Regulation and control target for treating axonal injury, medicine and application of lncRNAs
阅读说明：本技术 治疗神经轴突损伤的调控靶点及药物、lncRNAs的应用 (Regulation and control target for treating axonal injury, medicine and application of lncRNAs ) 是由 王东 郑铁妹 陈媛媛 于彬 于 2021-08-20 设计创作，主要内容包括：本发明提供一种治疗神经轴突损伤的调控靶点,涉及生物医学技术领域,包括一lncRNAs及调控下游的PI3K-Akt信号通路,所述lncRNAs为SEQ ID NO.1所示的RNA。(The invention provides a regulation and control target for treating axonal injury of nerves, relates to the technical field of biomedicine, and comprises lncRNAs and a PI3K-Akt signal channel for regulating and controlling the downstream, wherein the lncRNAs are RNAs shown in SEQ ID NO. 1.)
1. A modulatory target for treating axonal injury, comprising: comprises lncRNAs and a PI3K-Akt signal passage for regulating and controlling the downstream, wherein the lncRNAs are RNAs shown in SEQ ID NO. 1.
2. A medicament for treating axonal damage, comprising: comprises lncRNAs and an activator for regulating and controlling a downstream PI3K-Akt signal pathway, wherein the lncRNAs are RNAs shown in SEQ ID NO. 1.
3. A medicament for treating axonal damage according to claim 2, wherein: the activator comprises IGF-1 or SC 79.
4. An application of lncRNAs, wherein the lncRNAs are RNAs shown in SEQ ID NO.1, and the application is characterized in that: the lncRNAs are used as molecular targets to regulate and control the regeneration of nerve axons after peripheral and central nerve injuries.
Technical Field
The invention relates to the technical field of biomedicine, in particular to a regulation target for treating axonal injury, a medicament and application of lncRNAs.
Background
At present, the treatment means after nerve injury mainly depends on the transplantation of local nerves by the traditional microsurgery or simultaneously uses tissue engineering regeneration materials, but the treatment means cannot achieve good curative effect in patients with long-distance damage of peripheral nerves or central nerve damage. The inherent regeneration capacity of the neuron is a key factor for repairing nerve injury, and the research on key molecular targets and mechanisms for controlling the nerve injury is beneficial to developing new treatment means and strategies.
Disclosure of Invention
The purpose of the invention is to solve the problem that the nerve therapy of the prior art cannot achieve good curative effect in patients with long-distance damage of peripheral nerves or central nerve damage.
In order to achieve the purpose, the invention adopts the following technical scheme:
a regulation target for treating axonal injury comprises lncRNAs and a PI3K-Akt signal channel which regulates and controls the downstream, wherein the lncRNAs are RNAs shown in SEQ ID NO. 1.
The application also provides a medicine for treating axonal injury, which comprises lncRNAs and an activator for regulating and controlling a downstream PI3K-Akt signal pathway, wherein the lncRNAs are RNAs shown in SEQ ID NO. 1.
Preferably, the activator comprises IGF-1 or SC 79.
The application also provides application of lncRNAs, wherein the lncRNAs are RNAs shown in SEQ ID NO.1, and the lncRNAs are used as molecular targets to regulate and control the regeneration of nerve axons after peripheral and central nerve injuries.
The regulation target for treating the axonal injury proves that the regeneration and repair of the axonal injury of the neuron can be promoted by regulating the expression of ENSMUST00000156081 and the downstream PI3K-Akt signal channel, and development of a new treatment means and strategy is facilitated.
Drawings
FIG. 1 is a diagram of data showing the expression of ENSMUST00000156081 and the effect of DRG neuron axon growth according to the present invention;
FIG. 2 is a schematic diagram of data showing the effect of the expression of the interference ENSMUST00000156081 on axon regeneration after sciatic nerve injury according to the present invention;
FIG. 3 is a schematic diagram of data of the invention, wherein ENSMUST00000156081 regulates the activation of PI3K-Akt signaling pathway in neuronal axon regeneration;
FIG. 4 is a diagram of data showing the effect of ENSMUST00000156081 of the present invention on the axonal growth of embryonic cortical neurons.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments.
