CN111658776B - Application of miR-16 antagonist in preparation of drug for inhibiting non-alcoholic fatty liver disease - Google Patents

Abstract

The invention discloses an application of a miR-16 antagonist in the preparation of a drug for inhibiting non-alcoholic fatty liver disease. The sequence of the miR-16 is: AGCAGCAUUGUACAGGGCUAUGA. The inventors have proved through a large number of experiments that the highly expressed miR-16 in cells is related to the occurrence of NAFLD, and inhibiting the level of miR-16 can significantly reduce the expression of pathological-related molecules in NAFLD model cells; miR-16 regulates lipid metabolism synthesis and decomposition in NAFLD cell models The expression of key enzymes in the pathway, inhibiting miRNA-16 can alleviate the pathological changes of fatty liver; and miR-16 Antagomir, as an inhibitor of microRNA-16, specifically inhibits the expression of microRNA-16, can significantly inhibit NAFLD.

Description

Application of miR-16 antagonist in preparation of drug for inhibiting non-alcoholic fatty liver disease

Technical Field

The invention relates to application of an inhibitor of miR-16 in preparation of a drug for inhibiting non-alcoholic fatty liver disease.

Background

Non-alcoholic fatty liver disease (NAFLD) refers to the clinical pathological syndrome characterized by diffuse hepatocyte bullous fat becoming the main feature due to the exclusion of alcohol and other definite liver damage factors, including simple fatty liver and NASH evolved therefrom, IR and genetic susceptibility are closely related to the pathogenesis. With the improvement of living standard, the prevalence rate of NAFLD is increased year by year, which constitutes an increasingly serious public health problem.

Although simple lipidosis is a benign disease with a good prognosis, a small proportion of NASH can progress to liver fibrosis, cirrhosis and even HCC. Therefore, the research on the pathogenesis of NAFLD in various stages has become one of the current hotspots. NAFLD can increase the risk of cardiovascular disease, type 2 diabetes, and MS. Current molecular biology studies have shown that the development of NAFLD is a process involving multiple genes, multiple loops, multiple pathways, in which many genes are altered. The current understanding of the pathogenesis of NAFLD is still based on the hypothesis of "secondary hit", and the specific mechanism is not clear.

With the development of molecular biology technology, the molecular mechanism of NAFLD generation is gradually recognized. mirnas are newly discovered molecules that play an important role in lipid metabolism. mirnas are widely present in eukaryotes as a class of small non-coding RNAs of about 22 nucleotides in length. Its expression is highly tissue specific, conserved and time-ordered. miRNA degrades mRNA or represses gene translation through complementary pairing with target mRNA 3' -UTR, thereby playing an important role in regulation and control after gene transcription. miRNA has wide regulation function in life activity, and is closely related to life activity processes of development process, cell growth and differentiation, apoptosis, organ formation and the like of organisms.

Whether miRNA plays a regulating role in NAFLD or not is not completely clear at present as an important endogenous regulatory molecule.

Disclosure of Invention

The invention aims to provide application of a miR-16 antagonist in preparation of a medicine for inhibiting non-alcoholic fatty liver disease. The application provides a new way for inhibiting the non-alcoholic fatty liver disease and improves the effect. The sequence of miR-16 is as follows: AGCAGCAUUGUACAGGGCUAUGA (SEQ ID NO. 1).

The miR-16 antagonist is selected from small interfering RNA, dsRNA, shRNA, micro RNA and antisense nucleic acid which can reduce the expression content of the miR-16 antagonist: or a construct capable of expressing or forming the small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid.

In particular, the present invention provides an antagonist having the sequence: UCAUAGCCCUGUACAAUGCUGCU (SEQ ID NO. 2).

The inventor proves that miR-16 highly expressed in cells is related to NAFLD through a large number of experiments, and the inhibition of miR-16 level can obviously reduce the expression of related molecules of NAFLD model cell pathology; miR-16 regulates the expression of key enzymes of lipid metabolism synthesis and decomposition pathway in a NAFLD cell model, and inhibits miRNA-16 to relieve the physiological change of fatty liver; the miR-16Antagomir serving as an inhibitor of the microRNA-16 can specifically inhibit the expression of the microRNA-16 and can obviously inhibit the NAFLD.

