CN110484614B - Application of LncRNA XLOC _057528 inhibitor in preparation of medicine for promoting angiogenesis - Google Patents
Application of LncRNA XLOC _057528 inhibitor in preparation of medicine for promoting angiogenesis Download PDFInfo
- Publication number
- CN110484614B CN110484614B CN201910656142.6A CN201910656142A CN110484614B CN 110484614 B CN110484614 B CN 110484614B CN 201910656142 A CN201910656142 A CN 201910656142A CN 110484614 B CN110484614 B CN 110484614B
- Authority
- CN
- China
- Prior art keywords
- lncrna xloc
- lncrna
- xloc
- endothelial cells
- human umbilical
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Heart & Thoracic Surgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Cardiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Vascular Medicine (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses application of an inhibitor of lncRNA XLOC _057528 in preparation of a medicament for promoting angiogenesis. The overexpression and silencing of lncRNA XLOC _057528 prove that the promoter can influence the expression of p53 gene and protein in human umbilical vein endothelial cells, and simultaneously prove that the promoter participates in the promotion of the proliferation of the human umbilical vein endothelial cells, the inhibition of the apoptosis of the human umbilical vein endothelial cells and the inhibition of the tubule formation of the human umbilical vein endothelial cells, and further proves that lncRNA XLOC _057528 may participate in the inhibition of angiogenesis after myocardial infarction. Therefore, the inhibitor of lncRNA XLOC _057528 can be used for preparing a medicament for promoting angiogenesis, and particularly, the inhibitor of lncRNA XLOC _057528 can be used for preparing a medicament for promoting angiogenesis after myocardial infarction.
Description
Technical Field
The invention belongs to the technical field of cell engineering and genetic engineering, and particularly relates to application of an inhibitor of long-chain non-coding RNA XLOC _057528(lncRNA XLOC _057528) in preparation of a medicament for promoting angiogenesis.
Background
Myocardial Infarction (MI) is a cardiovascular disease that seriously compromises human health, based on the narrowing of coronary atherosclerosis, the formation of blood clots that block the lumen of coronary arteries, and ultimately lead to Myocardial necrosis due to prolonged ischemia, known as "first killer of human health".
Angiogenesis refers to the development of new blood vessels from existing capillaries or veins behind capillaries, primarily through the formation of well-functioning blood vessels by means of budding, primarily involving the proliferation, budding and migration of endothelial cells. In damaged hearts, effective vascular remodeling allows the focal zone to recover to some extent, and vascular endothelial cells play an important role in angiogenesis following injury to the adult heart.
Long Non-coding RNA (lncRNA) is a type of Non-coding RNA with the length of more than 200nt, and most lncRNA has no protein coding capacity. An increasing number of studies have shown that lncRNA, as an important regulator, is involved in the development and/or progression of various diseases, including MI, which plays a crucial role in cardiovascular physiology and pathology. In recent years, a lot of documents report that lncRNA influences angiogenesis by regulating Human Umbilical Vein Endothelial Cells (HUVECs) in tumor diseases, and angiogenesis after MI is rarely reported.
Disclosure of Invention
The invention aims to provide application of an inhibitor of long-chain non-coding RNA XLOC _057528(lncRNA XLOC _057528) in preparation of a medicament for promoting angiogenesis.
The application of lncRNA XLOC _057528 in angiogenesis after myocardial infarction is researched by using a molecular cell biology method, the application of lncRNA XLOC _057528 in human umbilical vein endothelial cells is proved for the first time, the overexpression and silencing of lncRNA XLOC _057528 prove that the application can influence the expression of mRNA and protein of a target gene p53 in the human umbilical vein endothelial cells, and meanwhile, the application is proved to participate in promoting the proliferation of the human umbilical vein endothelial cells, inhibiting the apoptosis of the human umbilical vein endothelial cells and inhibiting the tubule formation of the human umbilical vein endothelial cells, and further, the lncRNA XLOC _057528 is possibly involved in inhibiting the angiogenesis after myocardial infarction. Therefore, the inhibitor of lncRNA XLOC _057528 can be applied to preparation of a medicament for promoting angiogenesis, and particularly can be applied to preparation of a medicament for promoting angiogenesis after myocardial infarction.
The nucleotide sequence of the lncRNA XLOC _057528 is shown in SEQ ID NO. 1.
The inhibitor of lncRNA XLOC _057528 is siRNA of lncRNA XLOC _057528 or RNA interference vector of lncRNA XLOC _ 057528.
The invention also provides a medicament for promoting angiogenesis, which comprises an effective amount of an inhibitor of lncRNA XLOC _ 057528. Preferably, the medicament for promoting angiogenesis is a medicament for promoting angiogenesis after myocardial infarction.
