CN118222693A - Application of micro RNA for inhibiting expression of angiopoietin-like protein 3 with high activity - Google Patents
Application of micro RNA for inhibiting expression of angiopoietin-like protein 3 with high activity Download PDFInfo
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- CN118222693A CN118222693A CN202310061091.9A CN202310061091A CN118222693A CN 118222693 A CN118222693 A CN 118222693A CN 202310061091 A CN202310061091 A CN 202310061091A CN 118222693 A CN118222693 A CN 118222693A
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Abstract
The invention discloses application of human endogenous non-coding micro RNA or a bioactive functional fragment or a precursor or a variant thereof in preparing a medicament for diagnosing, preventing and/or treating diseases related to elevation of angiopoietin-like protein 3 (ANGPTL 3), wherein the micro RNA is miR-374c-5p. The invention discloses miR-374C-5p for the first time, which can be combined with a 3 '-end untranslated region (3' UTR) of mRNA transcribed by an ANGPTL3 gene, so that the mRNA and protein level of the ANGPTL3 can be reduced, the hydrolysis capability of lipoprotein lipase (LPL) to triglyceride can be obviously improved, the activity of Endothelial Lipase (EL) can be released, the serum LDL-C level can be reduced, and thus, the liver cell vacuole-like steatosis can be obviously improved, the liver disease and liver fibrosis can be inhibited and treated, the formation of atherosclerosis plaques can be effectively inhibited, and the cardiovascular disease can be prevented and treated.
Description
Technical Field
The invention belongs to the field of biomedical engineering, relates to human endogenous non-coding microRNA (microRNA, miRNA) and application thereof, and in particular relates to application of miR-374c-5p and a bioactive functional fragment or variant or a chemical derivative thereof in preparation of medicaments for diagnosing, preventing and/or treating cardiovascular diseases such as hypertriglyceridemia, hypercholesterolemia, hyperlipidemia, non-alcoholic fatty liver disease, liver fibrosis, vascular atherosclerosis and other diseases related to elevation of angiopoietin-like protein 3 (ANGPTL 3).
Background
Hyperlipidemia (HYPERLIPIDAEMIA) is a disorder in which plasma cholesterol or triglyceride exceeds a normal range due to abnormal fat metabolism or transport, and is an important risk factor for inducing cardiovascular and cerebrovascular diseases such as atherosclerosis and coronary heart disease (Expert opinion on therapeutic targets,2020,24 (1): 79-88). Hyperlipidemia is mainly expressed as abnormal lipid metabolism such as triglyceride, lipoprotein, cholesterol, etc. in plasma, and can be classified into hypercholesterolemia, hypertriglyceridemia, hyperchlorhydria, hyperlipidemia, and mixed hyperlipidemia, etc. (LIPIDS HEALTH DIS,2017,16 (1): 233). Epidemiological studies have confirmed that hypertriglyceridemia (TG) is closely related to metabolic diseases such as hyperlipidemia, non-alcoholic fatty liver disease, liver fibrosis, and the like. In addition, high plasma TG levels are closely related to the development of cardiovascular disease, and have become risk factors for inducing cardiovascular disease such as atherosclerosis (Nature reviews cardiology,2017,14 (7): 401-411). Therefore, the development of the medicine for reducing the hypertriglyceridemia has important significance for preventing and treating the related diseases such as hyperlipidemia, nonalcoholic fatty liver, liver fibrosis, atherosclerosis and the like.
The angiopoietin-like protein (ANGPTLs) family consists of 8 protein members, whose structure shares similarities with the vascular endothelial growth factor family. Since ANGPTLs has been an important regulator of Lipid and glucose metabolism (Expert opinion on therapeutic targets,2020,24(1):79-88;Lipids Health Dis,2017,16(1):233;Front Endocrinol(Lausanne),2020,11:504). lipoprotein lipase (LPL) which catalyzes the hydrolysis of triglycerides and ANGPTL3 converts LPL homodimers into monomers which lose enzymatic activity (Endocr Rev,2019,40(2):537-557;Trends in Endocrinology&Metabolism,2021,32(1):48-61;Clinica Chimica Acta,2020,503:19-34)., studies have shown that inhibiting angiopoietin-like protein 3 (ANGPTL 3) can release EL activity through the Endothelial Lipase (EL) pathway, reduce the Lipid content and size of Very Low Density Lipoprotein (VLDL) particles, allow them to become remnant particles to be cleared, ultimately limiting Low Density Lipoprotein (LDL) particle production, and thus reduce serum low density lipoprotein cholesterol (LDL-C) levels (J Lipid Res,2020,61 (9): 1271-1286). Thus, ANGPTL3 has an important role (Front Endocrinol(Lausanne),2020,11:504;Endocr Rev,2019,40(2):537-557;Global Cardiology Science and Practice,2017,2017(1):e201706), in regulating lipid and glucose metabolism and has become an important target for the treatment of hyperlipidemia and related diseases.
A variety of ANGPTL3 inhibitors are currently under development or marketed in batches for the treatment of Gao Chunge subfamily hypercholesterolemia, hypertriglyceridemia, obesity, etc., and mainly include the following: ① Monoclonal antibody Evinacumab (N Engl J Med,2020,383 (8): 711-720), which blocks binding of ANGPTL3 to LPL; ② Small molecule RNA (Sci Rep,2019,9 (1): 11866) was based on the principle of inhibiting the biosynthesis of ANGPTL3 and thus enhancing LPL activity. Thus, inhibiting ANGPTL3 protein levels may be an important tool in diagnosing, preventing, and/or treating these diseases associated therewith.
Endogenous non-coding micrornas (micrornas, mirnas) are single-stranded non-coding RNAs of about 22 nucleotides in length in eukaryotes, and play a broad and important regulatory role in life processes such as Cell proliferation, differentiation and apoptosis by acting on target gene mRNA to play a transcriptional and posttranscriptional inhibitory role (Cell, 2004,116 (2): 281-297). Several miRNAs have been reported to be involved in lipid metabolism regulation, for example miR-122 is the most abundant miRNA in the liver, and is also the first miRNA found to be involved in lipid metabolism regulation (Atherocelesis, 2013,227 (2): 209-215). miR-33, which is located in the intron region of the cholesterol regulatory element binding protein (Sterol-regulatory element binding proteins, SREBPs), binds to the 3' UTR of the target gene adenosine triphosphate binding cassette transporter A1 (ABCA 1) mRNA, inhibits ABCA1 translation, and thus inhibits cholesterol efflux (Journal of Biological Chemistry,2010,285 (44): 33652-33661). miR-181d-5p can bind to 3' UTR of ANGPTL3 mRNA, inhibit ANGPTL3 expression, and play a role in regulating lipid metabolism of obese patients (Sci Rep,2019,9 (1): 11866).
At present, no related report exists on the inhibition of the expression of ANGPTL3 by miR-374c-5 p.
