CN110551809A - Application of miR-124 in spinal cord injury repair - Google Patents

Application of miR-124 in spinal cord injury repair Download PDF

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CN110551809A
CN110551809A CN201910773307.8A CN201910773307A CN110551809A CN 110551809 A CN110551809 A CN 110551809A CN 201910773307 A CN201910773307 A CN 201910773307A CN 110551809 A CN110551809 A CN 110551809A
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mir
spinal cord
cord injury
repair
calpain1
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陈凌强
王静
王兵
董俊杰
杨晋
龚志强
赵学凌
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First Affiliated Hospital of Kunming Medical University
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First Affiliated Hospital of Kunming Medical University
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Abstract

The application of miR-124 in spinal cord injury repair. According to the invention, by constructing a miR-124 high expression/silencing model, the function of miR-124 in spinal cord injury repair is researched, and the action mechanism of regulating calpain1 to participate in spinal cord injury repair is disclosed for the first time from molecular, cell and overall levels, so that the influence and mechanism of miR-124 on spinal cord injury repair are disclosed, and a new thought and target point are provided for spinal cord injury treatment. The invention proves that calpain1 is a target gene of miR-124 for the first time, and a large number of experiments show that calpain1 and miR-124 play an important role in the process of spinal cord injury repair, and as a diagnostic marker for judging the degree of spinal cord injury, miRNA-124 can promote the repair of spinal cord injury, and can be used for preparing a medicine for treating spinal cord injury.