The invention changes the expression quantity of lncRNA XLOC _057528 in human umbilical vein endothelial cells by a genetic engineering technology, and proves that: under the in vitro environment, the lncRNA XLOC _057528 promotes the expression of mRNA and protein of a target gene p53 in endothelial cells; the lncRNA XLOC _057528 promotes the proliferation of human umbilical vein endothelial cells and inhibits the apoptosis of the human umbilical vein endothelial cells; lncRNA XLOC _057528 inhibited tubule formation by human umbilical vein endothelial cells.
The specific verification test and results are as follows:
1. the invention detects that the expression level of P53mRNA is increased (P <0.01) compared with a control group (pcDNA3.1(-)) after the lncRNA XLOC _057528 is over-expressed in human umbilical vein endothelial cells by a qRT-PCR method, and the expression level of P53mRNA is reduced (P <0.01) compared with the control group (NC) after the lncRNA XLOC _057528 is silenced (figure 1).
2. The invention detects that the expression level of P53 protein is increased (P <0.05) compared with that of a control group (pcDNA3.1(-)) after the lncRNA XLOC _057528 is over-expressed in human umbilical vein endothelial cells by a Western Blot method, and the expression level of P53 protein is reduced (P <0.01) compared with that of the control group (NC) after lncRNA XLOC _057528 is silenced (figure 2).
3. The invention detects that after lncRNA XLOC _057528 is over-expressed in human umbilical vein endothelial cells by an EdU method, the proliferation rate of the human umbilical vein endothelial cells is increased (P <0.01) compared with a control group (pcDNA3.1 (-))) (figure 3), and after lncRNA XLOC _057528 is silenced, the proliferation rate of the human umbilical vein endothelial cells is reduced (P <0.05) compared with the control group (NC) (figure 4).
4. The invention detects that after lncRNA XLOC _057528 is over-expressed in human umbilical vein endothelial cells by Annexin V-FITC/PI technology, the apoptosis rate of the human umbilical vein endothelial cells is reduced (P <0.01) compared with a control group (pcDNA3.1(-)) (FIG. 5), and after lncRNA XLOC _057528 is silenced, the apoptosis rate of the human umbilical vein endothelial cells is increased (P <0.01) compared with the control group (NC) (FIG. 6).
5. The invention detects that the tube forming number of the human umbilical vein endothelial cells is reduced (P <0.01) compared with a control group (Blank) after the lncRNA XLOC _057528 is over-expressed in the human umbilical vein endothelial cells by a Matrigel method (figure 7), and the tube forming number of the human umbilical vein endothelial cells is increased (P <0.01) compared with the control group (Blank) after the lncRNA XLOC _057528 is silenced (figure 8).
The invention has the beneficial effects that:
the invention detects the expression quantity of p53 at two levels of gene and protein after the overexpression/silencing of lncRNA XLOC _057528, and simultaneously detects the proliferation, apoptosis and tube formation conditions of the p53 in human umbilical vein endothelial cells, thereby laying a foundation for the molecular mechanism and action mechanism of the p53 in angiogenesis. Meanwhile, the result of the invention shows that lncRNA XLOC _057528 promotes the expression of p53 gene and protein in endothelial cells, participates in promoting the proliferation of human umbilical vein endothelial cells, inhibits the apoptosis of the human umbilical vein endothelial cells and inhibits the tubule formation of the human umbilical vein endothelial cells, and further shows that lncRNA XLOC _057528 may have an inhibiting effect on the post-infarction angiogenesis, which provides theoretical basis and clinical basis for researching a new target point and a new strategy for preventing and treating myocardial infarction. The inhibitor of lncRNA XLOC _057528 can be used for preparing medicaments for promoting angiogenesis, and particularly can be used for preparing medicaments for promoting angiogenesis after myocardial infarction.
Drawings
FIG. 1 is a graph showing the results of qRT-PCR method for detecting p53 gene expression after overexpression/silencing of lncRNA XLOC _ 057528. Wherein pcDNA3.1(-) -lncRNA XLOC _057528 represents overexpressed lncRNA XLOC _ 057528; pcDNA3.1(-) as control group; lncRNA XLOC _057528Silencer represents silent lncRNA XLOC _ 057528; NC is a control group.
FIG. 2 is a graph showing the result of Western Blot method for detecting the expression of p53 protein after the overexpression/silencing of lncRNA XLOC _ 057528. Wherein pcDNA3.1(-) -lncRNA XLOC _057528 represents overexpressed lncRNA XLOC _ 057528; pcDNA3.1(-) as control group; lncRNA XLOC _057528Silencer represents silent lncRNA XLOC _ 057528; NC is a control group.
FIG. 3 is a graph showing the result of EdU method detecting proliferation of endothelial cells in human umbilical vein after overexpression of lncRNA XLOC _ 057528. Wherein pcDNA3.1(-) -lncRNA XLOC _057528 indicates overexpressed lncRNA XLOC _ 057528; pcDNA3.1(-) was a control group.
FIG. 4 is a graph showing the results of EdU assay for human umbilical vein endothelial cell proliferation following silencing of IncRNA XLOC-057528. Wherein lncRNA XLOC _057528Silencer represents silent lncRNA XLOC _ 057528; NC is a control group.
