CN117646064A

CN117646064A - Application of FAF2 protein in preparation of medicines for treating myocardial hypertrophy

Info

Publication number: CN117646064A
Application number: CN202311643196.1A
Authority: CN
Inventors: 刘鄂湖; 陈乾乾; 戎晓玲; 代彩侠
Original assignee: Nanjing University of Chinese Medicine
Current assignee: Nanjing University of Chinese Medicine
Priority date: 2023-12-01
Filing date: 2023-12-01
Publication date: 2024-03-05

Abstract

The invention discloses an application of FAF2 protein in preparing a medicament for treating myocardial hypertrophy, and discloses an application of FAF2 protein serving as a target in screening and preparing a medicament for treating myocardial hypertrophy. The invention proves that the FAF2 protein is highly expressed in a cardiac hypertrophy cell/animal model for the first time; the over-expression experiment proves that the over-expression of the FAF2 protein can cause the up-regulation of the expression level of the myocardial cell center muscle hypertrophy marker, and the phenomenon similar to a myocardial hypertrophy cell model is presented; the knock-down experiment proves that inhibiting FAF2 protein can relieve myocardial cell hypertrophy in a myocardial hypertrophy cell model, and has no obvious influence on the size of normal myocardial cells. Therefore, the FAF2 protein can be used as a target spot for screening and preparing medicaments for treating myocardial hypertrophy, and the FAF2 protein inhibitor has the prospect of being developed into medicaments for treating myocardial hypertrophy.

Description

Application of FAF2 protein in preparation of medicines for treating myocardial hypertrophy

Technical Field

The invention belongs to the field of medicines, and relates to application of FAF2 protein in preparation of medicines for treating myocardial hypertrophy.

Background

Cardiac hypertrophy refers to an increase in heart mass and volume that occurs under the influence of various factors, including hypertrophy of the cardiomyocytes, hyperplasia of the cardiomyocytes, and alterations in the extracellular matrix of the myocardium. Myocardial hypertrophy is a major factor in predicting heart disease progression and poor prognosis, and is often associated with ischemic heart disease, heart valve disease, hypertension, heart failure, and the like. There are 2 types of myocardial hypertrophy: physiological cardiac hypertrophy and pathological cardiac hypertrophy. Pathological cardiac hypertrophy can lead to adverse cardiovascular events including heart failure, malignant arrhythmias, sudden death, and the like. With the improvement of the living standard of people and the increase of the aging degree of population, the incidence rate and the prevalence rate of myocardial hypertrophy and heart failure are obviously increased.

However, the pathogenesis of myocardial hypertrophy is not clear at present, and the clinical treatment effect on myocardial hypertrophy is not ideal. Therefore, it is important to find new therapeutic targets and medicines to find methods for treating myocardial hypertrophy.

FAF2 is an abbreviation for FAS-related factor 2, which exists as a hairpin-like protein anchored to the cytoplasm on the surface of the endoplasmic reticulum and lipid droplets, binding UBX and UBA domains via its p97/VCP and ubiquitinated proteins. FAF2 has been reported to be involved in regulating the synthesis of mature VLDL in hepatocytes by transporting lipidated and ubiquitinated ApoB from the lipid droplet surface to the surface of the proteasome. FAF2 protein was also identified as one of the tumor markers for melanoma.

At present, no report exists on the correlation of FAF2 protein and myocardial hypertrophy.

Disclosure of Invention

The invention aims to provide application of FAF2 protein in preparing a medicament for treating myocardial hypertrophy.

The above object of the present invention is achieved by the following technical scheme:

the FAF2 protein is used as a target spot in screening and preparing medicaments for treating myocardial hypertrophy.

Application of FAF2 protein inhibitor in preparing medicine for treating myocardial hypertrophy is provided.

Further, the medicine takes the FAF2 protein inhibitor as an active ingredient, and is prepared into a pharmaceutically acceptable dosage form through pharmaceutically acceptable auxiliary materials.

Still further, the auxiliary material is a solid, liquid or semi-solid auxiliary material.

Still further, the dosage forms include tablets, capsules, injections and oral liquids.

The beneficial effects are that:

the invention proves that the FAF2 protein is highly expressed in a cardiac hypertrophy cell/animal model for the first time; the over-expression experiment proves that the over-expression of the FAF2 protein can cause the up-regulation of the expression level of the myocardial cell center muscle hypertrophy marker, and the phenomenon similar to a myocardial hypertrophy cell model is presented; the knock-down experiment proves that inhibiting FAF2 protein can relieve myocardial cell hypertrophy in a myocardial hypertrophy cell model, and has no obvious influence on the size of normal myocardial cells. Therefore, the FAF2 protein can be used as a target spot for screening and preparing medicaments for treating myocardial hypertrophy, and the FAF2 protein inhibitor has the prospect of being developed into medicaments for treating myocardial hypertrophy.