Some nouns in the examples are paraphrased:
lncRNAs: long non-coding RNA
And (3) PCR reaction: polymerase chain reaction
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments of the present disclosure.
A regulatory target for the treatment of axonal injury, comprising: comprises lncRNAs and a PI3K-Akt signal passage for regulating and controlling the downstream, wherein the lncRNAs are RNAs shown in SEQ ID NO. 1. The sequence number of the RNA shown in SEQ ID NO.1 is RNA with the number of IncRNAs being ENSMUST 00000156081.
The present application also provides a medicament for treating axonal damage, characterized in that: comprises lncRNAs and an activator for regulating and controlling a downstream PI3K-Akt signal pathway, wherein the lncRNAs are RNAs shown in SEQ ID NO. 1. In one embodiment, the activator comprises IGF-1 or SC 79.
The application also provides application of lncRNAs, wherein the lncRNAs are RNAs shown in SEQ ID NO.1, and the lncRNAs are used as molecular targets to regulate and control the regeneration of nerve axons after peripheral and central nerve injuries.
The following are validation experiments of the present application:
example 1: examine the expression of ensust 00000156081 and the effect on DRG neuron axon growth:
s1: DRG sample RNA extraction and fluorescent real-time quantitative PCR reaction (qRT-PCR)
After preparing a mouse sciatic nerve clamp injury model, taking L4-L5 Dorsal Root Ganglion (DRG) tissues at the sciatic nerve clamp injury sides of 0h, 3h, 9h, 1d and 4d after operation, and extracting tissue RNA by a TRIZOL method.
RNA reverse transcription to synthesize cDNA: genomic DNA was digested using the Takara RR047A reverse transcription kit and 1. mu.g of RNA was inverted to cDNA. In the QRT-PCR experiment, the GAPDH gene is used as an internal reference and 2-3 compound holes are made. And (3) removing data with larger error, non-single dissolution curve and abnormal amplification curve from the subsequent analysis result, and performing analysis statistics by using a 2-delta CT method. The qRT-PCR results are shown as a graph showing that expression of ENSMUST00000156081 in DRG after sciatic nerve injury gradually recovered after a rapid increase at 3h compared to 0 d.
S2: effect of ensust 00000156081 on DRG neuron axon growth:
(1) and (3) separating and culturing DRG neurons:
placing the prepared dissecting liquid into a small dish, adding antibiotics, and placing on ice for precooling. Mice were anesthetized with abdominal cavity, the skin was cut open from the tail along the spine towards the head with surgical scissors, the entire spine was carefully removed, the vertebral plate was opened from the neck, and all DRG tissues were pulled out with micro-forceps and placed in the dissecting fluid. All DRG tissues were removed and the dissecting solution was discarded, the tissues were rinsed 2 times with cell-grade PBS, and excess tissue and blood traces were washed away. PBS was discarded, 2ml collagenase (3mg/ml) was added and the tissue and digest were transferred to a 5ml centrifuge tube. The tissue is cut into pieces by micro-scissors, and then placed in a cell culture box for digestion for 90 min. Centrifuging to remove collagenase, adding 1ml pancreatin digestive juice, blowing for about 1min with a gun until the tissue is dispersed uniformly, then placing into a cell culture box for digestion for 10min, taking out every 5min, blowing uniformly, and then placing back into the culture box. When the tissue is digested to no obvious tissue block, 3ml of digestion stop solution is added into the centrifuge tube to stop the digestion. Blow with gun for about 1min, then pass through screen (70 μm), filter off excess tissue and collect cell suspension into new 5ml centrifuge tube, 1200rpm × 5min, discard supernatant. 4ml of pre-warmed BSA solution was added to the centrifuge tube, the cells were resuspended, centrifuged at 900rpm for 5min, and the floating contaminating cells were carefully aspirated away. The above steps are repeated. Adding pre-warmed neuron cultureThe cells are blown and evenly inoculated into a cell culture plate pre-coated with polylysine, and 5 percent CO is added after the cells are evenly mixed in a cross way2And culturing in an incubator at 37 ℃.
