CN113633655A - Application of miR-574-3p inhibitor - Google Patents
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Abstract
The invention discloses an miR-574-3p inhibitor, and application thereof in the field of preparation of diabetes treatment medicines or treatment equipment. In combination with the data in the example section, qPCR detection indicates that the expression of miR-574-3p in HUVEC is significantly higher than that of a control group in a high-sugar state, and the expression of FIS1 gene of HUVEC is significantly reduced after the miR-574-3p inhibitor is added in the high-sugar state, so that the miR-574-3p inhibitor can be used for treating diabetes.
Description
Technical Field
The invention relates to the field of miRNA, in particular to application of an miR-574-3p inhibitor.
Background
Diabetes is a serious chronic disease in China, and vascular complications are lethal and disabling factors of the diabetes. Generally, hyperglycemia due to diabetes promotes the release of Reactive Oxygen Species (ROS) from vascular endothelial cells, destroys sensitive cellular components, leads to premature cell death, and reduces the availability of Nitric Oxide (NO), ultimately leading to vascular endothelial dysfunction. There are a number of sources of high-sugar-induced ROS, of which ROS produced by mitochondrial respiratory activity are key factors for endothelial dysfunction. Therefore, inhibition of mitochondrial-derived ROS is critical for protecting normal function of the vascular endothelial lining.
Mitochondrial dynamics, including mitochondrial fusion and division, are important regulatory mechanisms for mitochondrial homeostasis, closely related to cellular ROS, and imbalance thereof has been shown to contribute to diabetic vascular endothelial dysfunction. Mitochondrial fusion mainly involves MFN-1, MFN-2 and OPA-1 proteins, while mitochondrial fission-related proteins mainly include DRP1, FIS1 and MFF. Mitochondrial fusion can maintain genetic material, while mitochondrial fission can remove damaged mitochondria. Mitochondrial fusion and division are two opposite and unified regulatory processes, but excessive mitochondrial division can lead to damage of electron transport chains, mitochondrial fragmentation and increase of intracellular mitochondrial ROS.
mirnas are a class of non-coding single-stranded RNA molecules containing 20-25 nucleotides that can regulate gene expression by post-transcriptional level suppression or interference with mRNA. mirnas are involved in many cellular biological functions and the development of human diseases through the regulation of gene expression. A series of studies indicate that mirnas play an important role in the regulation of mitochondrial dynamics.
mirnas regulate gene expression at the post-transcriptional level, and are involved in cellular biological processes and disease development. At present, miRNA has been reported to regulate mitochondrial dynamics, for example, Wang et al reports that miR-484 can inhibit translation of mitochondrial fission protein FIS1 and inhibit cell FIS 1-mediated division and apoptosis, Lee et al finds that miR-200a-3p is a novel mitochondrial dynamics regulator targeting mitochondrial fission factor MFF, expression of MFF can be down-regulated through miR-200a-3p, mitochondrial elongation is positively regulated, Joshi et al finds that MFN1 is a direct target of miR-140, Guan et al reports that MFN2 is regulated by miR-106a, and Zhang et al finds that miR-21-5p/203a-3p can promote endothelial cell senescence by down-regulating DRP 1. These studies revealed the effect of different mirnas on the expression of genes associated with cellular mitochondrial dynamics (FIS1, MFN1, MFF, etc.).
However, related technical schemes and reports of miRNA inhibitors related to diabetes treatment are not available at present.
Disclosure of Invention
Based on the above, the need exists for a miR-574-3p inhibitor which can be used for treating diabetes.
An miR-574-3p inhibitor, and an application thereof in the field of preparation of diabetes treatment medicines or treatment equipment.
In combination with the data in the example section, qPCR detection indicates that the expression of miR-574-3p in HUVEC is significantly higher than that of a control group in a high-sugar state, and the expression of FIS1 gene of HUVEC is significantly reduced after the miR-574-3p inhibitor is added in the high-sugar state, so that the miR-574-3p inhibitor can be used for treating diabetes.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Wherein:
FIG. 1 is a graph comparing the expression of miR-574-3p in HUVEC in high-sugar and normal states in example 1.
