gctactctcc 180

tcgctccggg cccataactc gtgggggtag ctatacttga attgtatccg gcatctggtt 240

cttacctcag ggccatagca atcaagatcg cccacacgtt ccccttaaat aagacatctc 300

gatggatcac gggtctatca ccctattaac cagtcacggg aggtttccat gcatttggta 360

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atattctagc tccacgcaaa cttcagcaag gtgttattta attcatgctt gtaggacata 540

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cccaccccca tctccgacct tcatccaaac ccactcttgc caaaccccaa aaacaaaagt 660

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gaatattgta cggtaccata aatacttgac cacctgtagt acataaaaac ccaatccaca 180

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tcaactgcaa ctccaaagcc acccctcacc cactaggata ccaacaaacc tacccaccct 300

taacagtaca tagtacataa agccatttac cgtacatagc acattacagt caaatccctt 360

ctcgtcccca tggatgaccc ccctcagata ggggtccctt gaccaccatc ctccgtgaaa 420

tcaatatccc gcacaagagt gctactctcc tcgctccggg cccataacac ttgggggtag 480

ctaaagtgaa ctgtatccga catctggttc ctacttcagg gtcataaagc ctaaatagcc 540

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Claims

1. Use of a long non-coding RNA in any one of:

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

2. The use of the gene encoding the long non-coding RNA of claim 1 in any one of:

the coding gene is any one of the following:

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

3. Use of a substance that increases the content of the long non-coding RNA according to claim 1 or a substance that increases the expression of a gene that encodes the long non-coding RNA according to claim 2 in any one of:

4. The long non-coding RNA-associated biomaterial of claim 1, wherein: the biomaterial is any one of the following A1) -A4):

A1) an expression cassette comprising a gene encoding the long non-coding RNA of claim 2;

A2) a recombinant vector comprising a gene encoding the long non-coding RNA of claim 2 or the expression cassette of a 1);

A3) a recombinant microorganism prepared from the recombinant vector of a 2);

A4) a recombinant microorganism comprising the long non-coding RNA of claim 1, a gene encoding the long non-coding RNA of claim 2, A1) the expression cassette or A2) the recombinant vector.

5. The biomaterial of claim 4, wherein: A3) or A4) the recombinant microorganism is a recombinant adeno-associated virus or a recombinant lentivirus.

6. Use of the biomaterial of claim 4 or 5 in any one of the following:

7. A product characterized by: comprising the long non-coding RNA of claim 1, said product having at least one of the following functions:

8. The product of claim 7, wherein: the product is a medicament.

9. Use according to any one of claims 1-3, 6, the product according to claim 7, characterized in that: the symptom of the Alzheimer disease is improved by at least one of the following:

improving spatial cognitive ability;

reducing the content of A beta protein;

reducing phosphorylation levels of tau protein;

improving the morphology of mitochondria;

improving the autophagy function of mitochondria.

10. Use according to claim 9, the product according to claim 9, characterized in that: the improvement of the autophagy function of mitochondria is to increase the content of autophagy-related proteins of mitochondria.