CN114452294B - Small nucleic acid medicine for treating hepatic fibrosis - Google Patents

Small nucleic acid medicine for treating hepatic fibrosis Download PDF

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CN114452294B
CN114452294B CN202210322411.7A CN202210322411A CN114452294B CN 114452294 B CN114452294 B CN 114452294B CN 202210322411 A CN202210322411 A CN 202210322411A CN 114452294 B CN114452294 B CN 114452294B
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liver fibrosis
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fibrosis
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CN114452294A (en
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林楠
奉源
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Third Affiliated Hospital Sun Yat Sen University
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Abstract

The invention discloses a small nucleic acid drug, which discovers that miR-667-5p can be used for preventing and/or treating liver fibrosis for the first time, and can obviously inhibit the expression level of a liver fibrosis marker by inhibiting the activity of hepatic stellate cells, so that the effect of preventing and/or treating the liver fibrosis is achieved, and the small nucleic acid drug can be used as a drug for preventing and/or treating the liver fibrosis. The invention also provides a medicine containing miR-667-5p, which can be used for singly treating liver fibrosis or cooperatively treating liver fibrosis by combining with a common medicine for treating liver fibrosis, has obvious inhibition effect on the expression of liver fibrosis related indexes, and has a good application prospect when being applied to the liver fibrosis medicine.