Disclosure of Invention
The invention aims to: aiming at the defects of the prior art, the invention aims to provide the application of human endogenous non-coding micro RNA or a bioactive functional fragment or a precursor or a variant thereof in preparing medicaments for diagnosing, preventing and treating diseases related to the elevation of angiopoietin-like protein 3.
The technical scheme is as follows: in order to solve the technical problems, the invention provides application of human endogenous non-coding microRNA or a bioactive functional fragment or a precursor or a variant thereof in preparing medicaments for diagnosing, preventing and/or treating diseases related to the elevation of angiopoietin-like protein 3, wherein the microRNA is miR-374c-5p.
The invention discloses that human endogenous non-coding microRNA, namely miR-374c-5p can inhibit the expression of an ANGPTL3 protein by combining with a 3 '-end untranslated region (3' UTR) of mRNA transcribed from an ANGPTL3 gene, so that the activity of lipoprotein lipase (LPL) in hydrolyzing triglyceride is enhanced.
Further, the diseases associated with the elevation of angiopoietin-like protein 3 include one or more of hypertriglyceridemia, hypercholesterolemia, hyperchlorhydria, hyperlipidemia, nonalcoholic fatty liver disease, liver fibrosis or atherosclerotic cardiovascular disease.
Specifically, the method for effectively inhibiting the expression of the ANGPTL3 protein in liver cells and tissues by adopting miR-374C-5p provided by the invention can obviously improve the hydrolysis capability of lipoprotein lipase on triglyceride, thereby obviously reducing the serum T-CHO, TG, LDL-C level, obviously improving the cavitation-like steatosis of the liver cells of mice and effectively inhibiting the occurrence and development of vascular atherosclerosis plaques.
Further, the sequence of the miR-374c-5p is shown as SEQ ID NO. 1.
Further, the precursor sequence of the micro RNA is shown as SEQ ID NO. 5.
Specifically, the miR-374c-5p can be formed by processing a precursor miRNA (pre-miRNA), namely miR-374(Gene ID：100500807;NCBI Reference Sequence：NR_037511.1;Ensembl ID:ENSG00000283534.3;miRBase accession：MI0016684), in a host, and the miR-374c sequence (SEQ ID NO: 5) is provided by a miRBase (https:// www.mirbase.org) and has the sequence: 5'-ACACGGACAAUGAUAAUACAACCUGCUAAGUGCUAGGACACUUAGCAGGUUGUAUUAUAUCCAUCCGAGU-3'.
Further, the variant of the microRNA comprises one or more of methoxyl modification, thio modification, cholesterol modification and N-acetylgalactosamine modification of miR-374c-5 p.
Further, the use includes constructing a recombinant expression plasmid or virus containing miR-374c-5p or a biologically-active functional fragment or precursor or variant sequence thereof.
Further, the medicine contains miR-374c-5p sequence and a pharmaceutically acceptable carrier, wherein the carrier is one or more selected from gene expression carrier, virus, chitosan, cholesterol, liposome or nano-particle.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: the invention discloses miR-374C-5p for the first time, which can be combined with an untranslated region (3 'UTR) at the 3' end of mRNA transcribed from an ANGPTL3 gene to down regulate the mRNA and protein levels of ANGPTL3, remarkably improve the hydrolysis capability of lipoprotein lipase (LPL) to triglyceride, simultaneously release the activity of Endothelial Lipase (EL) and reduce the serum LDL-C level. Can obviously improve the cavitation-like fatty degeneration of liver cells, has the effects of inhibiting and treating fatty liver and liver fibrosis, effectively inhibiting the formation of atherosclerosis plaques, and has the effects of preventing and treating cardiovascular diseases.
Drawings
Fig. 1: miR-374c-5p down regulates the level of ANGPTL3 protein in human hepatoma cell HepG2 and mouse hepatoma cell Hepa 1-6. (FIG. 1A: targetScan7.2 software predicts 3 miR-374C-5p action targets in human ANGPTL 3' UTR, wherein target I has sequence conservation in human, chimpanzee (Chimpanzee) and Mouse (Mouse), and target II, III has sequence conservation in human, chimpanzee; FIG. 1B-C: western blot results show that miR-374C-5p and anti-miR-374C-5p respectively dose-dependently down-regulate and up-regulate ANGPTL3 protein levels in human liver cancer cells HepG2 in a concentration range of 25-100 nM; FIG. 1D: western blot results show that miR-374C-5p (100 nM) and anti-miR-374C-5p (50 nM) significantly down-regulate and up-regulate the ANGTL 3 protein levels in Mouse liver cancer cells Hepa1-6, respectively).
Fig. 2: miR-374c-5p down regulates the mRNA level of ANGPTL3 in human hepatoma cell HepG2 and mouse hepatoma cell Hepa 1-6. Fig. 2A: qRT-PCR results show that miR-374c-5p (100 nM) and anti-miR-374c-5p (50 nM) respectively down-regulate and up-regulate the level of ANGPTL3mRNA in human hepatoma cell HepG 2. Fig. 2B: qRT-PCR results show that miR-374c-5p (100 nM) and anti-miR-374c-5p (50 nM) respectively down-regulate and up-regulate the mRNA level of ANGPTL3 in mouse hepatoma cells Hepa 1-6. (n=3, mean±sem).
Fig. 3: miR-374c-5p binds to the 3 '-untranslated region (3' UTR) of human ANGPTL3 mRNA and inhibits luciferase expression. (FIG. 3A: schematic representation of the target Site of action of 3 miR-374C-5P in the mRNA 3'UTR of human ANGPTL 3. FIG. 3B: schematic representation of a double-luciferase recombinant expression vector pmirGLO-ANGPTL33' UTR obtained by cloning double-luciferase recombinant expression vector pmirGLO from human ANGPTL 3'UTR genes (ANGPTL 33' UTR fragments comprising Site I, site II and Site III, respectively, wherein Site I is positions 1-263 of human ANGPTL 3'UTR, site II is positions 247-582 of human ANGPTL 3' UTR, and SiteIII is positions 472-874 of human ANGPTL3 'UTR.) when the double-luciferase recombinant expression plasmid pmirGLO-ANGPTL 3' UTR comprises wild-type human ANGPTL 3'UTR sequences, respectively, the miR-374C-5P (100 nM) and anti-374C-5P (miR 50 nM) down-regulated the gene expression of miR); when the miR-374C-5P target sequence (seed sequence) in the recombinant expression plasmid pmirGLO-ANGPTL 3' UTR is mutated, the regulation and control effect of miR-374C-5P (100 nM) and anti-miR-374C-5P (50 nM) on the luciferase reporter gene expression is weakened, the miR-374C-5P target sequence in the pmirGLO-ANGPTL 3'UTR is wild type, and the miR-374C-5P target sequence in the MUT (mutant type): pmirGLO-ANGPTL 3' UTR is mutated, wherein P is less than 0.05, P is less than 0.01, and P is less than 0.001 (n=3, mean+/-SEM)).