FIG. 2 is a graph showing the change in expression of genes associated with cell mitochondrial morphology of HUVEC after miR-574-3p inhibition in example 2.
FIG. 3 is a photograph comparing fluorescence of HUVEC cells after miR-574-3p inhibition in example 3.
FIG. 4 is a graph of changes in the mitochondrial ROS levels of HUVEC cells following miR-574-3p inhibition in example 3.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention discloses an application of a miR-574-3p inhibitor in the field of preparation of diabetes treatment medicines or treatment equipment.
According to the invention, known public database transcriptomics data are consulted, miRNA with significant differences are screened and expressed by using a bioinformatics analysis method, and the target genes of the miRNA are predicted by using a biological platform.
At present, the two largest public databases in the world are: 1) NCBI/GEO of NIH in the United states and 2) Arrayexpress in Europe.
These two databases collect the expression data of RNAs (mRNA, miRNA, lncRNA, etc.) in almost all published articles and some unpublished articles in the world, respectively.
Sequence numbers in GEO for diabetic endothelial cell dysfunction and diabetic and normal samples versus total miRNA expression data are GSE74296, GSE21321, GSE74296 and GSE 74296.
The miRNA specifically screened is miR-574-3 p.
Whether the target Gene of the miR-574-3p and the database of mirTarbase are predicted by a bioinformatics platform miRecords is checked, the biological functions of Gene Ontology (GO) biological process and KEGG metabolic pathway for obviously enriching the target Gene and the target Gene of the miR-574-3p are shown to be closely related to mitochondrial dynamics.
In particular, the miR-574-3p can simultaneously target genes in both mitotic kinetic disruption and fusion directions (FIS1 and MFN1), suggesting that miR-574-3p may modulate the disruption and fusion bidirectional process. miR-574-3p may be involved in the regulation of biological processes associated with endothelial dysfunction in high-carbohydrate environments.
In combination with the data of example 1, the qPCR assay in the high glucose state suggested that the expression of miR-574-3p in HUVEC was significantly higher than the control group.
Combining the data of example 2, the FIS1 gene expression of HUVEC was significantly reduced after the addition of the miR-574-3p inhibitor in the high-sugar state.
In conclusion, the miR-574-3p inhibitor can be used for treating diabetes.
Specifically, the miR-574-3p inhibitor is miRNA complementary with miR-574-3p gene.
Preferably, the sequence of the miR-574-3p inhibitor is shown as SEQ ID NO: 1 is shown.
Combining the data of example 3, in a high-sugar state, MitoSox staining shows that the ROS derived from mitochondria of HUVEC cells are remarkably increased, which indicates that the oxidative stress of the cells is enhanced, and after the miR-574-3p inhibitor is added, the ROS level of HUVEC is remarkably reduced, which indicates that the miR-574-3p inhibitor is used for improving the oxidative stress state and dysfunction of endothelial cells.
The following are specific examples.
Wherein Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from north nah organism (BNCC), No.: 347734.
example 1
Expression of miR-574-3p in Human Umbilical Vein Endothelial Cells (HUVEC) under high-sugar and normal states
1. Cell culture: human Umbilical Vein Endothelial Cells (HUVEC) were thawed, and cultured in high-sugar DMEM medium (30mM glucose, DMEM basal medium, 10% fetal bovine serum) and low-sugar DMEM medium (5.5mM glucose, DMEM basal medium, 10% fetal bovine serum), respectively, after 48 hours, after digestion with Trypsin EDTA (0.05%) for 3 minutes, the cells were neutralized and collected in a centrifuge tube in complete medium containing 10% fetal bovine serum, and after 1000 cycles for 3 minutes, RNA extraction was performed.