Description

Small nucleic acid medicine for treating hepatic fibrosis
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to application of miR-667-5p in preparation of a medicine for preventing and/or treating hepatic fibrosis.
Background
Liver fibrosis is a common pathological process of various chronic liver diseases, and clinical treatment is very difficult. Hepatic stellate cell (hepatic stellate cells, HSC) activation and resulting in excessive deposition of extracellular matrix are central links in the development of liver fibrosis, inhibiting HSC activation or inducing HSC apoptosis is critical in the treatment of liver fibrosis. The prior art has studied various methods of treatment for HSCs, such as inhibiting HSC activation by interferon, promoting HSC apoptosis by using the fungal metabolite Gliotoxin, inhibiting collagen secretion by HSC by using angiotensin converting enzyme inhibitors, etc., but the efficacy is not obvious. Although the methods have certain curative effects, the side effects are too large to be applied to clinic.
The mechanism of the nucleic acid medicine acts on mRNA, and the expression of target protein is inhibited through gene silencing, so that the aim of treating diseases is fulfilled. The nucleic acid medicine treats from the post-transcriptional level, can realize breakthrough aiming at special protein targets which are difficult to prepare, and is hopeful to overcome diseases which are not treated by the medicine, including genetic diseases and other refractory diseases. Micrornas (mirnas) are endogenous non-coding regulatory RNAs of about 21-22 nt in length, can act on target mRNA to cause degradation or inhibit translation thereof, play an important negative regulatory role in gene expression, and are involved in numerous important biological processes in vivo such as development, cell differentiation, cell proliferation, cell death, and the like. Therefore, there is a need to develop new small nucleic acid drugs for treating liver fibrosis. At present, no research shows that miR-667-5p can be used as a target point for treating liver fibrosis.
Disclosure of Invention
The invention aims to provide a novel application of miR-667-5p in preparation of a medicament for preventing and/or treating liver fibrosis.
Therefore, the invention provides a novel small molecule ribonucleic acid miR-667-5p medicament, and in vitro HSC-T6 cell experiments prove that miR-667-5p can effectively inhibit HSC activation and down regulate the expression levels of hepatic fibrosis markers MMP2, collagen I and alpha-SMA in activated HSC, so that the medicament has better hepatic fibrosis prevention and treatment potential.
In a first aspect, the invention discloses an application of miR-667-5p in preparation of a medicament for preventing and/or treating hepatic fibrosis.
The sequence of miR-667-5p is shown in SEQ ID NO. 1: CGGUGCUGGUGGAGCAGUGAGCAC.
In a second aspect, the invention provides a medicament comprising a pharmaceutically acceptable carrier and miR-667-5p.
In some embodiments of the present invention, the drug further comprises a combination drug, wherein the combination drug comprises at least one of gulote and colchicine.
Preferably, the liver fibrosis treatment drug is gulote.
Preferably, the pharmaceutically acceptable carrier comprises a liposome, a nanoparticle.
Preferably, the administration of the drug includes oral administration, intravenous injection.
In a third aspect, the invention also provides a method of discovering and studying the mechanism of liver fibrosis treatment in an animal model, the method comprising studying high throughput sequencing and/or gene chip analysis with respect to the action target of miR-667-5p.
Compared with the prior art, the invention has the beneficial effects that:
the nucleic acid medicine does not need complex protein modification and CMC development in the research and development stage, the preparation process in the production stage is relatively simple, large-scale mammalian cell fermentation and protein purification are not needed, and the nucleic acid medicine has the advantages of abundant candidate targets, short research and development period, small side effect, durable medicine effect, high clinical development success rate and the like.
The invention provides a novel strategy for treating and preventing liver fibrosis by taking miR-667-5p as a core. In vitro cell experiments show that miR-667-5p small nucleic acid medicine can effectively inhibit HSCs activation, down regulate expression levels of hepatic fibrosis markers MMP2, collagen I and alpha-SMA in activated HSCs, and experiments in combination with marlotitide show that miR-667-5p can synergistically enhance a treatment effect of a commonly used medicine for treating hepatic fibrosis. miR-667-5p shows remarkable effect and potential for preventing and treating liver fibrosis.
Drawings
FIG. 1 is a graph of the activity of HSC-T6 cells from the CCK8 assay of example 3: wherein A is an influence diagram of inhibiting miR-667-5p on HSC-T6 cell activity, and B is an influence diagram of over-expressing miR-667-5p on HSC-T6 cell activity;
FIG. 2 is a graph of the expression level of HSC-T6 cell fibrosis markers of example 4: wherein A is qPCR map of HSC-T6 cell fibrosis marker after miR-667-5p inhibition, B is WB map of HSC-T6 cell fibrosis marker after miR-667-5p inhibition, C is qPCR map of HSC-T6 cell fibrosis marker after miR-667-5p over-expression, D is WB map of HSC-T6 cell fibrosis marker after miR-667-5p over-expression;
FIG. 3 is an IHC analysis of HSC-T6 cell fibrosis markers of example 4: wherein A is an IHC analysis chart of a HSC-T6 cell fibrosis marker after miR-667-5p inhibition, and B is an IHC analysis chart of a HSC-T6 cell fibrosis marker after miR-667-5p overexpression;
FIG. 4 is a qPCR map of fibrosis markers in rat liver tissue of example 5;
FIG. 5 is a qPCR map of HSC-T6 cell fibrosis markers of example 6.
Detailed Description
The present invention will be described in further detail with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The materials, reagents and the like used in this example are commercially available ones unless otherwise specified.
The reagents used in this example are all analytically pure grade reagents and are commercially available from regular sources.
EXAMPLE 1 cell culture, cell activation
(1) Experimental materials
Rat hepatic stellate cell line HSC-T6, purchased from Wohaze life technologies Co., ltd;
culture medium conditions: DMEM high sugar basal medium (purchased from Gibco) +10% foetal calf serum (purchased from Gibco) +double antibody (100U/mL penicillin and 100 μg/mL streptomycin, purchased from Gibco).
(2) HSC-T6 cell culture
HSC-T6 cells were cultured in high sugar (4500 mg/L) DMEM medium (containing 10% fetal calf serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin).
(3) HST-T6 cell activation
HST-T6 cells were activated by adding 10ng/ml transforming growth factor-beta 1 (TGF-. Beta.1) to the medium and incubating for 24 hours.
EXAMPLE 2 construction of HSC-T6 cell lines with up-regulated over-expression/down-regulated inhibition of miR-667-5p expression
HSC-T6 (5X 10) 5 Individual cells) were inoculated in six well plates and the cell culture medium was replaced with DMEM without serum and antibiotics. siRNA (rno-miR-667-5 p MIMIC/inhibitor) overexpressing/inhibiting miR-667-5p was transfected into cells using Lipofectamine 2000 transfection reagent (purchased from Invitrogen) to obtain HSC-T6 cell lines overexpressing and producing miR-667-5p, respectively. HSC-T6 cell lines transfected with non-potent siRNA (rno-miR-667-5 p mimic/inhibitor NC) were used as controls.
See table 1 for each siRNA sequence.
TABLE 1 siRNA sequences
EXAMPLE 3 experiments on the Effect of miR-667-5p on HSC Activity
1. Experimental method