Fig. 4: miR-374c-5p and anti-miR-374c-5p respectively enhance and weaken the intracellular and secretion of human hepatoma cell HepG2 and mouse hepatoma cell Hepa1-6 to extracellular lipoprotein lipase (LPL) activities. miR-374C-5p (100 nM) and anti-miR-374C-5p (50 nM) enhance and attenuate the intracellular (FIG. 4A, FIG. 4C) and extracellular (FIG. 4B, FIG. 4D) lipoprotein lipase (LPL) activities of human hepatoma cell HepG2 and mouse hepatoma cell Hepa1-6, respectively. (n=3, mean±sem).
Fig. 5: after administration of miR-374c-5p agomir tail vein, mice body weight, liver ANGPTL3 protein and mRNA levels, and lipoprotein lipase (LPL) activity in serum and biochemical index change. Fig. 5A: results of mice body weight. Fig. 5B: western blot results. Fig. 5C: qRT-PCR results. Fig. 5D: results of lipoprotein lipase (LPL) activity in mouse serum. Fig. 5E-H: results for T-CHO (FIG. 5E), TG (FIG. 5F), LDL-C (FIG. 5G) and HDL-C (FIG. 5H) levels in mouse serum. Pouring ：LFCD,low fat/cholesterol diet;HFCD,high fat/cholesterol diet;NC AG,negative control agomir;AG,miR-374c-5p agomir;*P<0.05;**P<0.01;***P<0.001;****P<0.0001;(n≥3,mean±SEM).
FIG. 6 effects on liver morphology, liver lipid deposition and liver fibrosis in mice following miR-374c-5p agomir tail vein administration. Fig. 6A: mouse liver morphology. Fig. 6B: results of mouse liver HE staining. Fig. 6C: results of mice liver oil red O staining. Fig. 6D: results of mice liver Masson staining.
Fig. 7: after miR-374c-5p agomir tail vein administration, the aortic root and abdominal aortic lipid deposition of the mice are obviously reduced. Fig. 7A-B: a graph of the general staining results of atherosclerotic plaques in the abdominal aorta. Fig. 7C: oil red O staining results. Fig. 7D: HE staining results plot. Fig. 7E: improved Movat five-color counterstain result graph. * P <0.05; * P <0.001; (n=4, mean±sem).
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
Example 1 miRNA prediction and target site homology analysis targeting the 3' utr of human ANGPTL3
The miRNA targeting human ANGPTL3 mRNA(Gene Aliases：ANG-5,ANGPT5,ANL3,FHBL2;Gene ID：27329;NCBI Reference Sequence：NM_014495.4;Ensembl:ENSG00000132855/ENST00000371129.3)3'UTR was predicted using online software such as TargetScan7.2 (http:// www.targetscan.org/vet_72 /), miRDB (http:// www.mirdb.org/miRDB /) and miRWalk (http:// mirwalk. Umm. Uni-heidelberg. De /), and the results showed that 3 miR-374c-5p action target seed sequences were present in the 3' UTR of human ANGPTL 3: 5'-GUAUUAA-3'; the miR-374c-5p sequence is as follows: 5'-AUAAUACAACCUGCUAAGUGCU-3', (SEQ ID NO: 1). On this basis, miR-374c-5p target site (seed sequence) homology was analyzed by using TargetScan7.2 software, and results show that miR-374c-5p action target I has sequence conservation in humans, chimpanzees (Chimpanzee) and mice (Mouse), and action targets II and III have sequence conservation in humans and chimpanzees (FIG. 1A).
EXAMPLE 2miR-374c-5p inhibits ANGPTL3 protein expression in human hepatoma cell HepG2 and mouse hepatoma cell Hepa1-6
2.1 Materials and instruments
2.1.1. 1. 1. IRNA and its control were synthesized by Shanghai Ji Ma pharmaceutical technologies Co., ltd. (GENEPHARMA):
(1)miR-374c-5p mimics(miR-374c-5p，cat#B02001)，
The sequence is as follows: 5'-AUAAUACAACCUGCUAAGUGCU-3' (SEQ ID NO: 1);
(2) MIRNA MIMICS negative control (Con miR, cat#B04002),
The sequence is as follows: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 2);
(3)miR-374c-5p inhibitor(anti-miR-374c-5p，cat#B03001)，
The sequence is as follows: 5'-AGCACUUAGCAGGUUGUAUUAU-3' (SEQ ID NO: 3);
(4) A negative control for miRNA inhibitor (ConInh, cat#B04003),
The sequence is as follows: 5'-CAGUACUUUUGUGUAGUACAA-3' (SEQ ID NO: 4).
2.1.2 Cell and plasmid sources
The human hepatoma cell HepG2, the mouse hepatoma cell Hepa1-6 and the human embryo kidney cell HEK-293T are purchased from the basic medical cell center of the basic medical institute of the national academy of medical science. The dual luciferase expression vector pmirGLO used in the present invention is a product of Promega company, USA.
2.1.3 Major reagents
The invention mainly uses the following reagents: fetal bovine serum (Fetal Bovine Serum, FBS, cat#f2442) was the sigm product; MEM medium (cat# 61100061), DMEM medium (cat# 12800017), opti-MEM medium (cat# 31985070) are Gibco products; lipofectamine 3000 (cat#L 3000015) is a Thermo product; beta-actin polyclonal antibody (cat#4970), ANGPTL3 polyclonal antibody (cat#d 320214) are products of bioengineering (Shanghai) company; HRP-labeled goat anti-rabbit IgG (H+L) (FMS-RB 01) was purchased from Nanjing Fumaisi Biotechnology Co., ltd; dual luciferase reporter assay System (Dual-Reporter ASSAY SYSTEM) (cat#e1910) is a product of Promega corporation, usa; total lipase assay kit (cat#A067-1-1), T-CHO assay kit (cat#A111-1), TG assay kit (cat#A110-1), LDL-C assay kit (cat#A113-1), HDL-C assay kit (cat#A112-1), HE staining kit (cat#D006), and oil red O dye solution (cat#D027) are Nanjing's institute of biological engineering; the modified Masson trichromatic staining kit (cat#G1346) is available from Beijing Soy Bao technology Co., ltd; russell modified Movat five-color counterstain kit (cat#BP-DL 318) is Nanjsen Bei Ga Biotech company.
2.1.4 Main instruments
The invention is mainly applied to the following instruments: PCR instrument, product of Eppendorf company; tanon 5200A full-automatic chemiluminescence image analysis system, shanghai Technical Co., ltd; fluorescence inverted microscope, zeiss product, germany; chemiluminescent microplate reader, thermo product of united states.
2.2 Method
2.2.1 Cell culture
HepG2 cells were cultured in MEM medium (complete medium) containing 10% FBS, penicillin (100U/mL) and streptomycin (100. Mu.g/mL); hepa1-6 and HEK-293T cells were cultured in DMEM medium (complete medium) containing 10% FBS, penicillin (100U/mL) and streptomycin (100. Mu.g/mL). Culturing at 37deg.C in 5% CO 2 incubator.