RNA extraction and reverse transcription: according to standard TRIzol experiments in the absence of ribonucleaseProtocol (Life Technologies), the pelleted HUVEC cell pellet was digested with TRIzol reagent. Total RNA from each set of HUVECs was then extracted using RNeasy kit (Qiagen, 74104). The quality of the extracted RNA was assessed by Optical Density (OD) (spectrophotometer ND-100, Nanodrop Technologies) and gel electrophoresis (1% (w/v) agarose). Qualified RNA for further analysis should meet the following OD value criteria: OD260/280 ratio>1.8 and OD260/230 ratio<2.0. Further purification of the isolated RNA by removal of genomic DNA and use of PrimeScript with genomic DNA removerTMRT kit (real time quantification) (TAKARA, Japan) for reverse transcription.
qPCR: the StepOnePlusTM real-time PCR system (applied biosystems, Foster City, Calif., USA) and
PremixEx-TaqTM II kit (TAKARA, Japan) was subjected to PCT amplification under the following conditions: denaturation was performed at 95 ℃ for 30 seconds followed by 15 seconds at 95 ℃ for 40 cycles, and extension was performed at 60 ℃ (34 seconds) with data collected at 72 ℃ (30 seconds). Three PCR reactions were performed for each RNA sample and internal control (. beta. -actin) to obtain FIG. 1.
Wherein the relative expression level of the target gene is normalized to the relative expression level of an endogenous reference beta-actin by a 2-delta Ct method.
Wherein the sequence of the forward primer of miR-574-3p is shown as SEQ ID NO: 2, the reverse primer sequence of the miR-574-3p is shown as SEQ ID NO: 3, the sequence of the forward primer of the beta-actin is shown as SEQ ID NO: 4, the reverse primer sequence of the beta-actin is shown as SEQ ID NO: 5, respectively.
As can be seen from FIG. 1, in the High Glucose (HG) state (30mM glucose), the qPCR assay indicates that the expression of miR-574-3P in HUVEC is significantly higher than that in the Control group (Control,5.5mM glucose) (P <0.01), confirming the prediction of the previous bioinformatics analysis.
Example 2
Cell mitochondrial morphology-related gene expression change of HUVEC after miR-574-3p inhibition
1. Cell culture: human Umbilical Vein Endothelial Cells (HUVECs) were thawed and the cells were cultured for 48 hours using the following formulas, respectively: (1) control group: low-sugar DMEM medium (5.5mM glucose, DMEM basal medium, 10% fetal bovine serum); (2) high sugar group: high-glucose DMEM medium (30mM glucose, DMEM basal medium, 10% fetal bovine serum) and miR inhibitor negative control; (3) experimental groups: high-glucose DMEM medium (30mM glucose, DMEM basal medium, 10% fetal bovine serum) and miR-574-3p inhibitor. According to the operating manual, Lipofectamine3000 is mixed with miRNA inhibitor and negative control respectively, then each group of cells is added, HUVEC cells are transfected for 48 hours, supernatant is discarded, Trypsin-EDTA (0.05%) is used for digestion for 3 minutes, then complete culture medium containing 10% fetal calf serum is used for neutralization and recovery to a centrifuge tube, and after 1000 turns for 3 minutes, RNA extraction is carried out.
Wherein, the sequence of the miR-574-3p inhibitor is shown as SEQ ID NO: 1, and the sequence of the negative control is shown as SEQ ID NO: and 6.