FIG. 5 is a graph showing the results of human umbilical vein endothelial cell apoptosis after the Annexin V-FITC method for detecting overexpression of lncRNA XLOC _ 057528. Wherein pcDNA3.1(-) -lncRNA XLOC _057528 indicates overexpressed lncRNA XLOC _ 057528; pcDNA3.1(-) was used as a control.
FIG. 6 is a graph showing the results of the Annexin V-FITC method for detecting human umbilical vein endothelial cell apoptosis after lncRNA XLOC _057528 silencing. Wherein lncRNA XLOC _057528Silencer represents silent lncRNA XLOC _ 057528; NC is a control group.
FIG. 7 is a graph showing the results of human umbilical vein endothelial cell tubule formation after Matrigel method detecting overexpression of lncRNA XLOC _ 057528. Wherein pcDNA3.1(-) -lncRNA XLOC _057528 represents overexpressed lncRNA XLOC _ 057528; blank is a control group.
FIG. 8 is a graph of the results of Matrigel assay for endothelial tubule formation in human umbilical vein following silencing of lncRNA XLOC _ 057528. Wherein lncRNA XLOC _057528Silencer represents silent lncRNA XLOC _ 057528; blank is a control group.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
lncRNA XLOC _057528Silencer in the following examples is synthesized by runbo biotechnology limited, guangzhou, with the ability to silence lncRNA XLOC _ 057528; lncRNA XLOC _057528Silencer NC does not silence lncRNA XLOC _057528, is a control for lncRNA XLOC _057528Silencer, provided by lunchbo biotechnology, ltd, guangzhou.
Example 1
1. Construction of Gene overexpression vectors
And screening the lncRNA related to the myocardial infarction XLOC _057528 from the differential lncRNA as a research object. DNAMAN software analysis shows that the full-length sequence of the lncRNA XLOC _057528 gene has no Kpn I restriction enzyme cutting sites and no Nhe I restriction enzyme cutting sites, while pcDNA3.1(-) vector (purchased from BioMed company, the product number is CL408-01) has Kpn I restriction enzyme cutting sites and Nhe I restriction enzyme cutting sites. primer premier 5.0 software designs gene full length primer, and its upstream and downstream primer add Kpn I and Nhe I enzyme cutting site sequence. Using human umbilical vein endothelial cell cDNA as a template, amplifying a target fragment by PCR (the amplified target fragment is a IncRNA XLOC _057528 sequence, and the nucleotide sequence of the amplified target fragment is shown as SEQ ID NO. 1), purifying, recovering, carrying out double digestion, connecting with a pcDNA3.1(-) vector, converting, screening, sequencing and identifying the correct sequence, and then extracting an endotoxin-free plasmid (an endotoxin-free plasmid small quantity extraction kit is purchased from Magen company) which is named as pcDNA3.1(-) -IncRNA XLOC _057528 (containing the IncRNA XLOC _057528 sequence).
2. Culture of human umbilical vein endothelial cells
(1) The endothelial cells were removed from the liquid nitrogen tank and thawed in a 37 ℃ water bath.
(2) Preparing an ECM complete medium: 93% ECM + 5% FBS + 1% ECGS + 1% double antibody.
(3) The thawed cells were added to an appropriate amount of complete medium and centrifuged at 1000rpm for 5min at room temperature.
(4) The supernatant was discarded, and the cells were washed with an appropriate amount of PBS buffer and centrifuged at 1000rpm at room temperature for 5 min.
(5) Discarding the supernatant, suspending the cells with the complete culture medium, and inoculating the cells in a 75mL culture bottle; standing at 37 deg.C and 5% CO 2 And (5) standing and culturing in an incubator.
The double-resistance is penicillin and streptomycin.
3. Cell RNA extraction and reverse transcription
The total RNA of the cells is extracted according to the TRIzol operating instruction of Takara company, and the specific operating steps are as follows:
(1) culturing human umbilical vein endothelial cells to a proper density, removing the culture medium, washing the cells twice with PBS, adding 1000. mu.L TRIzol according to the cell amount, and repeatedly blowing and beating for several times to fully lyse the cells.
(2) The lysate was transferred to a new 1.5mL RNase-free centrifuge tube and incubated on ice for 5min to allow complete separation of the nucleoprotein complex.
(3) Adding 200 μ L chloroform, shaking vigorously for 15s, incubating with ice for 5min, centrifuging at 4 deg.C and 11000rpm for 15min to obtain a mixture with three layers, wherein RNA is located in the upper water phase.
(4) The upper aqueous phase was transferred to a new 1.5mL RNase-free centrifuge tube, 500. mu.L isopropanol was added and mixed well, incubated on ice for 10min, and centrifuged at 1100rpm at 4 ℃ for 10 min.
(5) The supernatant was removed and the pellet carefully washed with 75% ethanol and centrifuged at 7500rpm for 3min at 4 ℃.