2.2.2 Cell transfection
Cells in the logarithmic growth phase were inoculated into 6-well plates and cultured overnight at 37℃in a 5% CO 2 incubator. When the cell confluency reached 70%, the complete medium was changed to Opti-MEM medium and incubated at 37℃in a 5% CO 2 incubator for 1h in 900ul per well. Diluting 5 mu L of Lipofectamine 3000 with 45 mu L of Opti-MEM culture medium, mixing, and standing at room temperature for 5min; mu.L of Con miR or miR-374c-5p or Con Inh or anti-miR-374c-5p stock solution with concentration of 20 mu M is diluted by 45 mu L of Opti-MEM culture medium, mixed gently and incubated for 5min at room temperature. Then, diluted miRNA was added to the diluted Lipofectamine 3000 reagent, gently mixed, incubated at room temperature for 15min to form a transfected complex, and the transfected complex was added dropwise to the cells to be transfected, and gently mixed. After culturing at 37℃in a 5% CO 2 incubator for 6 hours, the opti-MEM medium was changed to complete medium. Culturing the cells for 72 hours, and then detecting the expression level of the ANGPTL3 protein; ANGPTL3 mRNA level detection was performed after 48h of incubation.
2.2.3Western blot detection of ANGPTL3 protein expression levels in HepG2 and Hepa1-6 cells
(1) Total cell protein extraction
The medium in the cells was removed, the cells were rinsed three times with 4℃precooled PBS, the cells were collected into 1.5ml EP tubes, 100. Mu.L of RIPA cell lysate (PMSF: RIPA=1:100) was added to each tube, lysed on ice for 30min, and once every 10min, the lysed samples were centrifuged at 12000rpm at 4℃for 10min, and the supernatant was taken. Protein concentration of each sample was measured by BCA method, and sample loading volume was calculated according to experimental requirements. And respectively taking a proper amount of 5X Loading buffer and mixing with the corresponding protein sample uniformly, and carrying out denaturation in a water bath kettle at 100 ℃ for 10min, so as to carry out subsequent experiments.
(2) SDS-PAGE electrophoresis
SDS-PAGE gel is prepared according to 10% separating gel and 5% concentrating gel, the loading amount of each hole is 30 mug protein sample, and the sample is subjected to electrophoresis separation under the following conditions: electrophoresis was stopped after electrophoresis at 70V for 30min and at 110V for 60 min.
(3) Transfer film
After electrophoresis, PVDF membrane and filter paper with corresponding sizes are cut, and the PVDF membrane is soaked in methanol for 30s to activate surface groups, and then the PVDF membrane and the filter paper are transferred into a transfer buffer solution. The positive pole of commentaries on classics membrane splint is down, places from negative pole to positive pole in proper order: porous pad, 3 layers of filter paper, gel, PVDF membrane, 3 layers of filter paper and porous pad, and ensure that no bubbles exist between the layers, the membrane transfer clip is installed in a membrane transfer groove, and a membrane transfer buffer solution is added. And under the ice water bath condition, film transfer is carried out for 120min at a constant voltage of 100V.
(4) Closure
After the membrane transfer is finished, the PVDF membrane is taken out, is rinsed by TBST, is added with 5% skimmed milk powder, and is placed in a low-speed horizontal shaking table for sealing for 1h at room temperature.
(5) Film washing
After the completion of the blocking, the PVDF film was taken out, rinsed with TBST, and the residual blocking liquid was completely removed.
(6) Antibody incubation
PVDF membrane was placed in dilutions of ANGPTL3 polyclonal antibody (diluted 1:1000 with TBST containing 5% nonfat milk powder), beta-actin polyclonal antibody (diluted 1:1000 with TBST containing 5% nonfat milk powder) and incubated overnight at 4℃and then washed three times with TBST for 5 min/time. PVDF membrane was placed in HRP-labeled goat anti-rabbit IgG (H+L) (diluted 1:4000 with TBST containing 5% nonfat milk powder) dilution, which was placed on a constant temperature shaking table at 37℃and incubated slowly for 1H.
(7) Film washing
The PVDF membrane was taken out, and washed three times with TBST for 5 min/time.
(8) Color development imaging
And taking out the PVDF film, placing the PVDF film at a corresponding position of a gel imager, uniformly adding ECL color development liquid to the PVDF film, adjusting parameters of the gel imager, exposing, storing a result, and analyzing by using Image J software.
2.3 Results
The results are shown in FIGS. 1B-C: in the concentration range of 25-100 nM, miR-374c-5p and anti-miR-374c-5p respectively dose-dependently down-regulate and up-regulate the ANGPTL3 protein level in human hepatoma cell HepG 2. Fig. 1D shows: miR-374c-5p (100 nM) and anti-miR-374c-5p (50 nM) significantly down-regulate and up-regulate the level of ANGPTL3 protein in mouse hepatoma cells Hepa1-6, respectively.
EXAMPLE 3miR-374c-5p down-regulates angptl mRNA expression level in human hepatoma cell HepG2 and mouse hepatoma cell Hepa1-6
3.1 Cell culture and transfection methods were the same as in example 2
3.2QRT-PCR detection of angptl mRNA levels in HepG2 and Hepa1-6 cells
3.2.1 Total RNA extraction
Removing culture medium in cells, rinsing cells with PBS precooled at 4deg.C for three times, adding 1mL RNAiso Plus reagent, gently and repeatedly blowing, transferring lysate into RNase-free 1.5mL EP tube, standing at room temperature for 5min, adding 200 μl chloroform, shaking vigorously, standing at room temperature for 5min, and centrifuging at 12000g at 4deg.C for 15min. The colorless supernatant was aspirated into a fresh RNase-free 1.5mL EP tube, 500. Mu.L of isopropanol was added, mixed upside down, left standing at room temperature for 10min, centrifuged at 12000g at 4℃for 10min, and the supernatant was discarded to give a white RNA precipitate. 1mL of 75% ethanol was added, gently turned upside down, centrifuged at 7500g for 5min at 4℃and the supernatant discarded and the 75% ethanol wash step repeated. Drying at room temperature for 10min, adding 30 μl RNase-free water to dissolve the precipitate until the precipitate is transparent. The concentration and purity of RNA in each sample was determined by a Nanodrop2000/2000c spectrophotometer and stored at-80℃for further use.
3.2.2CDNA Synthesis
For experiments to quantify the mRNA level of the gene of interest, cDNA was synthesized by referring to the following procedure.
(1) The genomic DNA removal reaction system was prepared by sequentially adding the reagents to 200. Mu.L PCR tubes in an ice box, mixing the mixture, reacting at 42℃for 2min, and preserving the mixture at 4℃for later use, referring to Table 1-1.
TABLE 1-1 reaction System for removing genomic DNA
(2) Reverse transcription reaction System referring to tables 1-2, reagents were sequentially added to the above EP tube on an ice box, and gently mixed
Then, the reaction was carried out at 37℃for 15min and at 85℃for 5sec, and the reaction mixture was kept at 4℃for further use.