Description

Application of miR-124 in spinal cord injury repair
Technical Field
The invention relates to the technical field of biology, in particular to application of miR-124 in spinal cord injury repair.
Background
Spinal Cord Injury (SCI) is a common injury to the locomotor system, often resulting in paralysis of the patient, placing a heavy mental and economic burden on society and families. The treatment of the spinal cord injury is always a medical problem, and the research on the repair mechanism of the spinal cord injury provides theoretical basis for the treatment of the spinal cord injury. Research reports that miR-124 is expressed and down-regulated during spinal cord injury, and Calpain1 can induce spinal cord cell apoptosis, which suggests that both can participate in the spinal cord injury process. According to previous researches, miR-124 is low-expressed in neuronal cells at a spinal cord injury part, the spinal cord injury repair process is accelerated after miR-124 is over-expressed, and the target gene of miR-124 is predicted to be Calpain1, so that miR-124 is involved in the spinal cord injury repair process, but the action mechanism of the miR-124 is unclear, and the miR-124 can participate in the process by regulating the expression of a Calpain1 gene.
Whether miR-124 can regulate the expression of calpain1 gene in a targeted manner and influence the repair of spinal cord injury is not reported at present.
according to the invention, by constructing a miR-124 high expression/silencing model, the function of miR-124 in spinal cord injury repair is researched, and the action mechanism of regulation and control of calpain1 to participate in spinal cord injury repair is realized, so that the influence and mechanism of miR-124 on spinal cord injury repair are discussed from the molecular, cell and overall levels, and a new thought and target point are provided for spinal cord injury treatment.
Disclosure of Invention
Aiming at the problems, the invention provides a reagent or a kit for diagnosing the spinal cord injury prognosis, and the reagent or the kit can detect the expression condition of miR-124 in a sample, or detect the transcription condition of a target gene calpain1 regulated by miR-124 in the sample.
Preferably, the diagnostic reagent for the prognosis of spinal cord injury is used for detecting the transcription of miR-124 in a sample by adopting a high-throughput sequencing method and/or a quantitative PCR method and/or a probe hybridization method.
Preferably, the spinal cord injury diagnostic reagent is a reagent for detecting the transcription of the miR-129 target gene calpain1 in the sample by adopting a high-throughput sequencing method and/or a quantitative PCR method and/or a probe hybridization method or detecting the expression condition of the miR-129 regulated target gene calpain1 in the sample by adopting an immune method.
Preferably, the transcription of miR-124 in the sample is detected by a northern hybridization method, a miRNA expression profiling chip, a ribozyme protection analysis technology, a RAKE method, in-situ hybridization and microsphere-based flow cytometry.
Preferably, the transcription of the miR-124 regulated target gene calpain1 in the sample is detected by a northern hybridization method, a ribozyme protection analysis technology, a RAKE method, in situ hybridization and microsphere-based flow cytometry.
preferably, the quantitative PCR method comprises specific amplification of miR-124 primer; the probe-based hybridization methods include probes that hybridize to nucleic acid sequences of miR-124 and/or a precursor thereof.
Preferably, the quantitative PCR method comprises a primer for specifically amplifying the miR-124 regulated target gene; the probe-based hybridization method comprises a probe hybridized with a nucleic acid sequence of a miR-129 regulated target gene calpain 1.
The invention also provides a medicine for treating spinal cord injury, which comprises a miR-124 expression enhancer or a miR-124 target gene expression inhibitor.
The invention has the beneficial effects that: according to the invention, by constructing a miR-124 high expression/silencing model, the function of miR-124 in spinal cord injury repair is researched, and the action mechanism of regulating calpain1 to participate in spinal cord injury repair is disclosed for the first time from molecular, cell and overall levels, so that the influence and mechanism of miR-124 on spinal cord injury repair are disclosed, and a new thought and target point are provided for spinal cord injury treatment.
The invention proves that calpain1 is a target gene of miR-124 for the first time, and a large number of experiments show that calpain1 and miR-124 play an important role in the process of spinal cord injury repair, and as a diagnostic marker for judging the degree of spinal cord injury, miRNA-124 can promote the repair of spinal cord injury, and can be used for preparing a medicine for treating spinal cord injury.
Drawings
FIG. 1 is a schematic diagram of fluorescent quantitative RT-PCR according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of the Western blotting method for detecting protein expression according to the embodiment of the present invention;
FIG. 3 shows that miR-124 can inhibit luciferase reporter gene expression of calpain13' UTR vector according to the embodiment of the invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is within the scope of the present invention to modify or replace methods, steps or conditions of the present invention without departing from the spirit and substance of the present invention. Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1 construction of miR-124 high expression/silencing model
miR-124mimics, inhibitors, blank miRNA-FITC and BMSCs cells (ATCC brand, purchased from Shanghai enzyme research Biotechnology Co., Ltd.) are respectively cultured to a 24-well plate, a serum-free culture medium is changed 2 hours before transfection, the cells are transfected after the plasmid is wrapped by liposome 2000, the liquid is changed 6 hours later, FITC expression is observed under a fluorescence microscope after 24 hours, transfection efficiency is calculated by comparing a visible light field and a fluorescence field, the growth condition and activity of the cells are closely observed, on day 1, about 1 × 10 5 cells are respectively extracted from another group of cells and dripped to a 35 mm-well plate, the liquid is changed every 2-4 days, and a miR-124 overexpression cell line, a miR-124 low-expression cell line, a blank protein cell line and a blank control cell line are established through the treatment.
Example 2 fluorescent quantitative RT-PCR:
Total RNA (Trizol, In vitro) is extracted from each cell line group to be detected, 50 mu l of total RNA is extracted, a Nano Drop substance (Invitrogen) is subjected to PCR to observe the expression level of the target In the cell line, and the experiment is repeated for 3 times.
As a result: miR-124 is negatively associated with calpain1mRNA expression in different BMSCs cells, suggesting that calpain1 may be the target gene for miR-10b (FIG. 1).
example 3 western blot for protein expression:
The above cell lines are cracked and total protein is extracted, after centrifugation, supernatant SDS-PAGE electrophoresis is carried out, anode carbon plate exposure and development are carried out, GAPDH is used as a control, the expression quantity of the target protein after transfection is observed, and the experiment is repeated for 3 times.
As a result: as shown in FIG. 2, calpain1 protein expression showed significant down-regulation after miR-124 was overexpressed.
Example 4 Dual luciferase Gene reporter
Constructing reporter gene plasmid of calpain1 target protein, transfecting the reporter gene plasmid and target plasmid into miR-124 mics and a blank control cell line, fully mixing the reporter gene cell lysate, adding the reporter gene cell lysate, fully lysing cells, centrifuging for 3-5 minutes at 10000-15000g after fully lysing, taking supernatant for determination, melting firefly luciferase detection reagent and Renilla luciferase detection buffer solution, reaching room temperature, placing Renilla luciferase detection substrate on an ice bath or an ice box for standby, taking a proper amount of Renilla luciferase detection buffer solution according to the amount of 100 microliters required by each sample, adding the Renilla luciferase detection substrate according to the ratio of 1:100 to prepare Renilla luciferase detection working solution, starting a fluorescence determinator according to an instrument operation instruction, setting the determination interval to be 2 seconds, setting the determination time to be 10 seconds, and after completing the firefly luciferase determination step, adding 100 microliters of Renilla luciferase detection working solution, uniformly mixing, determining RLU, and under the condition that the Renilla luciferase is used as an internal reference, dividing the RLU value obtained by the firefly luciferase determination by the RLU value obtained by the Renilla luciferase determination, and checking the transcription activity of the target protein.
Culturing cells for 48h after transfection, removing old culture medium by sucking, washing twice by PBS, adding 100 mu l of PLB into each hole according to the instruction of a Dual-Luciferase Reporter Assay System kit, acting at room temperature for 20min, adding 20 mu l of LAR-II reagent into each hole, putting an instrument to read the Luciferase fluorescence value, then adding 20 mu l of Glo reagent, and putting the instrument to read the beta-gal to read the beta-galactosidase value; the fluorescence value and the relative activity value of beta galactosidase of the firefly Luciferase in each experimental group are measured by a Dual-Luciferase reporter gene detection system for numerical statistics, and as can be seen from FIG. 3, miR-124 can inhibit the Luciferase reporter gene expression of calpain13' UTR vector (FIG. 3).
Results show that calpain1 is a target gene of miR-124, miR-124 can be combined with calpain13' UTR to target and inhibit calpain1 expression, and miR-124 plays a negative regulation role in calpain 1.

Claims (2)

  1. Use of miR-124 as a regulator of Calpain1 target gene in preparation of a medicament for treating repair after spinal cord injury.
  2. 2. The use according to claim 1, wherein said miR-124 regulates the expression of the target gene, Calpain1, at both the RNA and protein levels.
CN201910773307.8A 2019-08-21 2019-08-21 Application of miR-124 in spinal cord injury repair Pending CN110551809A (en)

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Cited By (1)

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Application publication date: 20191210