(6) The supernatant was discarded, the RNA was dried to a wet state, and a suitable amount of DEPC water was used to dissolve the RNA precipitate.
mRNA reverse transcription was performed by reference to PrimeScript of TaKaRa TM RT Master Mix (Perfect Real Time) cDNA reverse transcription kit.
4. Seeding and transfection of human umbilical vein endothelial cells
(1) When the confluence degree of human umbilical vein endothelial cells reaches 90-95%, the culture medium is discarded, and the cells are washed for 2 times by PBS.
(2) Appropriate amount of trypsin is used for digestion, and the digestion is stopped by adding the same amount of complete culture medium immediately after the digestion is complete.
(3) PBS wash 2 times during 1000rpm room temperature centrifugation for 5 min.
(4) Gently resuspending the cell pellet with complete medium, evenly dividing into each well, supplementing volume with complete medium, gently shaking, and culturing in an incubator.
(5) The next day, the endothelial cell state is observed, and transfection can be carried out when the cell confluency reaches 70% -90%.
(6) Transfection method according to Invitrogen
3000 kit instructions; the groups were divided into 4 groups, each of which was a group overexpressing lncRNA XLOC _057528 (transfected pcdna3.1(-) -lncRNA XLOC _057528), a control group (transfected pcdna3.1(-)), a group silencing lncRNA XLOC _057528 (transfected lncRNA XLOC _057528 silener, said lncRNA XLOC _057528 silener having the function of silencing lncRNA XLOC _057528, which was composed of 6 sequences, 3 ASO sequences and 3 siRNA sequences), and a control group NC (transfected lncRNA XLOC _057528 silener NC, said lncRNA XLOC _057528 silener NC not having the function of silencing lncRNA oc _057528, which was composed of 2 sequences, 1 ASO sequence and 1 siRNA sequence), each of which was provided with 3 repeats.
(7) Transfected cells were incubated at 37 ℃ in 5% CO 2 And continuing culturing in the incubator.
(8) Cells were collected 24-48 h after transfection according to experimental objectives.
5. qRT-PCR detection of p53 Gene expression after overexpression/silencing of lncRNA XLOC _057528
In the present invention, the relative expression level of the gene was detected using Maxima SYBR Green qPCR Master Mix (2X) kit (Thermo Scientific Co.). The experimental result adopts a Ct value comparison method to detect the gene content of the sample, and the specific calculation formula is as follows:
relative gene expression level of 2 - { < gene Ct value of experimental group-Control group internal reference gene Ct value > - < - > (control group target gene Ct value > < - >
Wherein, the beta-Actin is used as an internal reference gene, and the qRT-PCR primer used by the invention is as follows:
qRT-PCR-p53Forward：5′-CAGCCTGGGCACAACAATTC-3′；
Reverse：5′-GTGCTCCTCGTGCTTACACT-3′；
qRT-PCR-β-Actin Forward：5′-TCCCTGGAGAAGAGCTACGA-3′；
Reverse：5′-AGCACTGTGTTGGCGTACAG-3′。
the invention exogenously synthesizes lncRNA XLOC _057528 overexpression vector (pcDNA3.1(-) -lncRNA XLOC _057528) and lncRNA function inhibition silencing reagent (lncRNA XLOC _057528Silencer) by gene engineering technology, transfects the lncRNA XLOC _057528Silencer into human umbilical vein endothelial cells, carries out qRT-PCR after 24h, and detects the p53mRNA expression condition. The results showed that the expression level of P53 gene was increased (P <0.01) compared to the control group (pcDNA3.1(-)) after overexpression of lncRNA XLOC _057528, and that the expression level of P53 gene was decreased (P <0.01) compared to the control group (NC) after silencing of lncRNA XLOC _057528 (FIG. 1).
6. Western Blot detection of p53 protein expression after overexpression/silencing of lncRNA XLOC _057528
(1) Extraction of Total cellular protein
a) 48h after cell transfection, cells were washed 3 times with PBS, 100-200. mu.L of protein lysate and 10. mu.L of 100mM PMSF were added, and lysed cells were transferred to 1.5mL EP tubes.
b) The cells were shaken until lysis was complete.
c) The mixture was centrifuged at 12000rpm at 4 ℃ for 15min, and the supernatant was transferred to a new 1.5mL EP tube and stored at a low temperature.
(2) BCA method for determining protein concentration
a) Preparation of protein standard: the standard protein stock solution of 2mg/mL was diluted with PBS to give a working solution of 0.5mg/mL, and stored at low temperature.
b) Preparing a BCA working solution: the BCA working solution was prepared in an amount of 200uL per sample.
c) Adding the standard substance 0, 2, 4, 8 and 16 μ L into the standard substance hole of a 96-well plate in sequence, and supplementing PBS to 20 μ L.
d) mu.L of diluted protein sample was added to each well of a 96-well plate standard well (protein sample PBS ═ 2: 18).
e) Add 200. mu.L BCA working solution to each well, mix well, incubate for 30min at 37 ℃.
f) The OD value of A570nm was measured for each porin. The extracted total protein is added with 5 xSDS protein loading buffer (in a ratio of 1: 4), heated to denature, and stored at-80 ℃ for later use.