TABLE 1-2 reverse transcription reaction System reagent Table
3.2.3 qRT-PCR
The primers required for qRT-PCR (see tables 1-3) were synthesized by Shanghai Bioengineering Co. The synthesized cDNA was diluted to 100 ng/. Mu.L with sterilized water, a reaction system was established according to tables 1 to 4, and quantitative PCR reactions were performed according to the procedures shown in tables 1 to 5. And analyzing the experimental result by using a delta Ct method, detecting the mRNA expression level of the target gene, and taking beta-actin as an internal reference.
TABLE 1-3 qRT-PCR primers
Tables 1-4 preparation of the reaction mixtures
TABLE 1-5 qRT-PCR reaction procedure
The results are shown in FIGS. 2A-B: in human liver cancer cells HepG2 and mouse liver cancer cells Hepa1-6, miR-374c-5p (100 nM) significantly down-regulates the mRNA level of ANGPTL3, and anti-miR-374c-5p (50 nM) significantly up-regulates the mRNA level of ANGPTL 3.
Example 4miR-374c-5p binds directly and acts on a target site in the 3' UTR of human ANGPTL3
Construction of 4.1pmirGLO-ANGPTL 3' UTR recombinant plasmid
Amplifying human ANGPTL 3'UTR fragment (874 bp, derived from the 1431-2304 bit sequence (figure 3A) in NCBI Reference Sequence:NM_014495.4 by PCR technology, specifically referring to the sequence table SEQ ID NO: 6), cloning to a double luciferase reporter gene expression vector pmirGLO, constructing a double luciferase recombinant expression plasmid pmirGLO-ANGPTL 3' UTR containing SiteⅠ,Position 1-263of Homo ANGPTL3 3'UTR;SiteⅡ,Position 247-582of Homo ANGPTL3 3'UTR;SiteIII,Position 472-874of Homo ANGPTL3 3'UTR) containing natural human ANGPTL 3'UTR sequence (respectively constructing double luciferase recombinant expression plasmid pmirGLO-ANGPTL 3' UTR containing SiteⅠ,Position 1-263of Homo ANGPTL3 3'UTR;SiteⅡ,Position 247-582of Homo ANGPTL3 3'UTR;SiteIII,Position 472-874of Homo ANGPTL3 3'UTR)), constructing double luciferase recombinant expression plasmid pmirGLO-ANGPTL 3'UTR-MUT (mutation site see figure 3B) with the miR-374c-5p acting target seed sequence in human ANGPTL 3' UTR sequence by gene site-specific mutation technology, transforming E.coli DH-5α competent cells, PCR screening cloning, delivering to biological engineering (Shanghai) company for gene sequencing, and comparing gene sequences by DNAMAN software.
4.2 Dual luciferase reporter analysis
Cells HEK-293T in logarithmic growth phase were inoculated in 12-well plates and cultured overnight at 37℃with 5% CO 2. When the cell confluency reached 70%, HEK-293T cells were co-transfected with 1. Mu. g pmirGLO-ANGPTL 3'UTR or pmirGLO-ANGPTL 3' UTR-MUT plasmid and 100nM Con miR or miR-374c-5p or 50nM Con Inh or anti-miR-374c-5p, respectively, using Lipofectamine 3000, 3 multiplex wells were set per group. After 24h transfection, the operation is carried out according to the specification of double-luciferase reporter gene detection, and the specific steps are as follows: (1) removing the medium from the cells; (2) rinsing the cells with PBS 2 times; (3) 250. Mu.L of 1 XPLB was added per well; (4) passive cleavage: fully lysing for 15min at room temperature, and transferring the cell lysate into a centrifuge tube; (5) Taking 20 mu L of cell lysate to a 96-well ELISA plate, adding 100 mu L of room temperature LARII reagent, uniformly mixing, and detecting the firefly luciferase reporter gene activity by a chemiluminescent microplate reader; (6) Adding 100 mu L of room temperature 1 xStop & Reagent into a 96-well ELISA plate, uniformly mixing, and detecting the activity of a Renilla luciferase reporter gene in a chemiluminescent microplate reader; (7) calculating a transcriptional activity value: firefly luciferase activity/Renilla luciferase activity.
As shown in FIGS. 3C-D, when the cloned human ANGPTL 3' UTR sequence contains a natural miR-374C-5p acting target site, miR-374C-5p (100 nM) significantly reduces the dual-luciferase reporter gene expression activity, while anti-miR-374C-5p (50 nM) can enhance the dual-luciferase reporter gene expression activity. When miR-374c-5p action target site is mutated in cloned human ANGPTL33' UTR sequence, miR-374c-5p (100 nM) and anti-miR-374c-5p (50 nM) have a significantly reduced regulation effect on the activity of the double-luciferase reporter gene. From this, it was demonstrated that miR-374c-5p binds directly to and acts on the target site in the 3' UTR of human ANGPTL 3.
EXAMPLE 5miR-374c-5p enhances Lipolipoprotein Lipase Activity in human hepatoma cell HepG2 and mouse hepatoma cell Hepa1-6
Cells in the logarithmic growth phase were inoculated into 6-well plates and cultured overnight at 37℃with 5% CO 2. When the cell confluency reached 70%, 10. Mu.L of transfection complex was prepared as described in example 2. The transfection complex is dripped into the cells to be transfected, and the cells are uniformly mixed (Con miR or miR-374c-5p final concentration is 100nM, con Inh or anti-miR-374c-5p final concentration is 50 nM), after 6 hours of transfection, opti-MEM culture medium is changed into complete culture medium, after 72 hours of culture, the cell culture medium is collected, meanwhile, PBS is used for rinsing the cells, cells are collected after pancreatin digestion, and 100ul RIPA cell lysate is added. The procedure was as per the instructions for lipoprotein lipase from the institute of biological engineering (cat#A 067-1-2) built in Nanjing.
The results show that: miR-374C-5p (100 nM) and anti-miR-374C-5p (50 nM) significantly increased and decreased lipoprotein lipase activity in HepG2 and Hepa1-6 cells (FIG. 4A, FIG. 4C) and secreted into cell culture media (FIG. 4B, FIG. 4D), respectively.
Example 6miR-374C-5p agomir significantly inhibits mouse liver ANGPTL3 expression, enhances serum lipoprotein lipase activity, reduces serum T-CHO, TG, LDL-C and HDL-C levels after administration
Agomir is designed according to miRNA mature body sequence, and double-stranded small RNA molecule with special mark and chemical modification is used for simulating endogenous mature body miRNA sequence. agomir includes a sequence identical to the target miRNA mature body sequence and a sequence complementary to the miRNA mature body sequence. The modification mode is as follows: agomir is modified only on the antisense strand, cholesterol modification is carried out at the 3' -end, 4 thio-skeleton modifications are carried out at the 3' -end, 2 thio-skeleton modifications are carried out at the 5' -end, and methoxy modification is carried out on the whole strand. miR-374c-5p agomir (based on sequence SEQ ID NO: 1) and NC agomir (based on sequence SEQ ID NO: 2) are synthesized by Shanghai Ji Ma pharmaceutical technologies Co., ltd.) in the modified manner described above.
