CN105254766B - Application of the Mecano growth factor MGF E domain peptides in regulation and control memory related gene and miRNA expression - Google Patents

Abstract

The invention discloses the application of mechanical growth factor MGF E domain peptide in regulating the expression of memory-related genes and miRNA. The present invention studies the regulatory effect of the MGF E domain peptide on the expression of memory-related igf-i gene, antioxidant enzyme gene and miRNA targeting memory-related protein in rats under simulated weightlessness conditions by intramuscularly injecting the MGF E domain peptide. It is proved by experiments that MGF E domain peptide can regulate the expression levels of igf-iea, mgf, sod1, sod2, miR-134 and miR-125b-3p in the hippocampus and/or cerebral cortex of rats under simulated weightlessness conditions, not only for further Studying the regulation mechanism of MGF E domain peptide on IGF‑I, antioxidant enzymes and miRNAs will provide a basis for understanding the memory-improving effect of MGF E domain peptide.

Description

Mechanical growth factor MGF E domain peptide regulates memory-related genes and miRNA application in expression

technical field

The invention belongs to the field of biotechnology, and in particular relates to the application of mechanical growth factor MGF E domain peptide in regulating the expression of memory-related genes and miRNA.

Background technique

The microgravity environment of spaceflight can have persistent effects on the nervous system, leading to space motion sickness, space adaptation syndrome (SAS) and cognitive impairment. Memory loss has also been found using human head-down bed rest (HDBR) and animals tail-suspended to simulate weightlessness. For example, declines in executive function and higher cognitive functions were seen during HDBR, along with changes in physiological function and working memory. In addition, hippocampus-related spatial memory function declines in tail-suspended mice and rats. However, the relevant mechanism is still unclear, and no effective protective measures have been formed.

In recent years, it has been discovered that antioxidant enzymes play an important role in the memory process. Antioxidant enzymes such as glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase (CAT) can repair brain damage in mice, improve Alzheimer's disease and aging resulting memory impairment. Alzheimer's disease and memory impairment due to aging are also accompanied by decreased levels of antioxidant enzymes. Traumatic brain injury (TBI) can cause spatial memory impairment, and overexpression of GPX can restore spatial memory impairment caused by TBI (Tsuru-Aoyagi, et al., 2009). Alzheimer's disease mouse model has a decline in spatial memory function, and overexpression of sod-2 can significantly improve this impairment (Massaad et al, 2009), while deletion of sod-1 can exacerbate this impairment (Murakami et al., 2011). Aging mice exhibit a decline in hippocampus-dependent conditioned fear memory, which was restored by subcutaneous injection of SOD/CAT analogues (Clausen et al, 2010).

miRNAs are a class of small non-coding RNAs that post-transcriptionally regulate gene expression. At present, a variety of miRNAs related to memory function have been screened, and they can target key proteins that regulate the memory process, and then play a vital role in synaptic plasticity, neuron activity and brain development related to memory formation. As shown in the table below.

Table 1. Memory-related miRNAs and their targeted memory-related proteins

miRNAs targeted memory-associated protein memory-related processes miR-124 CREB Synaptic long-term plasticity (Rajasethupathy et al, 2009) miR-134 CREB Synaptic plasticity and memory formation (Gao et al., 2010) miR-132 MeCP2 Synaptic Plasticity (Lusardi et al., 2010) miR-138 APT1 Morphogenesis of dendritic spines (Siegel et al., 2010) miR-125b NR2A Dendritic spine morphogenesis and synaptic physiology (Edbauer et al., 2010) miR-145 SOD2 Neuroprotection after cerebral ischemia (Dharap et al., 2009)

Insulin-like growth factor-I (IGF-I) is a class of polypeptides with multiple potentials, including regulating cell proliferation, apoptosis, differentiation, etc. The Igf-i gene has 6 exons, and exon 4-6 splicing can produce 3 mRNAs: igf-iea, igf-ieb and igf-iec, which encode a mature IGF-I peptide with the same structure (highly conserved Encoded by exons 3 and 4 of ) and structurally distinct E peptides: Ea (human and rat), Eb (rat) and Ec (human). These three types of E peptides all contain a common constitutive 16 amino acid sequences (encoded by exon 4) and different active E peptides, in which Ea, Eb and Ec are composed of 19, 61 and 24 amino acids, respectively. Encoded by exons 6, 5 and 5 and 8. Fragile X syndrome (Fragile X syndrome, FXS) mouse model has memory dysfunction. Administration of the IGF-I analog NNZ-2566 significantly improved this memory impairment (Deacon et al., 2015).