Drawings

FIG. 1: down-regulation of miR-16 can inhibit lipid droplet accumulation in NAFLD model cells. qRT-PCR (quantitative reverse transcription-polymerase chain reaction) for detecting the content of miR-16; B. oil red O staining detection results; data were counted using one-way analysis of variance with error bars representing SD. Denotes p <0.05, denotes p < 0.01.

FIG. 2: the miR-16 expression can be inhibited to obviously reduce the expression of related molecules of cell pathology of the NAFLD model. Data were counted using one-way analysis of variance with error bars representing SD. Denotes p <0.05, denotes p < 0.01.

FIG. 3: miR-16Antagomir regulates the expression of NAFLD cell lipid metabolism-related enzymes. A, detecting the expression conditions of lipid metabolism related proteins FASN, ACSL1 and ACOX1 by Western Blot; b, qRT-PCR detects mRNA levels of lipid metabolism related genes FASN, ACSL1 and ACOX 1; data were counted using one-way analysis of variance with error bars representing SD. Denotes p <0.05, denotes p < 0.01.

FIG. 4: the miR-16 in the liver tissue of the NAFLD model animal is highly expressed, and the miR-16Antagomir effectively inhibits the miR-16 level of the tissue. Detecting the miR-16 content by qRT-PCR; data were counted using one-way analysis of variance with error bars representing SD. Denotes p < 0.01.

FIG. 5: inhibiting the expression of miRNA-16, and relieving the pathological change of liver tissue of NAFLD model mouse. A. Oil red O staining results; he staining results.

FIG. 6: the inhibition of miR-16 expression can obviously relieve the expression of pathologically-related molecules in the serum of NAFLD model animals. Data were counted using one-way analysis of variance with error bars representing SD. Denotes p <0.05, denotes p < 0.01.

FIG. 7: miR-16 regulates and controls the expression of key enzymes of lipid metabolism pathways in liver tissues of a NAFLD animal model. Western Blot for detecting the expression of FASN, ACSL1 and ACOX1 which are key enzymes of lipid metabolism; qRT-PCR detects the mRNA expression of FASN, ACSL1 and ACOX1 which are key enzymes of lipid metabolism; data were counted using one-way analysis of variance with error bars representing SD. Denotes p < 0.01.

Detailed Description

1. Experimental methods

1.1 cell assay

Experimental cells: the normal human liver cell line L02 was purchased from Shanghai Fuxiang Biotech Ltd. DMEM medium containing 10% FBS (Gibco No.16000-044) at 37 ℃ with 5% CO₂Culturing under the condition of 2 × 10⁵Concentration in/mL.

Cell model construction and grouping

1) L02 cells;

2) l02 cell model group;

3) l02 cell model group + antagomir NC;

4) l02 cell model group + miR-16 antagomir.

The method adopts FFA to induce normal human liver cell line L02 cells to establish a NAFLD cell model, and the specific scheme is as follows: normal human liver cell line L02 cells were subcultured in DMEM medium containing 10% FBS until the cells reached 80-100% confluence. 1mM FFA palmitic acid treatment for 48h modeled successfully. After successful modeling, 3 and 4 groups of cells were treated with antagomir NC and miR-16antagomir transfection for 72h, respectively.

Cell transfection:

the confluency of the cells is about 70%, and the cells are replaced by a serum-free DEEM culture medium 2 hours before transfection; each transfection sample was prepared as follows: diluting miR-16antagomir (hsa-miR-16a-3p antagomir: UCAUAGCCCUGUACAAUGCUGCU, gemma gene without fluorescence) and antagomir NC (miR-16antagomir control: CAGUACUUUUGUGUAGUACAA, gemma gene without fluorescence) with 100 μ l of serum-free DMEM medium respectively, gently mixing, and standing at room temperature for 5 min; mu.l of Lipofectamine were also diluted separately with 100. mu.l of serum-free DMEM medium^TM2000, standing at room temperature for 5 min. Mixed Lipofectamine^TM2000 and diluent of miR-16antagomir or antagomir NC (total volume is 200 mu l), standing for 20min at room temperature; separately, add to the petri dish and gently shake the cell culture plate back and forth to mixMixing the culture solution with the culture solution in the culture plate; cells at 37 ℃ CO₂Culturing in an incubator, and replacing a new complete culture medium after 6 h; and (5) collecting cells for detection after 72 h.

