CN114276418B - RNA (ribonucleic acid) for targeted therapy of microtubule regulation and demyelinating lesions - Google Patents

Abstract

The invention discloses TubAR RNA and application of corresponding DNA molecules thereof in promoting microtubule assembly and maintaining myelin sheath integrity, wherein the sequence of TubAR RNA is shown as SEQ ID NO:1, the sequence of the corresponding DNA molecule is shown as SEQ ID NO: 2. The invention has great application and popularization value for the research of microtubule assembly and the treatment of demyelinating diseases.

Description

RNA (ribonucleic acid) for targeted therapy of microtubule regulation and demyelinating lesions

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to an application of TubAR RNA in maintaining functions of microtubule assembly and treating demyelinating lesions.

Background

Microtubules are a hollow tubular framework structure that is ubiquitous in eukaryotic cells. Microtubules are one of the important components of the cytoskeleton. The cytoskeleton is mainly composed of three components of microtubules, intermediate filaments and microfilaments. Microtubules are the coarsest of the three cytoskeleton and consist mainly of alpha-tubulin and beta-tubulin heterodimer subunits. Microtubules play an important role in maintaining cell structure, mediating intracellular material transport, participating in cell division and the like.

Microtubule assembly includes 2 stages: nucleation and extension. Nucleation refers to the function of tubulin interactions to form the microtubule core, the initial stage of microtubule assembly. Within the cell, the nucleation of microtubules occurs in the microtubule tissue center. Intracellular microtubule nucleation usually relies on gamma-tubulin, in which 10-13 gamma-tubulin first polymerize to form a complex of about 25nm in diameter, on which the alpha-tubulin and beta-tubulin heterodimers assemble to produce a linear fibrillar structure, and 10-15 fibrils (typically 13 in mammalian cells) then polymerize to form a 24nm version of hollow cylindrical single tube structure. This process is known as microtubule extension. When the microtubule assembling speed is consistent with the dissociation speed, the microtubule length is kept unchanged, and the equilibrium state of the microtubule is reached.

The role of microtubules in the central nervous system (central nervous system, CNS) mainly includes participation in the formation of nerve cell axons and dendrites, mediating neurotransmitter transport, participation in myelination, etc. Myelin (myelination) formation is a dynamically active process in which myelin-forming cells proliferate and align along neuronal axons, which are then surrounded by cell membranes, thereby forming myelin. Myelin ensures a rapid and stable signaling process on axons. In the central nervous system, oligodendrocytes (oligodendrocyte) are the only myelin-forming cells. During myelination, 2 different microtubule structures are produced within oligodendrocytes, radial microtubules (radial microtubule) and lamellar microtubules (lamellar microtubule). The radial microtubules are proximal microtubules extending along the branching protrusions towards the axons; lamellar microtubules are distal microtubules that spiral around the myelin sheath, starting from the outermost layer of the myelin sheath and extending all the way to the innermost layer of the myelin sheath. Lamellar microtubules mediate the transport function of vesicles to the myelin lining. The stable microtubule tissue process in oligodendrocytes ensures myelin extension and processing stability and provides a transport pathway for proteins involved in myelination.

Disclosure of Invention

The invention aims at providing a novel application of an RNA molecule and a corresponding DNA molecule thereof; wherein the RNA molecule is obtained from a mouse (Mus musulus), designated TubAR RNA, having the sequence set forth in SEQ ID NO:1 is shown in the specification; the sequence of the DNA molecule is shown in SEQ ID NO: 2.

In particular, the inventors have found that TubAR RNA can bind to tubulin TUBA1A and TUBB4A, promoting the interaction of TUBA1A-TUBB4A, i.e. promoting the assembly of tubulin dimers consisting of TUBA1A and TUBB4A, thereby promoting the microtubule assembly process. In vitro experiments, tubAR RNA microtubules were added with a higher polymerization rate than the control group without RNA (Mock Ctrl) and the treated group with negative control RNA (TubARAS).