(2) In vitro transfection of DRG neuronal siRNA:
after the DRG neuron cells are inoculated to the culture plate, the specific siRNA (Si 1 and Si 2) of the control siRNA (NC) and ENSMUST00000156081 and liposome transfection mixed liquor are added at the same time, and the mixture is placed in an incubator to be cultured for about 12 after being gently shaken. And replacing a fresh neuron culture medium, transfecting for 48 hours, then resuspending by using 0.025% pancreatin digested cells, re-inoculating into a 24-hole culture plate, and collecting cells within 12-18 hours after inoculation.
(3) In vitro neuronal axon growth experiments:
the cell culture medium was discarded and the cells were rinsed with 1 × PBS after preheating. Discard 1 XPBS, add pre-cooled 4% paraformaldehyde and fix on ice for 20 min. After discarding formaldehyde, the column was rinsed with 1 XPBS for 3 times at room temperature for 5 min. After the washing, the PBS was discarded, and the blocking solution was gently added dropwise to 200. mu.l/well, and allowed to stand at room temperature for 40 min. The blocking solution was discarded, and Anti-. beta. -Tublin III antibody (1:1000) diluted with primary antibody was gently added dropwise thereto in a volume of 200. mu.l per well, and incubated at 4 ℃ overnight with standing. The primary antibody was discarded, rinsed with 1 XPBS, and washed 3 times 5 min/time at room temperature. PBS was discarded, and Alexa fluor 647 goat anti-rabbitt (1:1000) diluted with a secondary antibody diluent was gently added dropwise thereto at 200. mu.l per well, followed by incubation at room temperature for 2 hours. The process is protected from light. The secondary antibody was discarded, rinsed with 1 XPBS, and washed 3 times 5 min/time at room temperature. The process is protected from light. After the washing, the round glass slide is taken out of the hole, the side with the cells faces downwards is covered on the glass slide on which the mounting liquid is dripped, and the glass slide is placed in a wet box and stored at 4 ℃. Care was taken not to generate bubbles in the process. The process is protected from light.
Referring to fig. 1, fig. 1A verifies that the expression change of ensust 00000156081 (GAPDH is an internal reference) in DRG tissues in sciatic nerve injury model by qRT-PCR, and the expression of ensust 00000156081 gradually decreases after nerve injury; figure 1b. neuronal axonal staining assay to examine the effect of interfering with ENSMUST00000156081 on neuronal axonal growth. FIG. 1C is a statistical plot of the longest axons of neurons. It can be seen in figure 1 that the growth of neuronal axons is significantly inhibited following the intervention of ENSMUST 00000156081.
Example 2: interference with the effect of expression of ENSMUST00000156081 on axon regeneration after sciatic nerve injury (sciatic nerve axon growth experiment in vivo):
(1) DRG in vivo injection of siRNA:
experimental 8-week-old male C57BL/6 mice were provided by the experimental animal center. Before operation, abdominal cavity anesthesia is firstly carried out, then the hair around the ilium on the back is disinfected, then the skin is exposed by a surgical scissors, muscle tissues are cut along the ilium by an ophthalmic scissors, the protuberant spinous process is cut off by a bone-changing scissors, L4 and L5 DRG tissues are found and exposed upwards along the sciatic nerve, and specific siRNA (Si 1 and Si 2) packaged with siRNA (including control siRNA (NC) and ENSMUST 00000156081) is injected into DRG at a constant speed by a microsyringe.