RNA extraction and qRT-PCR

First, appropriate amounts of cells were harvested, RNA extracted according to the instructions of the RNeasy MiniKit (Qiagen No.74106) kit and digested with DNase (RNase-Free DNase Set, Qiagen No. 79254). cDNA Synthesis Using a Large volume cDNA reverse transcription kit (Applied Biosystems No. 4368813); the qRT-PCR experiment was performed using the StepOneNus Real-Time PCR system in the SYBRGERREN method (Applied Biosystems) using comparative CT values (. DELTA.)^CT) And determining the relative expression of the genes in different samples by a method of normalization with U6 or GAPDH. The PCR primer sequences and related primer (both synthesized by Shanghai) sequences are as follows:

chart 1 primer sequence information

Antisense universal primer: 5'-CTCAAGTGTCGTGGAGTCGGCAA-3'

Chart 2 primer sequence information

Dyeing with oil red O:

fixing the cell slide for 15min, and washing with distilled water; 60% isopropanol, oil red O staining solution (Sigma, SLBP5248V) for 10 min; 60% isopropanol (national drug group, 80109218) was color-separated until the background was colorless, and washed with distilled water; mayer hematoxylin (Sigma, H9627) counterstain for several minutes, washing with tap water (bluing) for 1-3 min; and (5) sealing the water-soluble sealing agent by using distilled water. And (6) taking a picture. As a result: lipid droplets in the tissue cells appeared orange red, and the nuclei appeared blue.

Western Blot

Cells were lysed with lysis buffer containing PMSF (Nanjing Wako, 329-98-6) (Biyunyan, P0013B). The concentration of the protein lysate was measured by the Bradford method (Bio-Rad, 5000006). After mixing 30. mu.g of cell lysate obtained from the cells with 5 Xsample buffer (15g SDS, 15.6mL 2M Tris pH6.8, 57.5g glycerol, 16.6 mL. beta. -meriptoethanol), the sample was loaded on a 10% polyacrylamide gel, separated by SDS-PAGE and transferred onto PVDF membrane (Bio-Rad, 162-0177). After blocking with 4% milk containing 0.1% Tween-20, the following antibodies were added and incubated overnight at 4 ℃: FASN antibody (1:500, proteintech, 10624-2-AP), ACSL1antibody (1:1000, Affinity, DF9605), ACOX 1antibody (1:1000, Affinity, DF12046), GAPDH antibody (1:2500, Abcam, AB 9485). After washing the membranes 3 times with 0.1% Tween-20 in PBS, 4% milk containing 0.1% Tween-20 was added along with a secondary HRP antibody (1:4000, Dianova, Hamburg) and incubated for 2h at room temperature. The membrane was removed, washed 3 times with 0.1% Tween-20 in PBS, and then imaged by dropping ECL developer (Bio-Rad, 170-5060) on the membrane and by placing it in GelDoc imaging system (Bio-Rad). ImageJ software performs densitometric analysis.

Detection of content of blood lipid related factor

Collecting cells of each treatment group, digesting with 0.25% trypsin-EDTA digestive juice, incubating at 37 ℃ for 10min, adding serum to stop digestion, washing and centrifuging in multiple steps, taking supernatant, and detecting with Triglyceride (TG) determination kit (Nanjing, A110-1), alanine Aminotransferase (ALT) determination kit (Nanjing, C009-2), glutamic-oxaloacetic transaminase (AST/GOT) determination kit (Nanjing, C101-2), hydrogen peroxide (H2O2) determination kit (Nanjing, A064-1) and ATP content determination kit (Nanjing, A095).

1.2 animal experiments

Experimental animals: c57BL/6 mice were purchased from Liaoning Biotechnology Ltd.