6.1 Establishment of animal models
6 SPF-class C57BL/6 mice at 6 weeks of age, 24 SPF-class LDLR -/- mice at 6 weeks of age, (Jiangsu, changzhou, quality certificate No. 2002226971, license No. SYXK (Su) 2021-0013). After one week of adaptive feeding, 6 SPF-grade male C57BL/6 mice were set as background mice (C57 BL/6+LFCD+Saline) and fed a low-fat low-cholesterol control diet (LFCD, nantong Telofei diet technologies Co., ltd., cat#TP 28521). 24 LDLR -/- mice were randomly divided into 4 groups (n=6), LDLR -/- low-fat low-cholesterol control mice (LDLR -/- + LFCD +saline), fed low-fat low-cholesterol control feed; Mice in the LDLR -/- model group (LDLR -/-+HFCD+Saline)、LDLR-/- negative control group (LDLR -/- + HFCD + NC agomir) and the dosing group (LDLR -/- + HFCD + miR-374c-5p agomir) were fed a high-fat high-cholesterol model feed (HFCD, South Tong Telofei feed technologies Co., ltd., cat#TP 28528). 200 μL of saline was administered to C57BL/6 background mice, LDLR -/- low-fat low-cholesterol control mice and LDLR -/- model mice; the LDLR -/- negative control group was dosed at 20mg/kg (NC agomir in saline, 200ul in volume) and the LDLR -/- group was dosed at 20mg/kg (miR-374 c-5p agomir in saline, 200ul in volume). The tail vein is administrated, the administration frequency is 1 time per week, after continuous administration for 8 weeks, the mice are fasted and fed for 16 hours without water, and the eyeballs are picked up to collect tissue samples such as blood, liver, aortic root and the like, so as to carry out biochemical indexes and pathological detection.
6.2 Detection of Lipase Activity and Biochemical indicators of serum lipoproteins in mice
Collecting blood from eyeball, placing blood sample in a water bath at 37deg.C for 30min, standing in a refrigerator at 4deg.C for 4 hr, centrifuging at 4deg.C at 4000rpm for 15min, and collecting upper serum. The lipoprotein lipase activity in the serum of each group of mice was determined using the total lipase test box (Nanjing institute of biological engineering, cat#A 067-1-1); T-CHO test boxes (Nanjing institute of biological engineering, cat#A111-1-1), TG test boxes (Nanjing institute of biological engineering, cat#A110-1-1) and LDL-C test boxes (Nanjing institute of biological engineering, cat#A113-1-1) HDL-C test boxes (Nanjing institute of biological engineering, cat#A112-1-1) were used to determine T-CHO, TG, LDL-C and HDL-C levels in serum from each group of mice.
6.3Westen blot detection of the protein level of ANGPTL3 in mouse liver tissue
Following homogenization and RIPA lysate treatment of mouse livers, westen blot were tested for ANGPTL3 protein levels in the livers of each group of mice, and the procedure was as in example 2.
6.4QRT-PCR detection of mouse liver tissue angptl mRNA level
The qRT-PCR assay of the levels of ANGPTL3mRNA in the livers of each group of mice after homogenization and RNAiso lysate treatment was performed in the same manner as in example 3.
As shown in FIG. 5, after miR-374C-5p agomir tail vein administration, the mice in the LDLR -/- model group (LDLR -/- + HFCD +Saline) and the mice in the C57BL/6 background group (C57 BL/6+LFCD+Saline) showed a significant difference in body weight after 6 weeks of feeding. The mice in the model group of LDLR -/- (LDLR -/- + HFCD +Saline) and the mice in the control group of LDLR -/- (LDLR -/- + LFCD +Saline) have no significant difference in weight during the feeding process. No significant differences in body weight occurred between mice in LDLR -/- dosing group (LDLR -/- + HFCD +mir-374c-5p agomir) and LDLR -/- negative control group (LDLR -/- + HFCD + NC agomir) during feeding (fig. 5A). ANGPTL3 protein and mRNA levels were significantly down-regulated in liver tissue of LDLR -/- -dosed mice (LDLR -/- + HFCD +mir-374C-5p agomir) (fig. 5b, 5C), lipoprotein lipase activity was significantly increased in serum (5D), and T-CHO (5E), TG (5F), LDL-C (5G) and HDL-C (5H) levels were significantly reduced in serum.
Example 7miR-374c-5p agomir significantly reduces liver lipid deposition in mice after administration, significantly improves cavitation-like steatosis in hepatocytes of mice, and has inhibiting and treating effects on fatty liver
7.1MiR-374c-5p agomir for improving liver morphology of mice
Immediately after sacrificing the mice according to the method of example 6.1, the complete livers of the mice were removed, connective tissue was removed, and each group of liver morphology changes was observed.
As shown in FIG. 6A, the liver of the C57BL/6 background group mice (C57 BL/6+LFCD+Saline) is bright red, and the liver surface is ruddy and glossy; the liver of the LDLR -/- low-fat low-cholesterol control group mouse (LDLR -/- + LFCD +Saline) is bright red, and the liver surface is smooth; the liver of the mice of the model group of LDLR -/- (LDLR -/- + HFCD +Saline) and the mice of the negative control group of LDLR -/- (LDLR -/- + HFCD + NC agomir) are whitish, and the surface is greasy and rough; the LDLR -/- administration group mice (LDLR -/- + HFCD +miR-374c-5p agomir) have obviously improved liver pathologic hypertrophy and liver surface pathologic roughness, and the toughness of liver tissues is enhanced.
7.2HE staining method for detecting pathological changes of liver tissue cells of mice
After each group of fresh livers was fixed with paraformaldehyde for 48 hours, the tissues were dehydrated with water and waxed, and then serial sections were made into 5 μm paraffin sections, which were stained with hematoxylin-eosin staining (HE). The method comprises the following specific steps: (1) baking slices: baking at 60deg.C for 2 hr; (2) dewaxing, hydration: immediately feeding the slices into xylene I (10 min), xylene II (10 min), a xylene absolute ethanol mixed solution (1:1) (5 min), absolute ethanol I (2 min), absolute ethanol II (2 min), 90% ethanol (2 min), 80% ethanol (2 min) and 70% ethanol (2 min) for dewaxing; (3) Flushing for 3 times (5 min/time) with running water to rehydrate the paraffin sections; (4) hematoxylin staining for 1min; (5) washing with running water for 10min; (6) 0.1% ethanol hydrochloride differentiation for 10s; (7) flushing with running water for 2min; (8) 1% ammonia water for 30s; (9) flushing with running water for 5min; (10) 95% ethanol equilibration for 30s; (11) eosin staining for 2min; (12) flushing with running water for 5min; (13) dehydration: slicing, soaking in 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol for 30s; absolute ethyl alcohol I, absolute ethyl alcohol II, xylene I and xylene II are respectively soaked for 2min; (14) after airing, sealing the neutral resin; (15) acquiring an image.