Contents of the invention

One object of the present invention is to provide new uses of MGF E domain peptides.

The present invention provides MGF E domain peptides in the preparation and regulation of any of the following in the hippocampus and/or cerebral cortex of animals in a weightless state: the expression of igf-iea, mgf, sod1, sod2, miR-134 and miR-125b-3p level of product application;

The MGF E domain peptide is a) or b) or c):

a) The amino acid sequence is the protein shown in Sequence 1 in the sequence listing, and the C-terminal of the protein is amidated;

b) a fusion protein obtained by connecting a tag to the N-terminal or/and C-terminal of the protein shown in Sequence 1 in the sequence listing;

c) A protein with the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 1 in the sequence listing.

In the above application, the structural formula of a) is Tyr Gln Pro Pro Ser Thr Asn Lys Asn ThrLys Ser Gln Arg Arg Lys Gly Ser Thr Phe Glu Glu His Lys-NH ₂ .

Another object of the present invention is to provide new uses of biological materials related to the above-mentioned MGF E domain peptide.

The present invention provides any one of the following in the preparation and regulation of the hippocampus and/or cerebral cortex of an animal in a weightless state by the biological material related to the above-mentioned MGF E domain peptide: igf-iea, mgf, sod1, sod2, miR-134 and The application of miR-125b-3p expression level products.

In the above application, the regulation is to restore any of the following in the hippocampus and/or cerebral cortex of the animal after weightlessness treatment: the expression levels of igf-iea, mgf, sod1, sod2, miR-134 and miR-125b-3p to the state before weightlessness.

In the above-mentioned application, the state before the weightlessness treatment is recovered to be consistent or close to the state before the weightlessness treatment; specifically, the mgf mRNA expression level of the cerebral cortex of the animal after the weightlessness treatment, the igf-iea mRNA expression level of the hippocampus and the expression level of the hippocampus are reduced. mgf mRNA expression levels, sod1 mRNA expression levels and sod2 mRNA expression levels in the hippocampus, miR-134 expression levels and miR-125b-3p expression levels in the hippocampus, increased the expression levels of sod1 mRNA in the cerebral cortex and the cerebral cortex of animals after weightlessness treatment The expression level of miR-134.

In the above application, the method of weightlessness is tail suspension; the animal is human or rat.

In the above application, the animal is a rat.

Yet another object of the present invention is to provide a product with specific functions.

The active ingredient of the product with specific function provided by the present invention is a) or b) or c):

a) The amino acid sequence is the protein shown in Sequence 1 in the sequence listing, and the C-terminal of the protein is amidated;

b) a fusion protein obtained by connecting a tag to the N-terminal or/and C-terminal of the protein shown in Sequence 1 in the sequence listing;

c) a protein with the same function obtained by substituting and/or deleting and/or adding the amino acid sequence shown in Sequence 1 in the sequence listing by one or several amino acid residues;

The specific function is to regulate any of the following in the hippocampus and/or cerebral cortex of animals in a weightless state: the expression levels of igf-iea, mgf, sod1, sod2, miR-134 and miR-125b-3p;

The structural formula of a) is Tyr Gln Pro Pro Ser Thr Asn Lys Asn Thr Lys Ser GlnArg Arg Lys Gly Ser Thr Phe Glu Glu His Lys-NH ₂ .

In the above-mentioned product, the method of weightlessness is tail suspension; the animal is human or rat.

In the above product, the animal is a rat.

The application of the above products in regulating the expression levels of any of the following in the hippocampus and/or cerebral cortex of animals: igf-iea, mgf, sod1, sod2, miR-134 and miR-125b-3p also belongs to the protection of the present invention scope.

A final object of the present invention is to provide new uses of MGF E domain peptides.