(3) Preparation of SDS-PAGE gels
a) And cleaning the glass plate and drying for later use.
b) The glass plate is fixed on a rubber making frame after being aligned, and the bottom of the glass plate is sealed by a rubber strip.
c) Preparing 10% separation gel, uniformly mixing, pouring from the bottom of the glass plate, adding isopropanol, and standing until the gel is solid.
d) The isopropanol was discarded and blotted clean with filter paper, and 5% concentrated gum was dispensed and poured quickly to fill the remaining void.
e) Inserting a comb, and after the gel is solidified, beginning to sample for electrophoresis.
(4) Protein loading: the protein sample was added to the spot well using a pipette.
(5) Gel electrophoresis: the voltage of the foam concentrated glue is 70V, and the time is 30 min; the voltage of the running separation gel is 120V.
(6) Rotary film
a) Soaking the PVDF membrane in formaldehyde for 5min, washing with clear water, and soaking in a membrane transferring solution together with the adsorption filter paper.
b) Stacking the adsorption filter paper (4 sheets), the gel, the PVDF film and the adsorption filter paper (4 sheets) in sequence from the anode, and closing the cathode. Loading into electrophoresis tank.
c) The membrane conversion process is carried out on ice, the constant current is 300mA, and the membrane conversion time is selected according to different target proteins.
(7) Protein detection and result analysis
a) The membranes were rinsed in 1 × TBST and blocked with 5% skim milk at room temperature for 2 h.
b) Primary antibody incubation: discard blocking solution, wash membrane with 1 × TBST, dilute desired antibody with antibody diluent, incubate overnight at 4 ℃.
c) The primary antibody, 1 XTBST washed membrane, was recovered.
d) And (3) secondary antibody incubation: TBST was discarded, the secondary antibody was incubated, the antibody was diluted to the desired concentration with the antibody diluent, and the incubation was carried out at room temperature for 2 h. Discard the secondary antibody and wash the membrane with 1 × TBST.
e) Color development: detection was performed by ECL chemiluminescence, exposure was performed using a Bio-Rad exposure system and pictures were taken.
f) The electrophoretic band intensity value was measured using Image J.
The invention exogenously synthesizes lncRNA XLOC _057528 overexpression vector (pcDNA3.1(-) -lncRNA XLOC _057528) and a silencing reagent (lncRNA XLOC _057528Silencer) for inhibiting lncRNA function through a genetic engineering technology, transfects the silencing reagent into human umbilical vein endothelial cells, and carries out Wetern Blot for 48h to detect the expression condition of p53 protein. The detection results show that the expression level of P53 protein is increased (P <0.05) compared with the control group (pcDNA3.1(-)) after the lncRNA XLOC _057528 is overexpressed, and the expression level of P53 protein is reduced (P <0.01) compared with the control group (NC) after the lncRNA XLOC _057528 is silenced (FIG. 2).
7. Human umbilical vein endothelial cell proliferation assay
The experiment for detecting Cell proliferation by the EdU method in the invention refers to Cell-Light of Ruibo Biotech Co., Ltd, Guangzhou City TM EdU Apollo 567In vitro Kit. The method comprises the following specific steps (taking a 48-hole plate as an example):
(1) the Edu solution was diluted with complete medium at a ratio of 1000:1 to prepare an appropriate amount of Edu medium.
(2) mu.L of Edu medium per well was incubated for 2h and the medium was discarded.
(3) The cells were washed 2 times with PBS for 5min each.
(4) mu.L of cell fixative (80% acetone diluted with PBS) per well was incubated at room temperature for 30min and the fixative was discarded.
(5) Each well was washed with 200. mu.L PBS for 5min on a decolorization shaker, and PBS was discarded.
(6) 200 μ L of 1 XApollo staining reaction solution per well was incubated for 20min in dark at room temperature in a decolorized shaker, and the staining reaction solution was discarded.
(7) Washing with 200 μ L penetrant (PBS containing 0.5% Triton X) for 10min 2 times per well, discarding penetrant, and washing with PBS for 5 min.
(8) Diluting the reagent F with deionized water according to the ratio of 100:1, preparing a proper amount of 1 × Hoechst3342 reaction solution, and storing in the dark.
(9) 200 μ L of 1 × Hoechst3342 reaction solution per well, incubating for 20min in a dark place at room temperature and a decolorizing shaker, and discarding the staining reaction solution.
(10) Washing with 200. mu.L PBS for 2 times, and preserving with 200. mu.L PBS for each well after washing.
(11) After the staining was completed, a photograph was taken with a fluorescence microscope.