As shown in FIG. 6B, the liver cells of the mice of the C57BL/6 background group (C57 BL/6+LFCD+Saline) were aligned and had no balloon-like steatosis; the liver cells of the mice (LDLR -/- + LFCD +Saline) of the LDLR -/- low-fat low-cholesterol control group are orderly arranged, and the cavitation bubbles are small and sparse; the mice in the model group of LDLR -/- (LDLR -/- + HFCD +Saline) and the mice in the negative control group of LDLR -/- (LDLR -/- + HFCD + NC agomir) have disordered liver cell arrangement, stacked cell nuclei, large and dense vacuoles and multiple balloon-like steatosis; the mice (LDLR -/- + HFCD +miR-374c-5 pagomir) of the LDLR -/- administration group have relatively orderly hepatocyte arrangement, smaller vacuole and reduced balloon-like steatosis, which indicates that miR-374c-5p agomir has inhibiting and treating effects on fatty liver.
7.3 Detection of neutral fat content in liver by oil Red O staining method
And (3) quick-freezing and embedding each group of fresh livers by adopting an OCT embedding agent, then continuously slicing to prepare 7 mu m frozen slices, and observing lipid deposition conditions of the livers of the mice by using oil red O dye liquor for dyeing. The method comprises the following specific steps: (1) frozen sections were fixed in 10% neutral formaldehyde for 10min; (2) washing with distilled water for 5min; (3) soaking and washing with 60% isopropanol solution for 20-30s; (4) dyeing with oil red O working solution for 15min; (5) 60% isopropanol solution is differentiated until the matrix is clear; (6) washing with distilled water for 2min; (7) hematoxylin dye liquor is used for dyeing for 2min; (8) an aqueous caplet; (9) acquiring an image.
As a result, as shown in FIG. 6C, orange lipid droplets were hardly visible in the liver of the C57BL/6 background group mice (C57 BL/6+LFCD+Saline); the liver of the LDLR -/- low-fat low-cholesterol control group mice (LDLR -/- + LFCD +Saline) is occasionally provided with orange-red lipid drops; the livers of the mice in the LDLR -/- model group (LDLR -/- + HFCD +Saline) and the mice in the LDLR -/- negative control group (LDLR -/- + HFCD + NC agomir) have a large amount of orange lipid droplets to accumulate; the liver orange lipid drop of the mice (LDLR -/- + HFCD +miR-374c-5p agomir) in the LDLR -/- administration group is obviously reduced, which indicates that miR-374c-5pagomir can obviously reduce liver lipid deposition and has inhibiting and treating effects on fatty liver.
7.4Masson staining method for detecting liver fibrosis level
After each group of fresh livers was fixed with paraformaldehyde for 48 hours, the tissues were dehydrated and waxed, then serially sliced to prepare 5 μm paraffin sections, and Masson staining was performed using a modified Masson trichromatic staining kit (cat#g1346, beijing solebao corporation) to observe the degree of liver fibrosis in mice. The specific steps are carried out according to the specification of the product, and are briefly described as follows: (1) paraffin sections are routinely dewaxed to water; (2) Dip dyeing the slices in mordant liquid, mordant dyeing for 1h at 60 ℃ in an incubator, and washing for 10min with running water; (3) the Tianshiqing blue dye is dyed for 3min, and washed for 2 times; (4) The acidic differentiation liquid is differentiated for a plurality of seconds, the differentiation is stopped by washing, and the washing is performed for 10 minutes by distilled water; (5) Liquid dyeing of ponceau-fuchsin dye is carried out for 10min, and distilled water is used for washing for 2 times; (6) phosphomolybdic acid solution treatment for 10min; (7) Pouring the upper liquid, directly dripping aniline blue staining solution into the slices without washing the slices for 5min; (8) Washing off the aniline blue solution by using a weak acid solution, and then continuously dripping a weak acid working solution to cover the slices for 2min; (9) Dehydrating 95% ethanol for 30s, dehydrating absolute ethanol for 2 times, wherein the first time is 30s, and the second time is 1min; (10) xylene is transparent for 2 times, each time for 2min; (11) a neutral resin sealing piece; (12) acquiring an image.
As a result, as shown in FIG. 6D, the liver of the C57BL/6 background group mice (C57 BL/6+LFCD+Saline) hardly seen collagen fibers stained blue; the liver of the LDLR -/- low-fat low-cholesterol control group mice (LDLR -/- + LFCD +Saline) is slightly stained with blue collagen fibers; the livers of the mice in the LDLR -/- model group (LDLR -/- + HFCD +Saline) and the mice in the LDLR -/- negative control group (LDLR -/- + HFCD + NC agomir) have a large amount of collagen fibers dyed blue, which indicates that the liver fibrosis degree of the mice is higher; the blue-stained collagen fibers of the livers of the mice (LDLR -/- + HFCD +miR-374c-5p agomir) in the LDLR -/- administration group are obviously reduced, which indicates that the liver fibrosis degree of the mice is obviously improved.
Therefore, after miR-374c-5p agomir tail vein administration, the liver lipid deposition of the LDLR -/- mice can be obviously reduced, the cavitation-like fatty degeneration of the liver cells of the mice is obviously improved, and the anti-aging agent has inhibiting and treating effects on fatty liver.
Example 8MiR-374c-5p agomir significantly reduced aortic lipid deposition in mice and inhibited aortic root atherosclerotic plaque formation in mice following administration
8.1 General staining of the abdominal aortic atherosclerotic plaques
The abdominal aorta of each group of mice is stripped respectively, the length of the abdominal aorta is from the root of the aorta to the branch of the common iliac artery, and the abdominal aortic intima is dyed by oil red O, and the specific steps are as follows: (1) abdominal aortic blood vessel 4% paraformaldehyde fixation for 30min; (2) washing with running water for 10min, carefully peeling off adventitial connective tissue; (3) soaking in distilled water for 5min, and washing with physiological saline for 3 times; (4) dyeing with oil red O working solution for 30min; (5) The specimen is soaked by 70% ethanol until the plaque presents red color and the ground color presents white color (blood vessel is transparent); (6) acquiring an image.
As shown in fig. 7A, after the mice abdominal aortic atherosclerotic plaques were generally stained, C57BL/6 background group mice (C57 BL/6+lfcd+saline) had lipid deposition in the abdominal aorta; LDLR -/- low-lipid low-cholesterol control mice (LDLR -/- + LFCD +saline) had little lipid deposition in the abdominal aorta; the mice of the model group of LDLR -/- (LDLR -/- + HFCD +Saline) and the mice of the negative control group of LDLR -/- (LDLR -/- + HFCD + NC agomir) are dyed red by oil red O, and have a large amount of lipid deposition; the mice in the LDLR -/- dosing group (LDLR -/- + HFCD +miR-374c-5p agomir) had significantly reduced abdominal aortic lipid deposition.
Lipid deposition areas were calculated using Image-Pro Plus, which indicated that miR-374c-5pagomir significantly reduced lipid deposition at the abdominal aorta of mice following administration (FIG. 7B).