In addition, the inventors have found that TubAR RNA can also bind to other isoforms of tubulin, thereby facilitating assembly of tubulin dimers. Thus, the inventors have found TubAR RNA to promote microtubule assembly.

Since microtubule structures play a very important role in myelination and maintenance of myelination, tubulin abnormalities have been reported to cause myelination in patients. On this basis, the inventors concluded that TubAR RNA could also be used to maintain the integrity of myelin (as demonstrated by the instructions, e.g., example 3), and that TubAR RNA could be used to treat demyelinating diseases, as demyelinating diseases manifest in the damage and destruction of myelin.

Specifically, the invention provides the following technical scheme:

1. SEQ ID NO:1 or the RAN molecule shown in SEQ ID NO:2 in promoting microtubule assembly.

2. The use of item 1, wherein the RNA molecule or DNA molecule binds to tubulin, thereby promoting microtubule assembly.

3. A method of promoting microtubule assembly comprising inserting SEQ ID NO:1, or the RNA molecule shown in SEQ ID NO:2, or for increasing and/or promoting the DNA of SEQ ID NO:1 or the RNA molecule shown in SEQ ID NO:2 and/or the expressed substance is introduced into the cell.

4. A method of inhibiting microtubule assembly comprising reducing and/or silencing SEQ ID NO:1 or the RNA molecule shown in SEQ ID NO:2 and/or the expressed substance is introduced into the cell.

5. The method of item 4, wherein the method for reducing and/or silencing SEQ ID NO:1 or the RNA molecule shown in SEQ ID NO:2 is siRNA or shRNA; preferably, the siRNA has a sequence as shown in SEQ ID NO:3 or SEQ ID NO:4 is shown in the figure; preferably, the shRNA has a sequence as set forth in SEQ ID NO: 5-8.

6. SEQ ID NO:1 or SEQ ID NO:2 in maintaining myelin sheath integrity.

7. SEQ ID NO:1 or SEQ ID NO:2 in the preparation of a medicament for the treatment of demyelinating diseases.

8. The use of clause 7, wherein the demyelinating disease comprises hypomyelination with basal ganglia and cerebellum; autosomal dominant torsionally dystonia type-4; or isolated hypomyelination.

9. A method of maintaining myelin integrity comprising inserting SEQ ID NO:1, or the RNA molecule shown in SEQ ID NO:2, or for increasing and/or promoting the DNA of SEQ ID NO:1 or the RNA molecule shown in SEQ ID NO:2 and/or the expressed substance is introduced into the cell.

10. A method of reducing myelin integrity comprising reducing and/or silencing SEQ ID NO:1 or the RNA molecule shown in SEQ ID NO:2 and/or the expressed substance and/or the level of the DNA molecule shown in 2;

Optionally, wherein the agent is used to reduce and/or silence SEQ ID NO:1 or the RNA molecule shown in SEQ ID NO:2 is siRNA or shRNA; preferably, the siRNA has a sequence as shown in SEQ ID NO:3 or SEQ ID NO:4 is shown in the figure; preferably, the shRNA has a sequence as set forth in SEQ ID NO: 5-8.

It is known to those skilled in the art that tubulin comprises a plurality of subtypes, for example, 5 subtypes of α -tubulin, TUBA1A, TUBA1B, TUBA1C, TUBA4A, TUBA and 8 subtypes of β -tubulin, TUBB2A, TUBB2B, TUBB3, TUBB4A, TUBB4B, TUBB, TUBB6, the amino acid sequences of each of which are available from GenBank. In addition to the TubAR RNA shown in the examples of the present invention being able to bind tubulin TUBA1A and TUBB4A to facilitate microtubule assembly, the present inventors have also verified that TubAR RNA is able to bind at least the three subtypes TUBA4A, TUBB5 and TUBB6 to facilitate microtubule assembly. Based on the above results, it can be inferred that TubAR RNA can bind to other isoforms of tubulin to promote microtubule assembly.