The invention exogenously synthesizes lncRNA XLOC _057528 overexpression vector (pcDNA3.1(-) -lncRNA XLOC _057528) and a silencing reagent (lncRNA XLOC _057528Silencer) for lncRNA function inhibition through a genetic engineering technology, transfects the vector into human umbilical vein endothelial cells, and detects the proliferation condition of the endothelial cells after 24 hours by referring to an EdU kit instruction. The results showed that the proliferation rate of endothelial cells was increased (P <0.01) compared to the control (pcDNA3.1(-)) after overexpression of lncRNA XLOC-057528 (FIG. 3), and decreased (P <0.05) compared to the control (NC) after silencing of lncRNA XLOC-057528 (FIG. 4).
8. Human umbilical vein endothelial cell apoptosis assay
The invention uses Annexin V-FITC/PI technology to detect Apoptosis, and refers to the specification of FITC Annexin V Apoptosis Detection Kit with PI Kit of Guangzhou Korea Biotechnology Limited, the specific operation steps are as follows:
(1) taking out the cell culture plate, gently rinsing the cells in the culture plate by PBS at room temperature, and discarding the PBS;
(2) digesting with appropriate amount of trypsin, placing in incubator for about 3min, and adding equal amount of complete culture medium to terminate digestion;
(3) cells were harvested by centrifugation at 1000rpm for 5min, the supernatant discarded, and the cells were washed 2 times with pre-chilled PBS. Adjusting the number of cells per tube to 0.2-1.0X 10 6 Separately, 500. mu.L of 1 × Annexin V Buffer was added to resuspend the cells.
(4) mu.L Annexin V-FITC and 5. mu.L propidium iodide staining solution were added to each tube, mixed gently, and reacted at room temperature (25 ℃) in the dark for 15 min.
(5) Immediately after the reaction, the reaction solution was analyzed by flow cytometry.
The invention exogenously synthesizes lncRNA XLOC _057528 overexpression vector (pcDNA3.1(-) -lncRNA XLOC _057528) and lncRNA function inhibition silencing reagent (lncRNA XLOC _057528Silencer) through gene engineering technology, transfects the lncRNA into human umbilical vein endothelial cells, and detects the Apoptosis condition of the endothelial cells after 48 hours by referring to FITC Annexin V Apoptosis Detection Kit with PI specification. The results showed that the endothelial cell apoptosis rate was decreased (P <0.01) compared to the control group (pcDNA3.1(-)) after overexpression of lncRNA XLOC _057528 (FIG. 5), and increased (P <0.01) compared to the control group (NC) after silencing of lncRNA XLOC _057528 (FIG. 6).
9. Human umbilical vein endothelial cell tube formation detection
(1) Matrigel was melted overnight in a 4 ℃ freezer and a 48-well plate and tip were pre-cooled overnight in a 4 ℃ freezer.
(2) And (3) performing operation on ice: 100 μ L of matrigel per well was added to the 48 well plate to ensure complete coverage of the well bottom by the gel and to avoid the formation of air bubbles during the addition.
(3) Incubate at 37 ℃ for 60 min.
(4) The cells were digested to ensure 2 to 3 ten thousand cells per well.
(5) Incubating for 2-8 h at 37 ℃, observing under a fluorescence microscope at any time, and finishing photographing within 10 h.
The invention exogenously synthesizes lncRNA XLOC _057528 overexpression vector (pcDNA3.1(-) -lncRNA XLOC _057528) and a silencing reagent (lncRNA XLOC _057528 Silene) for lncRNA function inhibition through a genetic engineering technology, transfects the vector into human umbilical vein endothelial cells, and detects the tube forming condition of the endothelial cells after 24 hours by referring to a Matrigel matrix gel method instruction. The results of the assay showed that the number of tube formation of endothelial cells was decreased (P <0.01) after overexpression of lncRNA XLOC _057528 compared to endothelial cells without any treatment (Blank) (fig. 7), and increased (P <0.01) after silencing lncRNA XLOC _057528 compared to endothelial cells without any treatment (Blank) (fig. 8).
The invention takes lncRNA XLOC _057528 as a research object, proves that the lncRNA XLOC _057528 can influence the expression of p53 genes and proteins in human umbilical vein endothelial cells through overexpression and silencing of lncRNA XLOC _057528, simultaneously proves that the lncRNA XLOC _ 8925 participates in promoting the proliferation of the human umbilical vein endothelial cells, inhibiting the apoptosis of the human umbilical vein endothelial cells and inhibiting the tubule formation of the human umbilical vein endothelial cells, and further proves that the lncRNA XLOC _057528 possibly participates in inhibiting the angiogenesis after myocardial infarction. Therefore, the inhibitor of lncRNA XLOC _057528 can be used for preparing a medicament for promoting angiogenesis, and particularly, the inhibitor of lncRNA XLOC _057528 can be used for preparing a medicament for promoting angiogenesis after myocardial infarction.