(2) Sciatic nerve clamp injury:
sciatic nerve clamp injury was performed 2 days after DRG injection. First, abdominal anesthesia is performed, and then the left hind limb peritrichous is disinfected. Exposing skin with surgical scissors, separating muscle and basement membrane covering sciatic nerve with ophthalmic scissors, clamping sciatic nerve proximal end with clamping wound forceps 2mm wide, clamping wound for 20s, withdrawing sciatic nerve under muscle after clamping wound, and suturing wound. Care was taken after surgery.
(3) Tissue perfusion and dehydration:
and taking sciatic nerve tissue after 3d of pinching. The now prepared 4% formaldehyde solution is placed on ice for precooling for standby. Firstly, performing abdominal anesthesia, fixing an animal, cutting off abdominal skin in the direction of the heart by using surgical scissors, cutting off the abdominal skin along the edge after the diaphragm is exposed, exposing the heart, peeling off adipose tissues covered on the heart to expose the artery, inserting a needle tube into the artery from the apex of the heart, starting normal saline perfusion, cutting off the right auricle by using ophthalmic scissors, opening formaldehyde perfusion after all blood in the body is replaced by the normal saline, and observing that the four limbs of a rat are stiff and can perform material taking operation. Note that the pre-exposure procedure cannot cut into the viscera or otherwise affect perfusion.
(4) Sciatic nerve immunochemical staining:
soaking the tissue in 4% formaldehyde solution for 8 hr, removing formaldehyde solution, washing with 1 × PBS to remove residual formaldehyde, and dewatering with 30% sucrose solution. Observing that the tissue sinks in the sucrose solution, namely finishing dehydration, taking out the tissue, placing the tissue under a dissecting mirror, trimming redundant muscle tissue by using micro forceps, carefully straightening, placing the tissue on a freezing table attached with the sucrose solution, adding the sucrose solution for quick freezing, and storing the tissue after the freezing table is manufactured at-20 ℃ for processing slices. Before slicing, a slide coated with PLL is prepared, and slicing can be started after the thickness of the slice is set according to experiment needs. After the slicing is finished, the slices are placed in a 50 ℃ oven to be dried for 2h, and the slices are sealed after being dried and can be stored at minus 80 ℃. Taking out the tissue section from-80 ℃ 1h before staining, and placing the tissue section in room temperature for rewarming for 1 h. Selecting tissues without wrinkles, complete shape and bubbles, putting the tissues into a washing tank containing 1 XPBS, and placing the tissues on a shaking table to wash for 15min at a low speed. Taking out the section from the washing tank, wiping off water stain outside the tissue, separating the tissue by using a circle of a tissue forming pen, adding a proper amount of immunostaining sealing liquid into the circle, and standing for 1h at room temperature in a wet box. Before adding the sealing liquid, the tissue is kept moist as much as possible, otherwise, the dyeing effect is influenced. The blocking solution was discarded, the tissue edges were wiped off of water, SCG10 (1:400) diluted with primary anti-diluent was added dropwise to the ring, and the mixture was left to stand overnight in a wet box at 4 ℃. The next day, the wet box was left to stand at room temperature for 1 h. Primary antibody was discarded and the sections were placed in a wash tank containing 1 XPBS and washed 3 times 15 min/time on a shaker at low speed. The section is taken out from the washing tank, water stain outside the tissue is wiped dry, Alexa fluor 647 coat anti-rabbitt (1:1000) diluted by secondary antibody diluent is dripped into the combined ring under the condition of keeping out of the light, and the combined ring is placed in a light-proof wet box for incubation for 2 hours at room temperature. The secondary antibody was discarded and the sections were placed in a wash tank containing 1 × PBS and washed 3 times 15 min/time on a shaker at low speed. Taking out the slices from the washing tank under the condition of keeping out of the light, wiping water stain outside the tissues, dripping fluorescent sealing liquid into the grouping ring, sealing the slices by using a special cover glass, and storing the slices in a wet box without generating bubbles in the process.