In the present invention, by inhibiting the sequence of SEQ ID NO:1 to verify its effect on microtubule assembly and myelin sheath integrity; wherein the substance for reducing the level of the RNA molecule can be obtained by a conventional technique in the art, such as a specific siRNA or a specific shRNA, etc. For the present invention SEQ ID NO:1, the specific siRNA of the RNA molecule can be specifically shown as SEQ ID NO:3 or SEQ ID NO:4, the specific shRNA can be specifically shown as SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO: shown at 8. Similarly, in the present invention, the amino acid sequence used to reduce the amino acid sequence of SEQ ID NO:2 can also be obtained by techniques conventional in the art.

In embodiments of the present invention, the term "demyelinating disease" refers to a disease in which the axon is relatively intact with myelin damage, such as loss or thinning. The pathological change is demyelination of nerve fibers while nerve cells remain relatively intact, so that the transmission of nerve impulses is affected. Common symptoms are multiple sclerosis, acute inflammatory demyelinating polyneuropathy, acute disseminated encephalomyelitis, and the like. The invention has great application and popularization value for the research of microtubule assembly and the treatment of demyelinating lesions.

Drawings

FIG. 1 shows the results of the expression level TubAR RNA in step two of example 1.

FIG. 2 shows the results of the detection of tubulin dimer binding in step three of example 1.

FIG. 3 shows the result of microtubule extension in recombinant cells in step four of example 1.

FIG. 4 shows the results of in vitro microtubule assembly in example 2.

FIG. 5 shows the results of the myelin sheath structure of mice in step two of example 3; wherein shTubAR ^AA V refers to the result obtained by injecting an AAV virus into mice by coating a sequence capable of knockdown TubAR, and SHCTRL AAV refers to the result obtained by injecting a negative control shRNA sequence coated to produce an AAV virus into mice.

FIG. 6 is a map of the mCherry-EB3 plasmid used in step four of example 1.

Detailed Description

The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The test methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

PcDNA3.1 (+) vector: company addgene, U.S.

The plko.1 vector, the pRev plasmid, pGag/pol plasmid, pVSVG plasmid are all described in ：Wang J,Qiao M,He Q,et al.Pluripotency Activity ofNanog Requires Biochemical Stabilization by Variant Histone Protein H2A.Z[J].Stem Cells,2015,33(7)：2126-2134.., wherein plko.1 vector is a lentivirus shRNA expression vector and the remaining three plasmids are packaging plasmids. The plko.1 vector and the three packaging plasmids are used for transfecting cells together to obtain the lentivirus for expressing the target shRNA.

TubAR RNA is shown as a sequence 1 in a sequence table. In the genome DNA, the DNA corresponding to TubAR RNA is shown as a sequence 2 in a sequence table.

Example 1 Effect of TubAR RNA expression level on microtubule dimer binding and microtubule extension in neuroblastoma cell lines

1. Recombinant cell for inhibiting TubAR RNA expression and preparation of control recombinant cell thereof

The control siCtrl (purchased from ribobio) or the small interference RAN siTubAR of TubAR RNA (purchased from ribobio) was transfected into N2a cells (neuroblastoma cell line, commercially available, for example, from ATCC (CCL-131)) by means of lipofectamine 3000 (purchased from invitrogen) according to conventional methods, the transfection was followed by a transfer of the liquid, after which the cells were collected and designated recombinant cell I (transfected with siCtrl) and recombinant cell II (transfected with siTubAR), respectively.

2. Detection of TubAR RNA expression level

The test cells were recombinant cell I (transfected with siCtrl) and recombinant cell II (transfected with siTubAR) obtained in step one.

1. Total RNA of the test cells was extracted.

2. And (3) carrying out reverse transcription by taking the total RNA extracted in the step (1) as a template to obtain cDNA.