The above are only preferred embodiments of the present invention, and it should be noted that the above preferred embodiments should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Sequence listing
<110> Guangdong province laboratory animal monitoring station
South China Agricultural University
Application of <120> LncRNA XLOC _057528 inhibitor in preparation of medicine for promoting angiogenesis
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2085
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tttcacttct ctttctactc aggctggact cagagggagc agcagtttca gcttctcatt 60
cttggaggag atggcctgct accagtcgct tggccaaacc catcatgtga gtagcgttgc 120
tttacctgtt ccccggggga ctgagggaca gccagaggga acctgtccct cccagcacaa 180
ggggtaggtg gcacattctg cacctgccag ccctcccagt gtgtcgggag atgccgtgaa 240
ggggttgcca cctccctctg atatcctttc tgcatccttt ctggatgcaa gtggcttcat 300
gtccttgcta ttccatacct gcctgaagcc tcagtttctt catatttaaa aagcctagac 360
ctggagttcc cattgtgact cagcaggtta agaacctgac tggtatccat gaggatgcag 420
gttcgatccc tggcctcact caatgggtta aggatccggc gttgccgtga gctgtggtgt 480
atgttgcaga tgcagcttgc atcctgtgtt gctgtggctg tggtatagga tggcggcagc 540
agctttgatt tggccccgag tctgggaact cacatatgcc tcagatgtgg cccttaaata 600
gacaaaaaca aaacaaaaca aaacatcgat ttctccactg ggcatcctct gtgtaaccac 660
attaaaaaat cctccctata gattttgtcc ttgaaaagat ccagccaaag atgagaatta 720
ttttcaggtg tgctccttct ccgagtttca acagcgacaa ttagctggag atgtatggtt 780
tctccagaaa aacaggaccc ataggataca cgtatgaaga catttatttg aaggtagtgg 840
ctcacgcagt tatgggggcc gtctgatcga agctgggttt tgacattcaa agtaaaataa 900
ttcccaagtc atttatttat gaaattagct ctccttcaaa ggtaataacc atggccattt 960
tcacgtgtgg agggctaaat aacaccctct gtggtgtgat cccaaggaaa tctgcttatt 1020
cctcaaagta tctttttcag tgcacctgtc ctcgaggctg aaacgccaca gttactcctt 1080
gagcccatta taaggaaaat actctgagac tttgcgcacg gcaaggtcct gctgtaaatt 1140
tggaaaggat gtgaaggttg ttcccaggag gaggcttgag tggcgtcctt gggtggcggc 1200
tgagtggctc ccccccactt gctgagcaca ggaagtagga ggaaaaaaga agcaccaagg 1260
aaaaggcgca tgggagacaa tgaaagaaag gaggtgggca ggagagggat gccctgagga 1320
agctacgggt ctaggactgt ttgagcaaca cggaagcaca gaattttagt aacgtaacag 1380
ctggtctgag cccaaccaat ctgatctccg ctctgattac tggacgtgca ctccgccccc 1440
catccacctg gaccaatgac agtctcctgc tcctggcatg accagctcac cctggtatgc 1500
aggtcctttg atcaggggat cctctcagcc ttgggcaaac tgggatgatg agtgattgcc 1560
cccccccgcc cccaatacaa ccatcaccat ggaaacttag ggacgaattt cttttgtgag 1620
tttaccaaag gggggcattt tgcctggcca acttgagttt gggtgccagg actgagactc 1680
tcaaggccga gagccaggat gctcagtgga aatgaccact cccctgtttg gtcttctggg 1740
cagacagtgt gatttttgtg cgtcctgtta gctaattgta agtggtggga attcacgtgc 1800
aggtgctctc gtgttgggct tgtctgcata cgcttcgtaa ggaagctcag tctagcgcca 1860
agagtggctg agggtcagaa gaacctgggt tctagtctca gctcatccat tgaatttcgt 1920
gtgaccttcg gtagctgttt tttctcttct caatagcctc atctataaac tgtgatgttt 1980
tgtgttagat gttgcgttca gctccggtgt gaaccctccc tgcctgtttt ccctctagaa 2040
caaaatggaa gggcttcaga aggctgggga agacgcatgc gccac 2085
Claims (4)
- The application of an inhibitor of lncRNA XLOC _057528 in preparing a medicament for promoting angiogenesis, wherein the nucleotide sequence of lncRNA XLOC _057528 is shown as SEQ ID No. 1; the inhibitor of lncRNA XLOC _057528 is siRNA of lncRNA XLOC _057528 or RNA interference vector of lncRNA XLOC _ 057528.
- 2. The use according to claim 1, in the manufacture of a medicament for promoting angiogenesis after myocardial infarction.
- 3. A medicament for promoting angiogenesis, comprising an effective amount of an inhibitor of lncRNA XLOC _057528, wherein the nucleotide sequence of lncRNA XLOC _057528 is set forth in SEQ ID No. 1; the inhibitor of lncRNA XLOC _057528 is siRNA of lncRNA XLOC _057528 or RNA interference vector of lncRNA XLOC _ 057528.