Normal, control and miR-667-5p inhibited/overexpressed HSC-T6 cell lines (4 x 10) were plated in 96-well plates, respectively 3 Individual cells/well). CCK8 reagent (purchased from bi yun) was added to the cells for detection according to manufacturer's cell activity measurements. After culturing for 24, 48 and 72 hours respectively in the corresponding conditions, CCK8 reagent is added into cells for treatment according to the method of the kit instruction, and then absorbance values of 450nm wavelengths at 24, 48 and 72 hours after cell activation are recorded by an enzyme-labeling instrument.
2. Experimental results
FIG. 1A shows that by comparing the absorbance at various time points of the control (rno-miRNA inhibitor NC) and miR-667-5p inhibition (rno-miR-667-5 p inhibitor), a general increase in HSC-T6 activity following interference with miR-667-5p can be seen (FIG. 1A).
FIG. 1B shows that by comparing the absorbance at various time points of the control group (TGF-. Beta.1+rno-miRNA chemicals NC) after TGF-. Beta.1 activation and the miR-667-5p over-expression group (TGF-. Beta.1+rno-miR-667-5 p chemicals), the activity of HSC-T6 is generally reduced after over-expression of miR-667-5p.
Taken together, miR-667-5p can reduce HSC activity.
EXAMPLE 4 experiments on the Effect of miR-667-5p on the expression level of HSC fibrosis markers
1. Experimental method
(1) qRT-PCR analysis
Total RNA was extracted from HSC-T6 cells or liver tissue using TRIzol reagent (purchased from Invitrogen). cDNA was synthesized using Prime Script RT kit (purchased from Takara). Gene amplification was performed using Power SYBR Green PCR Master Mix (purchased from Bio-Rad) according to the manufacturer's instructions. qRT-PCR was performed on an ABI7500 real-time fluorescent quantitative PCR system and relative gene expression was calculated using the 2- ΔΔCT method. GADPH was used as an internal control. The siRNA sequences are shown in table 1.
(2) Immunohistochemical (IHC) analysis
Cell climbing plates were fixed with 4% paraformaldehyde, 0.5% Triton X-100 permeabilized and 3% H2O2, and then blocked in 2% goat serum. The slide was then incubated with primary antibodies such as MMP2 (from Abcam), alpha-SMA (from Boster) and collagen I (from Proteintech). Signals were detected after treatment using the VECTASTIN ABC Elite kit (from Vector Laboratories) and DAB substrate kit (from Vector Laboratories) according to the manufacturer's instructions.
(3) WB (Western blot) analysis
Total protein was extracted from HSC-T6 cells by RIPA supplemented with phenylmethylsulfonyl fluoride (purchased from Biyun). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to PVDF membranes. After blocking with 5% nonfat dry milk, membranes were incubated overnight at 4℃with primary antibodies such as MMP2 (1:1000, from Abcam), alpha-SMA (1:1000, from Boster), collagen I (1:1000, from Proteintech) and GAPDH (1:2000, from Proteintech). The next day, the membrane was incubated with secondary antibodies (1:5000, purchased from Zhongshan biotechnology) for 1 hour at room temperature. Proteins were observed using the enhanced chemiluminescent detection system (GE Healthcare). The relative expression of the protein was normalized to the expression of the internal control GAPDH.
2. Experimental results
(1) Results comparing the expression levels of the fibrosis markers MMP2, α -SMA and collagen I in HSCs using qPCR and WB analysis are shown in figure 2.
FIGS. 2A-B show that the expression level of the fibrosis marker of HSC-T6 is generally increased after interfering with miR-667-5p by comparing the expression levels of the fibrosis markers of the control group (rno-miRNA inhibitor NC) and the miR-667-5p inhibition group (rno-miR-667-5 p inhibitor).
FIGS. 2C-D show that by comparing the expression levels of the respective fibrosis markers of the control group (TGF-. Beta.1+rno-miRNA chemicals NC) and the miR-667-5p overexpression group (TGF-. Beta.1+rno-miR-667-5 p chemicals) after activation of TGF-. Beta.1, a general decrease in the expression level of the fibrosis marker of HSC-T6 after overexpression of miR-667-5p can be seen.
(2) The expression levels of the fibrosis markers MMP2, α -SMA and collagen I in HSCs were compared using IHC analysis, see figure 3.
FIG. 3A shows that by comparing the expression levels of each fibrosis marker in the control group (rno-miRNA inhibitor NC) and the miR-667-5p inhibition group (rno-miR-667-5 p inhibitor), an upregulation of the staining positive proportion of each fibrosis marker in HSC-T6 after interfering with miR-667-5p can be seen, indicating a general increase in the expression level of the fibrosis marker in HSC-T6.
FIG. 3B shows that by comparing the expression levels of each of the fibrosis markers in the control group (TGF-. Beta.1+rno-miRNA chemicals NC) and the miR-667-5p over-expression group (TGF-. Beta.1+rno-miR-667-5 p chemicals) after activation of TGF-. Beta.1, it can be seen that after over-expression of miR-667-5p, the staining positive proportion of each of the fibrosis markers in HSC-T6 is down-regulated and the expression level of the fibrosis marker in HSC-T6 is generally decreased.
Example 5 experimental investigation of miR-667-5p in the treatment of liver fibrosis rats
Rats (male SD rats, weighing about 130+ -10 g) were purchased from the medical laboratory animal center in Guangdong province. Hepatic fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl 4;1mL/kg; CCl4: olive oil, v/v=1:1) twice weekly over 8 weeks. To investigate the effect of miR-667-5p on liver fibrosis, the CCl described above 4 Eight weeks after injection, the resulting rno-miR-667-5p MImics siRNA and control rno-miRNA mimics NC siRNA (10 nmol/20g body weight/dose) were injected into rats via the tail vein. At week 11, liver tissues were collected and analyzed for the expression level of fibrosis markers by qPCR.
As can be seen from FIG. 4, the expression level of each fibrosis marker was generally decreased after treatment by injection of miR-667-5p siRNA (rno-miR-667-5 pmimics).
EXAMPLE 6 experiments of the effect of miR-667-5p in combination with Malotester (MLT) on HSC-T6
After TGF-. Beta.1 activation, each group of HSC-T6 was treated for a further 48h with or without 10. Mu.M MLT (purchased from selectk) and then compared to the expression levels of the fibrosis markers MMP2, α -SMA and collagen I in HSC using qPCR, the experimental results are shown in FIG. 5.
FIG. 5 shows that treatment of HSC with gulotester alone does not inhibit the expression of its fibrotic markers, whereas addition of gulotester further increases the effect of miR-667-5p in inhibiting HSC fibrosis, on the basis of upregulation of miR-667-5p expression in HSC, indicating that both have a synergistic effect in inhibiting liver fibrosis.
In conclusion, the experimental result shows that miR-667-5p can effectively inhibit HSCs from activating, and is a potential means for treating liver fibrosis. The invention provides a new strategy for preventing and treating liver fibrosis by using miR-667-5p as a core for the first time.
Although embodiments of the present disclosure have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations may be made therein without departing from the principles and spirit of the disclosure, the scope of which is defined in the appended claims and their equivalents.
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<210> 4
<211> 42
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
uucuccgaac gugucacgut tacgugacac guucggagaa tt 42
<210> 5
<211> 21
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
caguacuuuu guguaguaca a 21
<210> 6
<211> 20
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
ggagtgacag gtcccagtgt 20
<210> 7
<211> 20
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
agctttgatg gcccctatct 20
<210> 8
<211> 20
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
ggagagagca tgaccgatgg 20
<210> 9
<211> 20
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
gggacttctt gaggttgcca 20
<210> 10
<211> 20
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
accatcggga atgaacgctt 20
<210> 11
<211> 20
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
ctgtcagcaa tgcctgggta 20
<210> 12
<211> 19
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
gcaagagaga ggccctcag 19
<210> 13
<211> 20
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
tgtgagggag atgctcagtg 20