The template of the reaction system (20μl)：10μl 2×RT Mix、1μl Random Primers、1μl Oligo dT₂₃VN、2μl HiScript^RII Enzyme Mix,1μg RNA was made up to 20. Mu.l with RNase-free water. In the reaction system, the RNA template and RNase-free water were used as the components of HISCRIPT II One Step RT-PCR reverse transcription kit (China Vazyme Co.).

The reaction procedure: 25 ℃ for 5min,50 ℃ for 15min and 85 ℃ for 10min.

3. And (3) detecting the expression quantity of TubAR RNA by using the cDNA obtained in the step (2) as a template and GAPDH MRNA as an internal reference gene through fluorescent quantitative PCR.

Reaction System (10. Mu.l) 2.5. Mu.l RNase-free water, 5. Mu.l 2 XqPCR SYBR Green, 0.25. Mu.l forward primer, 0.25. Mu.l reverse primer, 2. Mu.l reaction product of step 2 (approximately 1. Mu.g cDNA). In the reaction system, F1 was at a concentration of 0.25. Mu.M, and R1 was at a concentration of 0.25. Mu.M.

The reaction procedure: 95 ℃ for 10min,1 cycle; 95℃for 15s, 60℃for 60s,40 cycles.

The primer pair for detection TubAR RNA consisted of F1 and R1.

The primer pair for detecting the reference gene consists of F2 and R2.

F1 (forward primer): 5'-GAGCAGGTAAGTGGCTTGGT-3' (SEQ ID NO: 9);

r1 (reverse primer): 5'-TTTGCTTGGGCTCTCACCTC-3' (SEQ ID NO: 10).

F2 (forward primer): 5'-CATGGCCTTCCGTGTTCCT-3' (SEQ ID NO: 11);

r2 (reverse primer): 5'-TGATGTCATCATACTTGGCAGGTT-3' (SEQ ID NO: 12).

TubAR RNA relative expression = expression of recombinant cells II TubAR RNA/expression of TubAR RNA in recombinant cells I. The detection results are shown in FIG. 1.

As can be seen from the results of fig. 1, the relative expression of TubAR RNA in recombinant cell II transfected with siTubAR was lower than 0.5, i.e., the efficiency of TubAR RNA interference in Neuro-2a cells (i.e., N2a cells) of TubAR siRNA used herein was 50% or more, compared to recombinant cell I transfected with siCtrl.

3. Detection of tubulin dimer binding in recombinant cells

The binding of tubulin dimer is detected by means of co-immunoprecipitation, which is a conventional technique, and the specific procedure is as follows:

1. Collecting the recombinant cells I and II obtained in the first step, and performing lysis by using NP-40 lysate, wherein protease inhibitors cocktail (purchased from Roche) and PMSF (purchased from Bio-Inc.) are added into the lysate when in use;

2. centrifuging the cell lysate at 12000rpm for 20min, and collecting supernatant;

3. To remove non-specific binding reactions of proteins to agarose beads in the experiment, therefore, a small amount of agarose beads are generally used to pre-wash the lysate, and after incubation is completed, the supernatant is collected by centrifugation at 12000rpm for 20 min;

4. Mouse anti-GFP antibody (purchased from proteintech company) was added to agarose beads and incubated to bind the antibody to protein A/G agarose beads;

5. adding the supernatant obtained in the step 3 into the antibody-agarose bead mixture obtained in the step 4, and selecting proper incubation temperature (4 ℃ or normal temperature) and incubation time (4-12 h) according to the characteristics of the protein to be detected;

6. Washing agarose beads for more than 6 times by using NP-40 lysate after incubation is completed, and placing a centrifuge tube on a rotating frame for rotating for 5mmin after adding the NP-40 lysate each time so as to thoroughly remove the background;

7. after the washing is completed, loading buffer is added into the centrifuge tube, and the centrifuge tube is placed in a metal bath at 100 ℃ for 10min;

8. After completion, the mixture was centrifuged at 12000rpm for 10min, and the supernatant was collected and stored at-20℃or directly subjected to western blot for detection. The detection results are shown in FIG. 2.