Referring to figure 2, figure 2a. sciatic nerve axon staining experiment in vivo the effect of interference ENSMUST00000156081 on sciatic nerve axon growth was examined. FIG. 2B is a statistical plot of the longest length of sciatic nerve axons; it can be seen from figure 2 that the inhibition of the sciatic nerve axon growth following the intervention of ENSMUST00000156081 significantly inhibited.
Example 3: the effect of activation of the PI3K-Akt signaling pathway in ENSMUST00000156081 in regulating neuronal axon regeneration:
s1: screening of downstream targets of ENSMUST 00000156081:
total RNA was extracted from the interfering DRG neurons (Si 1 and Si 2) and from the control (NC) and used for transcriptome sequencing. GO pathway analysis is carried out on the gene with differential expression, and the gene is found to mainly affect a PI3K-Akt signal pathway. qRT-PCR verification is carried out on the 4 differentially expressed genes in the channel, and the results show that the expression of ENSMUST00000156081 in DRG is interfered, and the expression of 4 genes (Fgf 2, Tlr4, Itgav and Tnxb) in the PI3K-Akt signal channel is obviously reduced.
S2: effect of ensust 00000156081 on PI3K-Akt signaling pathway in DRG neurons:
western Blot experiments were performed after total protein extraction on DRG neurons (Si 1 and Si 2) interfering with ENSMUST00000156081 expression and control (NC), and the results are shown in the figure, where Akt phosphorylation levels in DRG after interference with ENSMUST00000156081 were significantly reduced compared to control.
S3: the effect of activation of the PI3K-Akt signaling pathway in ENSMUST00000156081 in regulating neuronal axon regeneration:
after the DRG neuron cells are inoculated to the culture plate, the specific siRNA (Si 2) of the control siRNA (NC) and ENSMUST00000156081 and liposome transfection mixed solution are added at the same time, and the mixture is placed in an incubator to be cultured for about 12 after being gently shaken. Replacing a fresh neuron culture medium, after transfection for 48 hours, using 0.025% pancreatin to resuspend the cells, re-inoculating the cells into a 24-well culture plate, adding IGF-1 and SC79, and culturing for 12-18 hours for collecting the cells for staining.
Please refer to fig. 3, fig. 3a is a schematic diagram of the expression of the PI3K-Akt signaling pathway target gene after detecting the enb ust00000156081 in the interfering neuron by qRT-PCR. FIG. 3B is a graph showing the phosphorylation levels of Akt after detection of ENSMUST00000156081 in interfering neurons by Western Blot. FIG. 3C is a schematic diagram of a neuron axon staining experiment for detecting the effect of activation of PI3K-Akt signaling pathway on interfering with the regulation of neuron axon growth by ENSMUST 00000156081. FIG. 3D is a statistical plot of the longest axons of neurons. It can be seen that activation of the PI3K-Akt signaling pathway partially abolishes the inhibitory effect on neuronal axon growth following the intervention of ENSMUST 00000156081.
Example 4: effect of ENSMUST00000156081 on the growth of axons in embryonic cerebral cortical neurons
S1: separating and culturing embryonic cerebral cortex neurons:
placing the prepared dissecting liquid into a small dish, adding antibiotics, and placing on ice for precooling. And (3) taking out the E16 mouse after abdominal anesthesia, cutting off the brain by using surgical scissors, taking out the cerebral cortex tissue, placing the cerebral cortex tissue in an anatomical solution, rinsing the tissue for 2 times by using cell-grade PBS, and washing off redundant tissue and blood stains. PBS was discarded, 2ml collagenase (3mg/ml) was added and the tissue and digest were transferred to a 5ml centrifuge tube. The tissue is cut into pieces by micro-scissors, and then placed in a cell culture box for digestion for 90 min. Centrifuging to remove collagenase, adding 1ml pancreatin digestive juice, blowing for about 1min with a gun until the tissue is dispersed uniformly, then placing into a cell culture box for digestion for 10min, taking out every 5min, blowing uniformly, and then placing back into the culture box. When the tissue is digested to no obvious tissue block, 3ml of digestion stop solution is added into the centrifuge tube to stop the digestion. Blow with a gun for about 1min and then pass through a screen (70 μm), filter off excess tissue and collect the cell suspension into a new 5ml centrifuge tube at 1000rpm × 5 min. Adding preheated neuron culture medium, blowing and beating cells uniformly, inoculating to cell culture plate precoated with polylysine, cross mixing cells uniformly, and adding 5% CO2And culturing in an incubator at 37 ℃.