- 4. The angiogenesis promoting drug according to claim 3, wherein the drug is a drug that promotes angiogenesis after myocardial infarction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910656142.6A CN110484614B (en) | 2019-07-19 | 2019-07-19 | Application of LncRNA XLOC _057528 inhibitor in preparation of medicine for promoting angiogenesis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910656142.6A CN110484614B (en) | 2019-07-19 | 2019-07-19 | Application of LncRNA XLOC _057528 inhibitor in preparation of medicine for promoting angiogenesis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110484614A CN110484614A (en) | 2019-11-22 |
| CN110484614B true CN110484614B (en) | 2022-07-26 |
Family
ID=68547596
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910656142.6A Active CN110484614B (en) | 2019-07-19 | 2019-07-19 | Application of LncRNA XLOC _057528 inhibitor in preparation of medicine for promoting angiogenesis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110484614B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111286536B (en) * | 2020-04-24 | 2020-10-02 | 山东大学齐鲁医院(青岛) | Gene marker for diagnosing myocardial infarction and application thereof |
| CN112843083B (en) * | 2021-02-23 | 2021-09-07 | 青岛市中心医院 | Acute myocardial infarction treatment medicine |
| CN114958849B (en) * | 2022-05-26 | 2023-10-27 | 华南农业大学 | Application of lncRNACACF to miR-520b-3p adsorption in regulation of human umbilical vein endothelial cell cycle |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015162172A2 (en) * | 2014-04-22 | 2015-10-29 | Medizinische Hochschule Hannover | Incrnas for therapy and diagnosis of angiogenesis |
| EP3054012A1 (en) * | 2015-02-03 | 2016-08-10 | Johann Wolfgang Goethe-Universität, Frankfurt am Main | Long non-coding RNA for the treatment of diseases associated with endothelial dysfunction |
| US10400237B2 (en) * | 2016-03-04 | 2019-09-03 | The Administrators Of The Tulane Educational Fund | Compositions and methods for treating vascular disorders |
-
2019
- 2019-07-19 CN CN201910656142.6A patent/CN110484614B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN110484614A (en) | 2019-11-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110384800B (en) | Application of LncRNA XLOC _075168 in preparation of medicine for promoting angiogenesis | |
| CN110484614B (en) | Application of LncRNA XLOC _057528 inhibitor in preparation of medicine for promoting angiogenesis | |
| CN110403954B (en) | Application of LncRNA XLOC _110286 inhibitor in preparation of medicine for promoting angiogenesis | |
| CN110791501B (en) | Long-chain non-coding RNA and application of interference RNA thereof in treatment of atherosclerosis | |
| CN111154763B (en) | Application of long-chain non-coding RNA lncMGPF in regulation and control of pig muscle development function | |
| WO2018024034A1 (en) | Circular rna circ-nfatc3 and application thereof | |
| CN107663539B (en) | Use of circular RNA circ-PTGR1 | |
| CN106480037A (en) | A kind of long non-coding RNA and the application in diagnosis preeclampsia and target drug treatment is prepared | |
| CN104726500B (en) | Application of the MicroRNA26b 3p inhibitor in people's umbilical cord derived mesenchymal stem cell is prepared | |
| CN108913695B (en) | Application of ZEB1 in human heart fibroblast | |
| CN110846315B (en) | Long-chain non-coding RNA XLOC _102237 and application thereof in human umbilical vein endothelial cells | |
| CN109486816B (en) | Polynucleotide for treating tumor and its application | |
| CN111110691A (en) | Application of ginsenoside Rb2 in preparing medicament for preventing and/or treating atherosclerosis | |
| CN114836424B (en) | lncRNA IFFD and application thereof in porcine ovarian granulosa cells | |
| CN110846313B (en) | Long-chain non-coding RNA XLOC _025806 and application thereof in human umbilical vein endothelial cells | |
| CN114177298B (en) | Application of ADAR1 as medicine for treating pulmonary hypertension disease | |
| CN113774017B (en) | Application of circRNA XPO4 in osteogenic differentiation of interstitial cells of human aortic valve | |
| CN110157736B (en) | Method for promoting goat hair follicle stem cell proliferation | |
| CN113667733A (en) | Application of circRNA PRDM5 in diagnosis kit and therapeutic drug development of calcified aortic valve diseases | |
| CN114958849B (en) | Application of lncRNACACF to miR-520b-3p adsorption in regulation of human umbilical vein endothelial cell cycle | |
| CN112280859B (en) | Breast cancer marker and application thereof | |
| CN111549033B (en) | Lentiviral-infected human epidermal keratinocyte strain and construction method and application thereof | |
| CN114410589B (en) | Application of ETV4 gene in reprogramming Cheng Zeng-derived scar fibroblast | |
| CN113789340B (en) | Expression vector of circular RNA hsa_circ_0001741, recombinant engineering bacterium and application thereof | |
| EP4009051A1 (en) | Use of dkk1 inhibitor in prevention and/or treatment of tumor cachexia and diseases associated with diabetes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |