CN113577094B

CN113577094B - Application of miR-673-5P in preparation of preparation for promoting peripheral nerve regeneration

Info

Publication number: CN113577094B
Application number: CN202111052916.8A
Authority: CN
Inventors: 赵亚红; 杨宇民; 刘纪娜; 葛一凡
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2021-09-08
Filing date: 2021-09-08
Publication date: 2022-08-23
Anticipated expiration: 2041-09-08
Also published as: CN113577094A

Abstract

The invention discloses application of miR-673-5P in preparation of a preparation for promoting peripheral nerve regeneration. The invention provides a new application of an agonist miR-673-5P, and the effect is exact.

Description

Application of miR-673-5P in preparation of preparation for promoting peripheral nerve regeneration

Technical Field

The invention relates to application of miR-673-5P.

Background

The myelin sheath of peripheral nerves is formed by surrounding nerve axons with cell membrane layers of schwann cells, and its main component is lipid, and the lipid component in myelin sheath mainly includes Cholesterol (Cholesterol), Phospholipids (Phospholipids), glycosphingolipids (Glycosphing), and the like. The cholesterol accounts for about 30% of the myelin sheath lipid, a high proportion of cholesterol is beneficial for maintaining the stability and increasing the compactness of myelin sheath, the main role of cholesterol is to maintain the stability of myelin sheath by regulating the fluidity and permeability of the membrane, and in myeloblasts, the role of cholesterol is far beyond that of its biophysical properties, and the rate of cholesterol synthesis is also related to the rate of myelin biosynthesis. The phospholipid in the myelin sheath mainly comprises phosphatidylethanolamine (28-39%), phosphatidylcholine, depsiphosphatidylcholine, depsipolylcholine lipoylethanolamine, phosphatidylserine and sphingomyelin (10-35%). In most cells, these phospholipids can interact with different proteins, and these actions can regulate cellular physiological activities such as endocytosis, cytoskeletal remodeling, cell growth and polarization, etc. Myelin also has a higher proportion of glycosphingolipids in comparison to its biofilm, the most prominent class of glycosphingolipids being galactose glycosphingolipids, which play an important role in maintaining myelin stability. In summary, the cord sheath is a lipid-rich multilayer film structure, and the ratio of the lipid component in the cord sheath is significantly different from other biological membranes, and plays an extremely important role in promoting the production and maintenance of the cord sheath for long-term integrity and stability.

MicroRNAs (miRNAs) are an endogenous, regulatory-functional, non-coding RNA of a class found in eukaryotes with a very wide variety of biological functions. In recent years, tissue engineering developed based on the principles and techniques of life science and engineering opens up a new way for constructing nerve graft substitutes. The current research is more inclined to prepare nerve graft with nerve growth factor or functional nucleic acid such as loading miRNA, LncRNA and the like. Recent studies have shown that mirnas are involved in a wide variety of regulatory pathways including development, viral defense, hematopoietic processes, organogenesis, cell proliferation and apoptosis, fat metabolism, etc. At present, no research is available on the correlation between miR-673-5p and lipid synthesis in the nerve regeneration process.

Disclosure of Invention

The invention aims to provide application of miR-673-5P with a definite effect in preparation of a preparation for promoting peripheral nerve regeneration.

The technical solution of the invention is as follows:

application of miR-673-5P in preparation of a preparation for promoting peripheral nerve regeneration is provided.

The agent for promoting the regeneration of peripheral nerves is an agent for promoting the regeneration of peripheral nerves by increasing the expression level of lipids in the sciatic nerve.

The increase of the expression level of the lipid in the sciatic nerve is realized by increasing the expression of a cholesterol synthesis gene HMGCR.

The invention provides a new application of an agonist miR-673-5P, and the effect is exact.

Drawings

The invention is further illustrated by the following figures and examples.

FIG. 1 is a schematic diagram of miR-673-5p sequence.

(http://www.mirbase.org/textsearch.shtmlq＝miR-132-5p&submit＝submit)

Fig. 2 is a graph showing the expression levels of relevant lipid components after subcutaneous injection of miR-673-5p Agomir/Agomir-NC in SD mice 1-15d ([ p ] <0.05, [ p ] <0.01, [ p ] <0.001vs.

Figure 3 is a lipid sequencing heatmap.

FIG. 4 is a schematic diagram of the expression of mTOR downstream transcription factor SREBP2 and its downstream genes detected by qRT-PCR after SD suckling mice 1-15d are injected with miR-673-5p Agomir/Agomir-NC subcutaneously. A: SREBP2 expression level; b: HMGCR expression level ([ p <0.05, [ p <0.01, [ p ] 0.001vs.

FIG. 5 is a schematic diagram of expression conditions of a mTOR downstream transcription factor SREBP2 and downstream genes thereof detected by qRT-PCR after miR-673-5p mimics/inhibitor is transfected in vitro. A: SREBP2 expression level; b: HMGCR expression levels ([ p ] 0.05, [ p ] 0.01, [ p ] 0.001, [ p ] 0.0001).

Detailed Description

Experiment:

1. construction of miR-673-5p mimics and inhibition substance particles

The construction of the miR-673-5p simulant and inhibitor in the experiment is to directly purchase a MirONTMmiRNA series kit of the Ruibo company, directly provide the mature sequence of miR-673-5p for the company, and synthesize the recombinant plasmid with correct sequence.

2. Culture and purification of primary Schwann cells and miR-673-5p treatment

SD suckling mice born for 1d were first sterilized with 75% ethanol, the sciatic nerve was dissected off in a clean bench and placed in a petri dish (on ice) poured with basal medium. Then removing the culture medium, adding 1mL of 3mg/mL collagenase, rapidly cutting sciatic nerve with micro scissors, gently blowing, mixing, placing at 37 deg.C and 5% CO ₂ Digesting in the incubator for 30 min. The collagenase is removed after 30min, 1ml of 0.25% trypsin is added, the mixture is gently blown, beaten and mixed evenly, and then digested in an incubator for 5 min. After 5min, three times the amount of complete medium was added to stop digestion, and centrifuged at 1200rpm for 5 min. Then, the supernatant is discarded and added with fresh complete culture medium, the mixture is blown and evenly mixed and then passes through a screen, and cell seeds are placed on a culture dish coated with a proper amount of Poly L Lysine (PLL) in advance and are placed in an incubator for culture. After 16hThe method comprises the following steps of 1: 1000 to complete medium Cytarabine (working concentration 10. mu. mol/L) was added. After 24h of cultivation, the medium was changed to complete medium and then to complete medium containing 2. mu. mol/LForskolin and 25ng/mLHRG for 24h, the medium was changed every 2 days.

After the cell density reached more than 80%, the cells were washed once with PBS. 1mL of 0.25% trypsin was added, the trypsin was discarded after 1min of digestion, 3mL of complete medium was added to stop the digestion and the cells were collected in a 15mL centrifuge tube. Centrifuging at 1200rpm for 5min, removing supernatant, adding fresh complete culture medium, and then adding the mixture into a reactor according to the volume ratio of 1: 1000 to complete medium was added fibroblast-specific antibody thy 1.1. After 2h on ice, the supernatant was centrifuged off and mixed with 1: 3, adding complement into the basic culture medium, blowing, uniformly mixing, and putting into an incubator for 30 min. After taking out, the supernatant was centrifuged and washed 1 time with complete medium. The supernatant was centrifuged again, fresh complete medium was added and the cells were seeded in petri dishes previously coated with PLL. The next day the medium was changed to complete medium containing 2. mu. mol/LForskolin and 25ng/mLHRG, and the medium was changed every 2 days and the well plates were used after the cells had grown.

By using riboFECT ^TM CP transfection reagent, 100nM transfection concentration, according to the instructions, miR-673-5p mimetics and inhibitors were transfected into purified Schwann cells.

3. Animal model construction and miR-673-5p treatment

60 newborn (P0) SD suckling mice were prepared and divided into two main groups: and the number of the groups is 30 respectively in an agomir-NC group and a miR-673-5pagomir group. Agomir is miRNA mimics of in vivo overexpression, the group of the Agomir-NC is a control group, and the group of miR-673-5pagomir is an experimental group. The two are in a freeze-dried powder state after being purchased, and are dissolved by DEPC water, and the solution is dissolved at present when being used, so that repeated freeze thawing is avoided.

In the case of SD suckling mouse 1d, 30 mu Lagomir-NC and miR-673-5pagomir are injected into the back of the suckling mouse close to the two hind limbs by a micro-injector, and are injected 5nmol each time and are injected once again at intervals of 2 d. Setting corresponding time points, taking materials on days 2, 5, 7, 11 and 15, and taking sciatic nerves of two hind limbs according to different experimental purposes.

4. Quantitative PCR detection of gene expression level

Total cellular RNA was extracted using Trizol reagent (Invitrogen) and transcribed into cDNA using a cDNA synthesis kit (TaKaRa). Using this cDNA as a template, the gene expression level was analyzed by SYBR Green Supermix (Bio-Rad). The Detection apparatus was an iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad). Results 2 ^-ΔCt The method calculates mRNA levels. mRNA levels were calibrated with the 18S housekeeping gene. The primer sequences used were as follows:

5'-AGTCCCTGCCCTTTGTACACA-3' as 18S rRNA forward primer;

5'-CGTTCCGAGGGCCTCACT-3' as 18S rRNA negative primer.

Srebp2 forward primer: 5'-CTGTCGGGTGTCATGGG-3', respectively;

srebp2 negative primer: 5'-GCTCGCTGTTCTCATCCA-3' are provided.

Hmgcr forward primer: 5'-GGCTGAAGATGTGTCCAAG-3', respectively;

hmgcr negative primer: 5'-AGCCAAAAGCAACGCTAA-3' are provided.

The sequence of miR-673-5p is shown in figure 1. To investigate the effect of miR-673-5p on lipid synthesis, we constructed miR-673-5p agomir by injecting it subcutaneously and locally into SD suckling mice born at 1 d. The lipid level expression in sciatic nerve is observed after injection every two days, and the materials are taken on days 2, 5, 7, 11 and 15, and the lipid level expression in sciatic nerve can be improved by over-expressing miR-673-5p (figure 2). Further lipid sequencing results show that the miR-673-5p is over-expressed, so that the expression content of each component in the lipid is increased (figure 3). In order to further verify the phenomenon of experimental miR-673-5p and carry out qRT-PCR detection on the sciatic nerve, the result shows that the over-expression of miR-673-5p increases the expression of the cholesterol synthesis gene HMGCR, and the expression level of the HMGCR is obviously increased on days 2, 7 and 11 (figure 4). In order to further confirm the influence of miR-673-5p on the expression level of HMGCR, miR-673-5p mimics and inhibitor are respectively transfected into Schwann cells through in vitro experiments, and the change of the expression level of the gene HMGCR is further verified, so that compared with a control group, the expression of SREBP2 and HMGCR is increased after miR-673-5p mimics are transfected, and the expression of SREBP2 and HMGCR is decreased after miR-673-5p inhibitor is transfected, and both the expression are significantly different (FIG. 5). In conclusion, in vivo and in vitro experiments prove that the over-expression miR-673-5p can regulate and synthesize a large amount of lipid required in the peripheral nerve myelination process, so that the myelination is promoted, and the nerve regeneration capacity is further enhanced.

Claims

1. Application of miR-673-5P in preparation of a preparation for promoting peripheral nerve regeneration is provided.

2. The use of miR-673-5P according to claim 1, wherein the use of miR-673-5P for the preparation of a preparation for promoting peripheral nerve regeneration is characterized by: the agent for promoting the regeneration of peripheral nerves is an agent for promoting the regeneration of peripheral nerves by increasing the expression level of lipids in the sciatic nerve.

3. The use of miR-673-5P according to claim 2, wherein the use of miR-673-5P for the preparation of a preparation for promoting peripheral nerve regeneration is characterized by: the increase of the expression level of the lipid in the sciatic nerve is realized by increasing the expression of a cholesterol synthesis gene HMGCR.