Animal model construction and grouping

1) A normal group;

2) NAFLD group;

3) NAFLD group + antagomir NC;

4) NAFLD group + miR-16 antagomir.

NAFLD model establishment:

after 8-week-old C57BL/6 mice were acclimatized for 1 week, they were randomly divided into 2 groups according to body weight, the control group was fed with basal diet (3), and the NAFLD model group was fed with high-fat diet (80.5% normal diet, 2% cholesterol, 7% lard, 10% egg yolk powder and 0.5% bile salt) (9). The breeding environmental conditions are as follows: the temperature range is 20-22 ℃, the humidity range is 50-55%, and the light and shade are 12h respectively. The experimental animals had free access to food and water, and were fed in 3 cages. The latter two groups were treated in groups 4 weeks later, with miR-16antagomir control or antagomir NC (directly injected without a carrier) injected in the tail vein at a dose of 15mg/kg in 0.2ml pbs, supplemented once 2 weeks later, and each group of mice (6 per group) was sacrificed 8 weeks after the start of the experiment.

Oil red O dyeing

After the animals were anesthetized, blood was removed with normal saline, liver tissue was rapidly removed by focusing with 4% paraformaldehyde, the 4% paraformaldehyde was fixed after continuing, then dehydrated with 30% sucrose, embedded with OCT embedding medium, and then prepared into sections with a cryomicrotome and adhered to slides. Freezing and slicing, fixing with formaldehyde-calcium for 10min, and washing with distilled water; dipping and washing with 60% isopropanol, and dyeing with oil red O dye liquor for 10 min; color separation is carried out on 60% isopropanol until the background is colorless, and then washing is carried out by distilled water; mayer hematoxylin counterstain for several minutes, washing (bluing) for 1-3 min; after the distilled water is washed, the water-soluble sealing agent is used for sealing and photographing.

RNA extraction and qRT-PCR

First, a suitable amount of minced tissue was harvested, RNA extracted according to the instructions of the RNeasy MiniKit (Qiagen No.74106) kit and digested with DNase (RNase-Free DNase Set, Qiagen No. 79254). cDNA Synthesis Using a Large volume cDNA reverse transcription kit (Applied Biosystems No.4368813) (Note: mRNA was reversed with oligo dT only, miRNA was also reversed with a specific loop primer); the qRT-PCR experiment was performed using the StepOneNus Real-Time PCR system in the SYBRGERREN method (Applied Biosystems) using comparative CT values (. DELTA.)^CT) And determining the relative expression of the genes in different samples by a method normalized by GAPDH/U6. The PCR primer sequences and related primer (both synthesized by Shanghai) sequences are as follows:

chart 1 primer sequence information

Sequence information of Chart 2miRNA-16 primer

Antisense universal primer: 5'-CTCAAGTGTCGTGGAGTCGGCAA-3'

Western Blot

The tissue blocks were built into small blocks and lysed with lysis buffer containing PMSF (Nanjing Wako, 329-98-6) (Biyunyan, P0013B).

The concentration of the protein lysate was measured by the Bradford method (Bio-Rad, 5000006). After mixing 30. mu.g of cell lysate obtained from the cells with 5 Xsample buffer (15g SDS, 15.6mL 2M Tris pH6.8, 57.5g glycerol, 16.6 mL. beta. -meriptoethanol), the sample was loaded on a 10% polyacrylamide gel, separated by SDS-PAGE and transferred onto PVDF membrane (Bio-Rad, 162-0177). After blocking with 4% milk containing 0.1% Tween-20, the following antibodies were added and incubated overnight at 4 ℃: FASN antibody (1:500, proteintech, 10624-2-AP), ACSL1antibody (1: 1000;, Affinity, DF9605), ACOX 1antibody (1:1000, Affinity, DF12046), GAPDH antibody (1:2500, Abcam, AB 9485). After washing the membranes 3 times with 0.1% Tween-20 in PBS, 4% milk containing 0.1% Tween-20 was added along with a secondary HRP antibody (1:4000, Dianova, Hamburg) and incubated for 1h at room temperature. The membrane was removed, washed 3 times with 0.1% Tween-20 in PBS, and then scanned and photographed by dropping ECL developer (Bio-Rad, 170-5060) on the membrane and by placing it in GelDoc imaging system (Bio-Rad).