S2: in vitro transfection of embryonic cerebral cortical neuron siRNA:
after the embryonic cerebral cortical neuron cells are inoculated on the culture plate, the specific siRNA (Si 1 and Si 2) of the control siRNA (NC) and ENSMUST00000156081 and liposome transfection mixed solution are added at the same time, and the mixture is put in an incubator to be cultured for about 12 after being gently shaken. And replacing a fresh neuron culture medium, transfecting for 48 hours, then resuspending by using 0.025% pancreatin digested cells, re-inoculating into a 24-hole culture plate, and collecting cells within 12-18 hours after inoculation.
S3: in vitro neuronal axon growth experiments:
the cell culture medium was discarded and the cells were rinsed with 1 × PBS after preheating. Discard 1 XPBS, add pre-cooled 4% paraformaldehyde and fix on ice for 20 min. After discarding formaldehyde, the column was rinsed with 1 XPBS for 3 times at room temperature for 5 min. After the washing, the PBS was discarded, and the blocking solution was gently added dropwise to 200. mu.l/well, and allowed to stand at room temperature for 40 min. The blocking solution was discarded, and Anti-. beta. -Tublin III antibody (1:1000) diluted with primary antibody was gently added dropwise thereto in a volume of 200. mu.l per well, and incubated at 4 ℃ overnight with standing. The primary antibody was discarded, rinsed with 1 XPBS, and washed 3 times 5 min/time at room temperature. PBS was discarded, and Alexa fluor 647 goat anti-rabbitt (1:1000) diluted with a secondary antibody diluent was gently added dropwise thereto at 200. mu.l per well, followed by incubation at room temperature for 2 hours. The process is protected from light. The secondary antibody was discarded, rinsed with 1 XPBS, and washed 3 times 5 min/time at room temperature. The process is protected from light. After the washing, the round glass slide is taken out of the hole, the side with the cells faces downwards is covered on the glass slide on which the mounting liquid is dripped, and the glass slide is placed in a wet box and stored at 4 ℃. Care was taken not to generate bubbles in the process. The process is protected from light.
Referring to fig. 4, fig. 4a neuronal axonal staining experiments examined the effect of interference ensust 00000156081 on the axonal growth of embryonic cerebral cortical neurons. FIG. 4B is a statistical plot of the longest axons of embryonic cortical neurons. It can be seen from fig. 4 that ensust 00000156081 significantly inhibited the growth of embryonic cerebral cortical neuronal axons following the interference.
The verification shows that the in vivo and in vitro interference on the expression of the ENSMUST00000156081 gene can obviously inhibit the growth of the neuron axon. Further through functional analysis of differential genes after ENSMUST00000156081 interference, an ENSMUST00000156081 is found to regulate and control a downstream key PI3K-Akt signal channel, and a biological macromolecule (IGF-1) or a small molecule compound (SC 79) activates the PI3K-Akt signal channel to detect the influence of the ENSMUST00000156081 on neuron axon regeneration after the interference. The experiment proves that ENSMUST00000156081 can influence the growth of neuron axons by regulating a PI3K-Akt signal channel, so that the ENSMUST00000156081 becomes an important molecular target for repairing nerve injury.
The above description is only an example of the present invention, and is not intended to limit the present invention, and it is obvious to those skilled in the art that various modifications and variations can be made in the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.
Sequence listing
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