The present invention provides the application of MGF E domain peptide in the preparation of products with at least one function in the following 1)-6):

1) Repair brain damage;

2) Improve memory impairment;

3) Improve spatial memory ability;

4) Regulating synaptic plasticity associated with memory formation;

5) Regulating neuron activity;

6) Regulate brain development;

The MGF E domain peptide is a) or b) or c):

a) The amino acid sequence is the protein shown in Sequence 1 in the sequence listing, and the C-terminal of the protein is amidated;

b) a fusion protein obtained by connecting a tag to the N-terminal or/and C-terminal of the protein shown in Sequence 1 in the sequence listing;

c) a protein with the same function obtained by substituting and/or deleting and/or adding the amino acid sequence shown in Sequence 1 in the sequence listing by one or several amino acid residues;

The structural formula of a) is Tyr Gln Pro Pro Ser Thr Asn Lys Asn Thr Lys Ser GlnArg Arg Lys Gly Ser Thr Phe Glu Glu His Lys-NH ₂ .

The present invention studies the regulating effect of the MGF E domain peptide on the expression of memory-related antioxidant enzyme genes and miRNAs targeting memory-related proteins in rats under simulated weightlessness conditions by intramuscularly injecting the MGF E domain peptide. It is proved by experiments that MGF E domain peptide can regulate the expression levels of igf-iea, mgf, sod1, sod2, miR-134 and miR-125b-3p in the hippocampus and/or cerebral cortex of rats under simulated weightlessness conditions, not only for further Studying the regulation mechanism of MGF E domain peptides provides a basis for antioxidant enzymes and miRNAs, and will help to understand the memory-improving effect of MGF E domain peptides.

Description of drawings

Figure 1 shows the expression of igf-iea and mgf mRNA in the hippocampus and cerebral cortex of male SD rats in each treatment group. Wherein, Fig. 1A is the expression of igf-iea and mgf mRNA in the hippocampus of male SD rats of each treatment group in 7 days; Fig. 1 B is igf-iea and mgf in the cerebral cortex of male SD rats of each treatment group in 7 days The expression of mRNA; Figure 1C is the expression of igf-iea and mgf mRNA in the hippocampus of male SD rats of each treatment group in 14 days; Figure 1D is the igf-iea in the cerebral cortex of male SD rats of each treatment group in 14 days The expression of iea and mgf mRNA.

Figure 2 shows the expression of memory-related antioxidant enzyme genes in the hippocampus and cerebral cortex of male SD rats in each treatment group. Wherein, Fig. 2A is the expression situation of antioxidant enzyme gene in the hippocampus of the male SD rat of each treatment group in 7 days; Fig. 2B is the expression situation of antioxidant enzyme gene in the cerebral cortex of the male SD rat of each treatment group in 7 days Figure 2C is the expression of antioxidant enzyme genes in the hippocampus of male SD rats in each treatment group for 14 days; Figure 2D is the expression of antioxidant enzyme genes in the cerebral cortex of male SD rats in each treatment group for 14 days.

Figure 3 shows the expression of miRNAs targeting memory-related proteins in the hippocampus and cerebral cortex of male SD rats in each treatment group. in. Figure 3A is the expression of miRNA targeting memory-related proteins in the hippocampus of male SD rats of each treatment group in 7 days; Figure 3B is the expression of miRNAs targeting memory-related proteins in the cerebral cortex of male SD rats of each treatment group in 7 days The expression of miRNA; Figure 3C is the expression of miRNA targeting memory-related proteins in the hippocampus of male SD rats in each treatment group on 14 days; Figure 3D is the target in the cerebral cortex of male SD rats in each treatment group on 14 days Expression of miRNAs to memory-related proteins.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The common reagents in the following examples are domestic analytical grades.

Example 1, Application of mechanical growth factor (MGF) E domain peptide in regulating memory-related igf-iea, antioxidant enzyme gene and miRNA expression

1. Preparation of MGF E domain peptide

(1) Artificially synthesized the amino acid sequence of human mechanogrowth factor (MGF) E domain peptide as shown in Sequence 1 in the Sequence Listing, with a purity of more than 90% as detected by HPLC, meeting the test requirements.