Detection of content of blood lipid related factor

The mouse is treated according to the groups, the eyeball artery is blood-taken, and the contents of biochemical indexes H2O2, ATP, TG, ALT and AST in the serum of the mouse are detected by adopting a Triglyceride (TG) determination kit (Nanjing was constructed, A110-1), an alanine Aminotransferase (ALT) determination kit (Nanjing was constructed, C009-2), an aspartate aminotransferase (AST/GOT) determination kit (Nanjing was constructed, C101-2), a hydrogen peroxide (H2O2) test kit (Nanjing was constructed, A064-1) and an ATP content test kit (Nanjing was constructed, A095).

HE staining

The experimental mice were taken, the vertebras were dislocated, the desired tissue mass was removed from the abdomen, placed in 1PBS, then the tissue was transferred to ice 1PBS, fixed overnight with 4% paraformaldehyde, washed three times with 1PBS for 10 min/time, then passed through 30%, 50% and 70% Ethanol for 20min each time. The treated tissue was treated with xylene for 30min to prepare a wax block, followed by preparation of a section. The slices are stained in Mayer hematoxylin staining solution for 5-7min, and washed by tap water and returned to blue; adding 1% hydrochloric acid alcohol, differentiating for 2-5s (if microscopic examination shows that the background is over-dyed, the step is required for differentiation, if the background is clean, the step is not required), washing with tap water, and washing to obtain blue. Absolute ethyl alcohol I for 5 min; anhydrous ethanol II for 5 min; xylene I for 5 min; xylene II for 5 min; after air drying, the gel is sealed by neutral gum, and finally microscopic examination is carried out.

2. Results and analysis of the experiments

2.1 the down regulation of miR-16 can inhibit lipid drop accumulation in NAFLD model cells;

l02 cells were treated with palmitic acid (FFA,1mM) for 48h to construct NAFLD cell models, and qRT-PCR assay showed significant increase in miR-16 content (P <0.05) in NAFLD cells (fig. 1A); meanwhile, the oil red O staining result shows that NAFLD cells have obvious lipid droplet accumulation compared with the control group (fig. 1B); the result shows that the NAFLD cell model is successfully constructed by FFA treatment, and miR-16 is highly expressed in the NAFLD cell.

The miR-16antagomir is adopted to effectively reduce the miR-16 content in the NAFLD cell (figure 1A), and meanwhile, the phenomenon of lipid drop accumulation of the NAFLD cell (figure 1B) is remarkably relieved.

The above results indicate that high expression of miR-16 in cells is associated with NAFLD development.

2.2 the inhibition of miR-16 expression can obviously reduce the expression of related molecules of cell pathology of the NAFLD model;

the biochemical detection result shows that compared with the control group, the NAFLD group cell hydrogen peroxide (H)₂O₂) The contents of Triglyceride (TG), alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) are obviously increased, and AThe TP content is obviously reduced; the NAFLD model cell is simultaneously added with miR-16Antagomir to treat and inhibit miR-16 expression, and compared with an Antagomir NC group, the NAFLD model cell has a cell H₂O₂The contents of TG, ALT and AST were significantly reduced, and the ATP content was significantly increased (fig. 2).

The results show that the inhibition of miR-16 level can obviously reduce the expression of the molecules related to the cell pathology of the NAFLD model. The miR-16 antagonist can realize the inhibition of the non-alcoholic fatty liver disease.

2.3miR-16 regulates and controls the expression of key enzymes of lipid metabolism pathway in NAFLD model cells;

western Blot detection results show that mRNA levels and protein levels of fatty acid synthesis key enzymes FASN and ACSL1 in NAFLD model cells are remarkably increased, and mRNA levels and protein levels of fatty acid oxidative degradation key enzymes ACOX1 are remarkably reduced; the NAFLD model cell is simultaneously treated by miR-16Antagomir to inhibit miR-16 expression level, the expression level of FASN and ACSL1 of the cell is obviously reduced, and the expression level of ACOX1 is obviously increased (figure 3).