Claims (1)

1. Application of miR-667-5p in preparation of medicines for treating hepatic fibrosis rats is provided, wherein the nucleotide sequence of miR-667-5p is shown as SEQ ID NO.1.
CN202210322411.7A 2022-03-29 2022-03-29 Small nucleic acid medicine for treating hepatic fibrosis Active CN114452294B (en)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
JP2016101153A (en) * 2014-11-28 2016-06-02 コリア・インスティテュート・オブ・サイエンス・アンド・テクノロジー Microrna for identifying presence or absence of exposure against hexanal, and identification method using the same
KR102242639B1 (en) * 2020-01-28 2021-04-21 고려대학교 산학협력단 Biomaker miRNA4668-5p for diagnosing liver fibrosis
KR102256680B1 (en) * 2020-04-09 2021-05-27 가톨릭대학교 산학협력단 Composition using mirna-101 for differntiation of mesenchymal stem cell into hepatocyte-like cell and composition using mirna-101 for diagnosis, prevention or treatment of hepatic fibrosis
CN113730427A (en) * 2021-08-17 2021-12-03 华南理工大学 Application of miRNA-6766-3p in preparation of medicine for preventing and/or treating hepatic fibrosis

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* Cited by examiner, † Cited by third party
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JP2016101153A (en) * 2014-11-28 2016-06-02 コリア・インスティテュート・オブ・サイエンス・アンド・テクノロジー Microrna for identifying presence or absence of exposure against hexanal, and identification method using the same
KR102242639B1 (en) * 2020-01-28 2021-04-21 고려대학교 산학협력단 Biomaker miRNA4668-5p for diagnosing liver fibrosis
KR102256680B1 (en) * 2020-04-09 2021-05-27 가톨릭대학교 산학협력단 Composition using mirna-101 for differntiation of mesenchymal stem cell into hepatocyte-like cell and composition using mirna-101 for diagnosis, prevention or treatment of hepatic fibrosis
CN113730427A (en) * 2021-08-17 2021-12-03 华南理工大学 Application of miRNA-6766-3p in preparation of medicine for preventing and/or treating hepatic fibrosis

Non-Patent Citations (2)

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Title
CircPan3 Promotes the Ghrelin System and Chondrocyte Autophagy by Sponging miR-667-5p During Rat Osteoarthritis Pathogenesis;Jing Zeng等;《Front Cell Dev Biol.》;第9卷;第1-15页 *
Identifying the Biomarkers of Spinal Cord Injury and the effects of Neurotrophin-3 Based on MicroRNA and mRNA Signature;Shuang Qi等;《Research Square》;第8页第1段 *

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