(2) Carry out C-terminal amidation of the amino acid sequence synthesized in step (1) to obtain a stable MGF E peptide with the following structure: Tyr Gln Pro Pro Ser Thr Asn Lys Asn Thr Lys Ser Gln Arg Arg Lys Gly SerThr Phe Glu Glu His Lys-NH ₂ , and dissolved in physiological saline (the concentration of MGF E domain peptide is 1 μg/0.1ml), and set aside.

2. Test object and treatment

In the present invention, male SD rats (110-130 g, Beijing Experimental Animal Center) are used as test objects. The male SD rats are raised at 23±2° C., with 12-hour light and 12-hour dark cycle, free to eat and drink.

Male SD rats were randomly divided into the following 6 groups (8 rats in each group) according to different treatment methods:

1) Control 7-day group (C7): male SD rats were reared in a single cage for 7 days, and intramuscularly injected with normal saline on the second day of feeding;

2) Tail suspension for 7 days group (S7): male SD rats were subjected to tail suspension at 30 degrees for 7 days, and intramuscularly injected with normal saline on the second day of tail suspension;

3) Tail suspension for 7 days + MGF injection group (S7M): Male SD rats were suspended by tail at 30 degrees for 7 days, and intramuscularly injected with MGF E domain peptide on the second day of tail suspension;

4) Control 14-day group (C14): male SD rats were reared in a single cage for 14 days, and intramuscularly injected

injection of saline;

5) Tail suspension 14 days group (S14): male SD rats were subjected to tail suspension at 30 degrees for 14 days, and intramuscularly injected normal saline on the second day of tail suspension;

6) Tail suspension for 14 days + MGF injection group (S14M): Male SD rats were tail suspended at 30 degrees for 14 days, and MGF E domain peptide was injected intramuscularly on the second day of tail suspension.

3. RT-qPCR

After treatment in each group, male SD rats were anesthetized, and RNA from the hippocampus and cerebral cortex of male SD rats was extracted, and RT-qPCR was performed directly to detect the expression levels of igf-iea, mgf, antioxidant enzyme genes and miRNA. Specific steps are as follows:

(1) Extraction of RNA

TRIzol (Invitrogen) was used to extract total RNA from the hippocampus and cerebral cortex of the male SD rats in each treatment group.

The RNA content was detected by a spectrophotometer (Pharmacia Biotech), and the RNA purity was identified by analyzing A260/A280 after 1.5% agarose gel EB staining.

(2) reverse transcription

Using the total RNA obtained in the above step (1) as a template, cDNA was obtained by reverse transcription.

(3) PCR amplification

Using the cDNA obtained in step (2) as a template, the primers in Table 1 were used for PCR amplification, and the igf-iea, mgf, and memory-related antioxidant enzyme genes sod1, sod2, gpx1, and cat were detected by RT-qPCR. Expression levels of miR-124, miR-134, miR-132, miR-138, miR-125b-3p, miR-125b-5p and miR-145 to memory-related proteins. Among them, igf-iea, mgf, sod1, sod2, gpx1 and cat use gapdh as an internal reference gene; miR-124, miR-134, miR-132, miR-138, miR-125b-3p, miR targeting memory-related proteins -125b-5p and miR-145 use 5s rRNA as internal reference gene.

The PCR reaction conditions were as follows: pre-denaturation at 95°C for 1 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 15 s, and a total of 45 cycles.

Table 2. qPCR primer sequences

4. Test results

Differences between groups were analyzed by one-way analysis of variance (ANOVA), and the test results were expressed as mean ± standard error. P<0.05 means significant difference. The specific test results are as follows:

(1) Effects on the expression levels of igf-iea and mgf

The detection results of the expression levels of igf-iea and mgf in male SD rats treated in each group are shown in Figure 1: after 7 days of tail suspension, the expression level of mgf in the cerebral cortex increased, and the treatment with MGF E domain peptide significantly restored the expression of mgf in the cerebral cortex Levels; After 14 days of tail suspension, the expression levels of igf-iea and mgf in the hippocampus were significantly increased, and MGF E domain peptide treatment significantly restored the expression level of igf-iea in the hippocampus. It shows that MGF E domain peptide can regulate the expression level of mgf and igf-iea in rat hippocampus and cerebral cortex.