The above results demonstrate that miR-16 regulates the expression of key enzymes in lipid metabolism synthesis and degradation pathways in NAFLD cell models.

2.4 the miR-16 expression of mouse liver cells in the NAFLD group is high, and the physiological change of the fatty liver can be relieved by inhibiting miRNA-16;

in order to verify the correlation of miR-16 and fatty liver occurrence at the whole animal level, a mouse NAFLD model is constructed by feeding the mouse with high-fat feed for 8 weeks, and miR-16 expression level is interfered by tail vein injection of miR-16 Antagomir. qRT-PCR results show that miR-16 in liver tissues of NAFLD model mice is highly expressed; tail vein injection of miR-16Antagomir effectively down-regulated miR-16 levels in liver tissues of NAFLD model mice (figure 4).

The oil red O staining result shows that the liver cells of the control mouse are arranged regularly, and orange fat drops are not observed in the cells; a large amount of red fat drops can be seen in liver tissues of mice in a NAFLD model group and a NAFLD + Antagomir NC group; the liver tissue red lipid drop number of mice in NAFLD + miRNA-16Antagomir group is obviously reduced (FIG. 5A).

HE staining results show that the hepatocytes of the normal group are arranged neatly and have no obvious pathological changes; the liver tissues of mice in a NAFLD model group and a NAFLD + Antagomir NC group can show obvious diffuse fat vesicles, liver cells have balloon-like degeneration, cell nuclei are extruded and deformed, liver cell cords are disordered, and liver sinuses narrow or disappear; the extent of liver tissue pathology was reduced in mice of the NAFLD + miRNA-16Antagomir group (FIG. 5B).

In conclusion, miR-16 in liver tissues of the NAFLD model mouse is highly expressed, and miRNA-16Antagomir can obviously inhibit miR-16 level, improve pathological changes of fatty liver tissues, and show that miR-16 participates in promoting fatty liver generation.

2.5 inhibition of miR-16 expression can significantly alleviate the expression of pathologically-related molecules in NAFLD model animal serum

The serum biochemical detection result shows that: compared with the control group, NAFLD group mouse serum H₂O₂The contents of TG, ALT and AST are obviously increased, and the content of ATP is obviously reduced; h in serum of miR-16 Antagomir-treated group₂O₂The contents of TG, ALT and AST were significantly reduced, and the ATP content was significantly increased (fig. 6).

The results show that the expression of the molecules related to the liver histopathology of the mice in the NAFLD group is abnormal, and the expression of the miRNA-16 can be inhibited by the miR-16 antagonist, so that the expression of the molecules related to the pathology in the model can be relieved.

2.6miR-16 regulates and controls the expression of key enzymes of lipid metabolism pathway in liver tissue of NAFLD animal model

Western Blot detection results show (figure 7), mRNA levels and protein levels of fatty acid synthesis key enzymes FASN and ACSL1 in liver tissues of NAFLD model animals are remarkably improved, and mRNA levels and protein levels of fatty acid oxidative degradation key enzymes ACOX1 are remarkably reduced; the expression level of FASN and ACSL1 in liver tissues of the miR-16Antagomir treated group is remarkably reduced, and the expression level of ACOX1 is remarkably increased.

The results are completely consistent with cell experiments, and show that miR-16 regulates and controls the expression of key enzymes of lipid metabolism pathways in liver tissues of the NAFLD animal model.

Sequence listing

<110> first-person hospital in Hangzhou city

Application of <120> miR-16 antagonist in preparation of drug for inhibiting non-alcoholic fatty liver disease

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> RNA

<213> Unknown (Unknown)

<400> 1

agcagcagac agggcaga 18

<210> 2

<211> 23

<212> RNA

<213> Unknown (Unknown)

<400> 2

ucauagcccu guacaaugcu gcu 23

Claims (1)

1. The application of a miR-16 antagonist in the preparation of a drug for inhibiting non-alcoholic fatty liver disease, the sequence of the miR-16 is: AGCAGCAUUGUACAGGGCUAUGA; the sequence of the antagonist is: UCAUAGCCCUGUACAAUGCUGCU.

Images