From the analysis of the results of FIG. 2, it was found that TUBA1A binds to GFP-TUBB4A in the Neuro-2a cell line after interfering TubAR with expression using siRNA.

4. Detection of microtubule extension in recombinant cells

1. With the aid of lipofectamine 3000, mCherry-EB3 plasmids (map see fig. 6) and siCtrl or siTubAR are transfected into N2a cells (neuroblastoma cell line), the liquid is changed during transfection, and the culture is continued for 48h after transfection;

2. EB3 fluorescence (Delta Vision microscope, GE HEALTHCARE) was collected 48 hours after transfection using a living cell workstation. Every 4 seconds, a total of 100 photographs were taken for each cell sample.

3. EB3 movement velocity was calculated using ImageJ software, completing statistics. The detection result is shown in FIG. 3.

From the results of FIG. 3, it can be seen that mCherry-EB3 plasmid was transfected into the Neuro-2a cell line, while siRNA interference TubAR was used. Since mCherry-EB3 fusion proteins are able to specifically bind microtubule ends, mCherry-EB3 localization can be used to identify microtubule extension. From the results in the figure, the decreased EB3 particles movement rate in recombinant cell II transfected with siTubAR compared to recombinant cell I transfected with siCtrl, showed a decrease in microtubule extension rate after interfering TubAR RNA with siRNA.

Example 2, tubAR RNA effect on microtubule assembly in vitro.

In vitro microtubule assembly is a common assay that detects the effect of a protein or small molecule compound on microtubule assembly. The test kit is derived from Cytoskeleton, and the test operation is performed in strict compliance with the operation instructions.

1. Opening the microplate reader, setting a program (emission wavelength is 360nm, excitation wavelength is 420nm, constant temperature is 37 ℃, starting the front vibrating plate for 1min, detecting 1 time per minute, and detecting 200min altogether);

2. placing a 96-hole opaque blackboard for detection in an instrument, and preheating for 10min at 37 ℃;

3. thawing paclitaxel, and diluting to 30 μm for use;

taking out the Tubulin solution from the refrigerator at the temperature of-80 ℃, rapidly placing the solution in a normal-temperature water bath kettle for thawing, and immediately placing the solution on ice after thawing;

5.Buffer 1,GTP stock,tubulin glycerol buffer taking out and thawing;

6. Microtube assembly reaction solution (Buffer 1 243. Mu.l, tubulin glycerol Buffer. Mu.l, GTP 4.4. Mu.l, tubulin 85. Mu.l) was prepared, and the solution was quickly placed on ice after completion of the preparation;

7. adding 50 μl of microtube assembly reaction solution into a 96-well plate, and placing the well plate into an enzyme-labeling instrument for preheating for 1min;

8. Taking out the 96-well plate, rapidly adding 5 μl of the molecular solution to be detected (positive control taxol, tubAR RNA, blank control H2O and negative control TubAR AS respectively), mixing uniformly, and placing into an enzyme-labeling instrument for starting the procedure;

9. If the detection program is abnormal within 10 minutes, the detection can be restarted;

10. After the detection is completed, the data is exported, and then is further analyzed and processed by GRAPHPAD PRISM software.

The detection results are shown in FIG. 4. As can be seen from the results of FIG. 4, the addition of TubAR RNA to the in vitro microtubule assembly solution increased the microtubule assembly rate, whereas the addition of negative controls TubARAS RNA or H2O did not.

Example 3, tubAR RNA effects of loss of mouse brain sheath structure.