8.2 Evaluation of aortic root lipid deposition by aortic root oil red O staining
After the fresh aortic root of each group is quickly frozen and embedded by adopting an OCT embedding agent, serial sections are prepared into 7 mu m frozen sections, and the sections are dyed by oil red O dye solution to observe lipid deposition of the aortic root of the mice. The procedure is as in example 7.3, and the results are shown in FIG. 7C, with no lipid deposition at the aortic root of the C57BL/6 background group mice (C57 BL/6+LFCD+Saline); LDLR -/- low-fat low-cholesterol control mice (LDLR -/- + LFCD +saline) had a small amount of lipid deposited around aortic valve roots; the aortic valve roots of the mice of the model group of LDLR -/- (LDLR -/- + HFCD +Saline) and the mice of the negative control group of LDLR -/- (LDLR -/- + HFCD + NC agomir) can be provided with obvious plaque lipid deposition and local bulge; aortic root lipid deposition was significantly reduced in mice of the LDLR -/- dosing group (LDLR -/- + HFCD +mir-374c-5p agomir).
8.3 Evaluation of aortic root atherosclerotic plaque in mice by HE staining
In addition, the root tissues of the aorta of each group of mice were serially sectioned, paraffin sections were prepared, and hematoxylin-eosin (HE) staining was performed, and the specific procedure was as in example 7.2. As shown in FIG. 7D, the C57BL/6 background group mice (C57 BL/6+LFCD+Saline) have clear aortic root vascular wall structure, smooth and complete intima, tight endothelial cell connection, no shedding, no lipid streak or fibrous plaque, and small subintimal gap; LDLR -/- low-fat low-cholesterol control mice (LDLR -/- + LFCD +saline) had a small amount of plaque attached to the vascular endothelium; the aortic intima thickening is remarkable in the mice of the model group of LDLR -/- (LDLR -/- + HFCD +Saline) and in the mice of the negative control group of LDLR -/- (LDLR -/- + HFCD + NC agomir), obvious plaques and local bulge are visible; the aortic root intima thickness of the mice in the LDLR -/- administration group (LDLR -/- + HFCD +miR-374c-5p agomir) is reduced, the intima tends to be smooth, the endothelial cell connection is relatively tight, and the plaque area is remarkably reduced.
8.4Movat five-color counterstaining method for evaluating stability of atherosclerosis plaque at aortic root of mice
The aortic root tissues of each group of mice were also taken, serially sectioned, paraffin sections were prepared, and stained as described in Russell modified Movat's five-color kit (cat#BP-DL 318, nanjsen Bei Ga Biotech). The method comprises the following specific steps: (1) paraffin sections are conventionally dewaxed and serial ethanol is hydrated; (2) staining the tissue section with an elastic staining solution for 20min; (3) fully flushing with running water; (4) Immersing the slide glass into ferric chloride solution for 10 times, and flushing with running water; (5) Checking under a microscope whether the tissue is differentiated, and repeating step 4 if necessary; (6) washing with distilled water for 2 times; (7) Placing the glass slide into a sodium thiosulfate solution, and incubating for 1min; (8) washing with tap water for 2min and washing with distilled water for 2 times; (9) The slide glass is put into acetic acid solution (3%) and incubated for 2min; (10) Placing the glass slide into an aliskiren blue staining solution, and staining for 25min; (11) washing with tap water for 2min and washing with distilled water for 2 times; (12) Placing the glass slide into a solution of bivalirudin-acid fuchsin, and incubating for 2min; (13) washing the glass slide with distilled water for 2 times; (14) placing the slide glass in acetic acid solution (1%) for 10s; (15) distilled water rapid flushing; (16) Dropping phosphotungstic acid solution on the glass slide tissue, distinguishing the tissue for 2 times, and 5min each time; (17) washing the slide glass with distilled water; (18) dropwise adding acetic acid solution (1%) to the glass slide for 3 times; (19) Shaking up acetic acid dropwise added on a glass slide, and then dropwise adding acid golden G dye liquor for dyeing for 15min; (20) absolute ethyl alcohol for 2 times, each time for 1min; xylene is transparent for 2 times, each time for 1min; (21) a neutral resin sealing piece; (22) acquiring an image.
As shown in FIG. 7E, the C57BL/6 background group mice (C57 BL/6+LFCD+Saline) have clear aortic root vascular wall structure, smooth and complete inner membrane and large ratio of yellow collagen to black elastic fiber; the mice of the LDLR -/- low-fat low-cholesterol control group (LDLR -/- + LFCD +Saline) have a small amount of plaque attached to vascular endothelium, and the yellow collagen and black elastic fiber occupy larger proportion; the mice in the LDLR -/- model group (LDLR -/- + HFCD +Saline) and the mice in the LDLR -/- negative control group (LDLR -/- + HFCD + NC agomir) have obvious aortic intima thickening, obvious plaques are visible, cholesterol ester and cholesterol crystallization are obviously increased, and the surface of the intima is uneven, so that vulnerable plaque characteristics are shown; the thickness of the aortic root part of the mice (LDLR -/- + HFCD +miR-374c-5p agomir) in the administration group of LDLR -/- is reduced, the intima tends to be smooth, the endothelial cells are connected relatively tightly, the plaque area is obviously reduced, cholesterol ester and cholesterol crystallization are also obviously reduced, yellow collagen and black elastic fiber components are obviously increased, the surface of the intima is relatively flat, and the plaque stability is increased.
Therefore, after miR-374c-5p agomir tail vein administration, the aortic lipid deposition of the mice can be obviously reduced, the formation of atherosclerosis plaques at the root of the aorta of the mice can be inhibited, and the preparation method has the effects of preventing and treating cardiovascular diseases.
Claims (7)
1. Use of a human endogenous non-coding microrna or a biologically active functional fragment or precursor or variant thereof for the manufacture of a medicament for the diagnosis, prevention and/or treatment of a disease associated with elevated angiopoietin-like protein 3 ANGPTL3, wherein the microrna is miR-374c-5p.
2. The use according to claim 1, wherein the diseases associated with elevated angiopoietin-like protein 3 ANGPTL3 include one or more of hypertriglyceridemia, hypercholesterolemia, hyperchlorhydria, hyperlipidemia, nonalcoholic fatty liver disease, liver fibrosis or atherosclerotic cardiovascular disease.
3. The use of claim 1, wherein the sequence of miR-374c-5p is set forth in SEQ ID No. 1.
4. The use according to claim 1, wherein the precursor sequence of the microRNA is shown in SEQ ID NO. 5.
5. The use of claim 1, wherein the variant of the microrna comprises one or more of a methoxy modification, a thio modification, a cholesterol modification, an N-acetylgalactosamine modification to miR-374c-5 p.
6. The use according to any one of claims 1 to 5, comprising constructing a recombinant expression plasmid or virus comprising miR-374c-5p or a biologically-active functional fragment or precursor or variant sequence thereof.
7. The use according to any one of claims 1 to 6, wherein the medicament comprises a miR-374c-5p sequence and a pharmaceutically-acceptable carrier selected from one or more of a gene expression vector, a virus, chitosan, cholesterol, a liposome or a nanoparticle.
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