RNA extraction and reverse transcription: the spun-pelleted HUVEC cell pellet was digested with TRIzol reagent under ribonuclease-free conditions according to standard TRIzol protocol (Life Technologies). Total RNA from each set of HUVECs was then extracted using RNeasy kit (Qiagen, 74104). The quality of the extracted RNA was assessed by Optical Density (OD) (spectrophotometer ND-100, Nanodrop Technologies) and gel electrophoresis (1% (w/v) agarose). Qualified RNA for further analysis should meet the following OD value criteria: OD260/280 ratio>1.8 and OD260/230 ratio<2.0. Further purification of the isolated RNA by removal of genomic DNA and use of PrimeScript with genomic DNA removerTMRT kit (perfect real time) (TAKARA, Japan) for reverse transcription.
qPCR: the StepOnePlusTM real-time PCR system (applied biosystems, Foster City, Calif., USA) and
premix Ex-TaqTM II kit (TAKARA, Japan) was subjected to PCT amplification under the following conditions: denaturation was carried out at 95 ℃ for 30 seconds followed by 15 seconds at 95 ℃ for 40 cycles, and extension was carried out at 60 ℃ (34 seconds), and several collections were collected at 72 ℃ (30 seconds)Accordingly. Three PCR reactions were performed for each RNA sample and internal control (. beta. -actin) to obtain FIG. 2.
Wherein the relative expression level of the target gene is normalized to the relative expression level of an endogenous reference beta-actin by a 2-delta Ct method.
Wherein the sequence of the forward primer of the FIS1 is shown as SEQ ID NO: 7, the reverse primer sequence of FIS1 is shown as SEQ ID NO: 8, the sequence of the forward primer of the beta-actin is shown as SEQ ID NO: 4, the reverse primer sequence of the beta-actin is shown as SEQ ID NO: 5, respectively.
As can be seen from FIG. 2, in the high sugar (Inh NC + HG) state (30mM glucose), qPCR detection suggests that the gene expression of HUVEC cell mitogen FIS1 is significantly increased, and the gene expression of HUVEC FIS1 is significantly decreased after the miR-574-3P inhibitor is added (Inh miR-574-3P) (P < 0.01).
Example 3
HUVEC cell mitochondrial ROS level change after miR-574-3p inhibition
1. Cell slide and culture: human Umbilical Vein Endothelial Cells (HUVECs) were thawed by placing slides in 24-well plates, plated on slides in 24-well plates, and cultured for 48 hours using the following formulations, respectively: (1) control group: low-sugar DMEM medium (5.5mM glucose, DMEM basal medium, 10% fetal bovine serum); (2) high sugar group: high-glucose DMEM medium (30mM glucose, DMEM basal medium, 10% fetal bovine serum) and miR inhibitor negative control; (3) experimental groups: high-glucose DMEM medium (30mM glucose, DMEM basal medium, 10% fetal bovine serum) and miR-574-3p inhibitor. After mixing with the miRNA inhibitor and the negative control using Lipofectamine3000, respectively, according to the manual, the cells of each group were added, and HUVEC cells were transfected for 48 hours, and then the supernatant was discarded and stained.
Wherein, the sequence of the miR-574-3p inhibitor is shown as SEQ ID NO: 1, and the sequence of the negative control is shown as SEQ ID NO: and 6.
MitoSox cell staining: mitochondrial ROS production was measured by staining with a MitoSox Red probe (5mM,4000X, Cat No.: M36008, ThermoFisher) and preparing 50ml of mixed solution of MitoSox Red12.5ul in PBS to a final concentration of 1.25. mu.M. Fibronectin was coated on the coverslips of 24-well plates at least 4 hours prior to plating the cells. After high-sugar treatment of the cells, the complete medium was discarded and the cells were washed with PBS. Next, HUVEC were incubated with Mitosox Red solution at 37 ℃ for 15 minutes in the absence of light. After the incubation was complete, the solution was discarded and incubated at 37 ℃ for a further 20 minutes with complete medium. Cells were gently washed twice with warm PBS for 5 minutes each, and then incubated with 4% paraformaldehyde (4% PFA, Cat No.:199431LT, USB) for 10 minutes. After fixation, cells were gently washed in PBS, then mounted for microscopic observation, resulting in fig. 3, and fig. 4 from fig. 3.
Wherein, in order to evaluate the signal intensity of ROS, ROS was verified in mitochondria, and relative fluorescence intensity was calculated from the fold change of the high-pool group and the experimental group from the control group. Similarly, an average intensity value was calculated for 30 cells per group.