1. Construction of TubAR RNA deletion mouse model

1. The method comprises the steps of 1, carrying out anesthesia treatment on a mouse by using 0.5% pentobarbital sodium solution through an intraperitoneal injection mode, and fixing the mouse to be injected on a stereotactic instrument after anesthesia is finished;

2. A microinjection pump was used to inject 0.5. Mu.l of packaged AAV (i.e., adeno-associated virus AAV, obtained by coating a shRNA sequence capable of specifically knocking down TubAR (any of SEQ ID NOs: 5-8), purchased from Shanghai and metazoan) on both sides of a mouse brain (A/P: -7.1mm, M/L: +/-1.0mm, D/V: -3.0 mm) or secondary motor cortex region (A/P: +1.3mm, M/L: +/-0.7mm, D/V: -0.8 mm), respectively, at a rate of 50nl/min;

3. after injection is completed, the mice are properly arranged, water and food are timely supplemented, and the states of the mice are detected at regular time;

4. After injection is completed for 1-2 months, the mice recover well, namely TubAR RNA deletion mice model is completed, and related experiments can be carried out.

2. TubAR RNA-deleted mouse myelin sheath structure

All Magnetic Resonance Imaging (MRI) experiments were performed using a 7.0Tesla 20-cm horizontal bore MR spectrometer (BRUKER BioSpec 70/20 USR), a 20cm (diameter) birdcage coil, and a 20mm (diameter) surface coil to transmit or receive nuclear magnetic resonance signals. The mice were placed in a prone position on a specially designed cradle and placed in a magnet and secured with two ear bars and one dental bar. Mice were anesthetized with isoflurane during the scan (4% injection, 1.0% -1.2% air/02 7:3 maintenance). T2-weighted MR images were obtained through the sagittal plane using a fast acquisition with a relaxation enhancement (RARE) sequence with a repetition Time (TR) of 3000ms, echo interval = 22.5ms, RARE factor = 4; furthermore, local proton imaging was obtained from the cerebellum region outlined on the T2 weighted image using the following parameters: effective echo Time (TE) =45 ms, field of view (FOV) =20×20mm ², matrix size=256×256, slice thickness=0.6 millimeters (15 slices, gap=0), bandwidth (BW) =50 kHz, average=4. The detection results are shown in FIG. 5.

As can be seen from the results of fig. 5, in TubAR knock-down mice (i.e., shTubAR ^AV V), the white matter portion of the cerebellum showed abnormal signals, and demyelination occurred, so that the loss of TubAR could lead to demyelination lesions.

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims (3)

1. A method for promoting microtubule assembly comprising introducing into a cell an RNA molecule as set forth in SEQ ID No. 1, or a DNA molecule as set forth in SEQ ID No. 2, or a substance for increasing and/or promoting the level and/or expression of an RNA molecule as set forth in SEQ ID No. 1 or a DNA molecule as set forth in SEQ ID No. 2, said method being for non-therapeutic purposes.

2. A method of inhibiting microtubule assembly to construct a demyelinating model comprising introducing into a cell a substance for reducing and/or silencing the level and/or expression of an RNA molecule represented by SEQ ID No. 1 or a DNA molecule represented by SEQ ID No. 2;

Wherein the substance for reducing and/or silencing the level and/or expression of the RNA molecule shown in SEQ ID NO.1 or the DNA molecule shown in SEQ ID NO. 2 is shRNA;

Wherein the sequence of the shRNA is shown in any one of SEQ ID NO 5-8.

3. A method of reducing myelin integrity to construct a demyelination model comprising introducing into a cell a substance for reducing and/or silencing the level and/or expression of an RNA molecule represented by SEQ ID No.1 or a DNA molecule represented by SEQ ID No. 2;

Wherein the substance for reducing and/or silencing the level and/or expression of the RNA molecule shown in SEQ ID NO.1 or the DNA molecule shown in SEQ ID NO. 2 is shRNA;

Wherein the sequence of the shRNA is shown in any one of SEQ ID NO 5-8.