(2) Effects on the expression level of antioxidant enzyme genes

The detection results of antioxidant enzyme gene expression levels in male SD rats treated in each group are shown in Figure 2: after 7 days of tail suspension, the expression levels of hippocampus sod1 and sod2 increased, and MGF E domain peptide treatment significantly reduced hippocampal sod1 and sod2 levels. sod2 expression level; after 14 days of tail suspension, the expression levels of sod1 and sod2 in the hippocampus increased, and MGF E domain peptide treatment significantly reduced the expression levels of sod1 and sod2 in the hippocampus; after 14 days of tail suspension, the expression level of sod1 in the cerebral cortex decreased significantly, and the MGF E domain peptide Treatment significantly increased the level of Sod1 expression in the cerebral cortex. It shows that MGF E domain peptide can regulate the expression levels of antioxidant enzyme genes sod1 and sod2 in rat hippocampus and cerebral cortex.

(3) Effects on miRNA levels targeting memory-related proteins

The detection results of miRNA levels targeting memory-related proteins in male SD rats treated in each group are shown in Figure 3: after 7 days of tail suspension, the level of miR-125b-5p in the cerebral cortex was significantly increased; The levels of miR-134 and miR-125b-3p increased, and the treatment with MGF E domain peptide significantly decreased the levels of miR-134 and miR-125b-3p in the hippocampus; the level of miR-134 in the cerebral cortex decreased after 14 days of tail suspension, and the treatment with MGF E domain peptide significantly Increased cortical miR-134 levels. It shows that MGF E domain peptide can regulate the levels of miR-134 and miR-125b-3p targeting memory-related proteins in rat hippocampus and cerebral cortex.

Based on the above results, it can be seen that the MGF E domain peptide can regulate the expression levels of igf-iea mRNA, mgf mRNA, sod1, sod2, miR-134 and miR-125b-3p in the hippocampus of rats in weightlessness, and restore them to Expression levels before weightlessness.

Claims (8)

1.MGF E domain peptides are preparing animal hippocampus i of the regulation and control in state of weightlessnessgf-iea、 sod1、sod2、miR- M in 134 and miR-125b-3p and/or cerebral cortexgf、sod1With the application in the product of the expression of miR-134；

The MGF E domain peptides are a）Or b）：

a）Amino acid sequence is protein shown in sequence 1 in sequence table, and the C-terminal amidation of the protein is modified；

b）The fused protein that the N-terminal of protein shown in sequence 1 or/and C-terminal connection label obtain in sequence table；It is described dynamic Object is rat.

2. application according to claim 1, it is characterised in that：It is described to be regulated to make weightless treated rat hippocampus igf- iea、 sod1、sod2, m in miR-134 and miR-125b-3p and/or cerebral cortexgf、sod1With the expression water of miR-134 The flat state being restored to before weightless processing.

3. application according to claim 1 or 2, it is characterised in that：The expression is transcriptional level.

4. application according to claim 1 or 2, it is characterised in that：The mode of the weightlessness is hung for tail.

5. a kind of product with specific function, active constituent a）Or b）：

a）Amino acid sequence is protein shown in sequence 1 in sequence table, and the C-terminal amidation of the protein is modified；

b）The fused protein that the N-terminal of protein shown in sequence 1 or/and C-terminal connection label obtain in sequence table；

The specific function is rat hippocampus i of the regulation and control in state of weightlessnessgf-iea、 sod1、sod2, miR-134 and miR- M in 125b-3p and/or cerebral cortexgf、sod1With the expression of miR-134.

6. product according to claim 5, it is characterised in that：The hippocampus for being regulated to make weightless treated rat igf-iea、 sod1、sod2, m in miR-134 and miR-125b-3p and/or rat cerebral cortexgf、sod1And miR-134 Expression be restored to the state before weightless processing；

The expression is transcriptional level.

7. product according to claim 5 or 6, it is characterised in that：The mode of the weightlessness is hung for tail.

8. product described in claim 5 or 6 is in regulation and control rat hippocampus igf-iea、 sod1、sod2, miR-134 and miR- M in 125b-3p and/or cerebral cortexgf、sod1With the application in the expression of miR-134.