As can be seen from FIGS. 3 and 4, in the high sugar (Inh NC + HG) state (30mM glucose), mitoSox staining showed significant increase of mitochondrial-derived ROS in HUVEC cells, suggesting that oxidative stress of cells is enhanced, and after adding miR-574-3P inhibitor (Inh miR-574-3P), ROS level of HUVEC is significantly reduced (P <0.05), suggesting that inhibition of expression of miR-574-3P contributes to improvement of oxidative stress state and dysfunction of endothelial cells.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the claims. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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<110> Shenzhen Hospital of Beijing university
<120> application of miR-574-3p
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<213> Artificial sequence
<400> 1
ugugggugug ugcaugagcg ua 22
<210> 2
<211> 30
<212> DNA
<213> Artificial sequence
<400> 2
acactccagc tgggcacgct catgcacaca 30
<210> 3
<211> 16
<212> DNA
<213> Artificial sequence
<400> 3
tggtgtcgtg gagtcg 16
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence
<400> 4
agcctcgcct ttgccgatcc 20
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence
<400> 5
acatgccgga gccgttgtcg 20
<210> 6
<211> 24
<212> RNA
<213> Artificial sequence
<400> 6
ucacaaccuc cuagaaagag uaga 24
<210> 7
<211> 23
<212> DNA
<213> Artificial sequence
<400> 7
gtctctatcc tctgtggcct tca 23
<210> 8
<211> 23
<212> DNA
<213> Artificial sequence
<400> 8
ccccgtttta tttacactca tcc 23
Claims (4)
1. An miR-574-3p inhibitor is characterized by being applied to the field of preparation of diabetes treatment medicines or treatment equipment.
2. The inhibitor of miR-574-3p according to claim 1, wherein the inhibitor of miR-574-3p is a miRNA complementary to the miR-574-3p gene.
3. The miR-574-3p inhibitor of claim 2, wherein the sequence of the miR-574-3p inhibitor is shown in SEQ ID NO: 1 is shown.
4. The miR-574-3p inhibitor according to any one of claims 1 to 3, wherein the miR-574-3p inhibitor is used for improving the oxidative stress state and dysfunction of endothelial cells.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009149182A1 (en) * | 2008-06-04 | 2009-12-10 | The Board Of Regents Of The University Of Texas System | Modulation of gene expression through endogenous small rna targeting of gene promoters |
| CN111904974A (en) * | 2020-08-28 | 2020-11-10 | 中国人民解放军北部战区总医院 | medical application of miR-574-5p in diabetes and related diseases thereof |
-
2021
- 2021-09-28 CN CN202111143666.9A patent/CN113633655A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009149182A1 (en) * | 2008-06-04 | 2009-12-10 | The Board Of Regents Of The University Of Texas System | Modulation of gene expression through endogenous small rna targeting of gene promoters |
| CN111904974A (en) * | 2020-08-28 | 2020-11-10 | 中国人民解放军北部战区总医院 | medical application of miR-574-5p in diabetes and related diseases thereof |
Non-Patent Citations (4)
| Title |
|---|
| TIANYU FENG等: "Weighted Gene Coexpression Network Analysis Identified MicroRNA Coexpression Modules and Related Pathways in Type 2 Diabetes Mellitus", 《OXIDATIVE MEDICINE AND CELLULAR LONGEVITY》 * |
| XIAOHUI GONG等: "Adrenomedullin regulated by miRNA-574-3p protects premature infants with bronchopulmonary dysplasia", 《BIOSCIENCE REPORTS》 * |
| 韦登文等: "瑞芬太尼通过调控miR-574-3p对缺氧/复氧诱导的心肌细胞损伤的影响及机制研究", 《解放军医药杂志》 * |
| 高雁鸿等: "血管内皮细胞功能与糖尿病的关系研究进展", 《山西大同大学学报(自然科学版)》 * |
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