Drawings

FIG. 1 shows the target protein and RNA expression levels in a cardiac hypertrophy cell model, wherein: A. western blot detects FAF2 and BNP protein expression, tubulin is an internal reference protein, and n=3/group; B. relative expression levels of FAF2, BNP protein relative to Tubulin protein, n=3/group; C. RT-PCR detects the expression of Faf2 and cardiac hypertrophy marker Nppa, nppb, myh7 at mRNA level, n=3/group; D. immunofluorescence detection of Faf2 protein expression level;

FIG. 2 shows the target protein and RNA expression levels in a cardiac hypertrophy animal model, wherein: A. western blot detects FAF2 and BNP protein expression, tubulin is an internal reference protein, and n=3/group; B. relative expression levels of FAF2, BNP protein relative to Tubulin protein, n=3/group; C. RT-PCR detects the expression of Faf2 and cardiac hypertrophy marker Nppa, nppb, myh7 at mRNA level, n=3/group; D. immunohistochemical detection of Faf2 protein expression levels;

fig. 3 is a graph of FAF2 overexpression induced H9c2 cardiomyocyte hypertrophy wherein: A. western blot detects FAF2 protein expression, tubulin is an internal reference protein, n=3/group; B. RT-PCR detects Faf2 expression at mRNA level, n=3/panel; C. RT-PCR detects expression of cardiac hypertrophy marker Nppa, nppb, myh at mRNA level, n=3/group.

FIG. 4 shows FAF2 knockdown inhibition H ₂ O ₂ Induced H9c2 cardiomyocyte hypertrophy wherein: A. western blot detects FAF2 protein expression, tubulin is an internal reference protein, n=3/group; B. RT-PCR detects Faf2 expression at mRNA level, n=3/panel; C. detection of FAF2 knock-down pair H by phalloidin staining ₂ O ₂ Induced H9c2 cardiomyocyte hypertrophy effect, n=3/group.

Detailed Description

The following describes the essential aspects of the present invention in detail with reference to examples, but is not intended to limit the scope of the present invention.

Example 1: FAF2 protein high expression in cardiac hypertrophy cell/animal model

1. Experimental materials

H9c2 rat cardiomyocytes were purchased from Shanghai Life cell Bank of China academy of sciences. SPF-grade male, 7-8 week old C57BL/6J mice purchased from Shanghai Sipuler-BiKai laboratory animals Co. Sodium pentobarbital and angiotensin II (Ang II) were purchased from Sigma. DMEM high sugar medium was purchased from kemel biosystems. Fetal Bovine Serum (FBS) was purchased from Gibco corporation. Antibodies used include: ant-FAF2 antibody (Cat No.16251-1-AP, proteintech), ant-BNP antibody (Cat No. sc-271185, santa), beta-Tubulin antibody (Cat No. #2146, CST), alexaFluor 594 goat anti-rabbit antibody (Cat No.33712ES60, yeasen).

2. Experimental method

1. Myocardial hypertrophy cell model modeling (Ang II induction method) and target protein and RNA determination

1.1H9c2 cell culture and Ang II drug treatment

(1) Cell resuscitation: rapidly shaking and thawing a freezing tube containing 1mLH9c2 cell suspension in a water bath at 37 ℃, and adding 4mL of culture medium to uniformly mix; centrifuging at 1000rpm for 5min, discarding supernatant, adding 1mL of culture medium, and blowing uniformly. All H9c2 cell suspensions were then added to a 6cm dish, approximately 4mL of complete medium (90% DMEM+10% FBS) was added, and incubated overnight in incubator (95% air+5% carbon dioxide; temperature: 37 ℃). The next day the fluid was changed and the cell density was checked.

(2) Cell plating: after the H9c2 cell density reaches 80% -90%, the cells are digested by trypsin-EDTA digestion solution, resuspended and spread in six-well plates, each well containing about 1×10 ⁶ Individual cells. Cell grouping: control (Ctrl), model (Ang II), three duplicate wells were set up per group.

(3) Cell modeling: after the H9c2 cell density reaches 60% -70%, changing the culture medium into a serum-free culture medium for 6 hours, adding Ang II with the final concentration of 10 mu M, molding for 48 hours, and collecting cell proteins and RNA for corresponding detection.

1.2 target protein assay

(1) The protein expression is detected by Western blot, and the specific steps are as follows: after H9c2 cell molding is finished, using RIPA cell lysate to lyse cells on ice for 30min, and centrifuging at 12000rpm for 10min; taking the supernatant, and carrying out protein quantification according to the instructions of the BCA protein assay kit; adding protein loading buffer solution, heating at 99deg.C for 10min to denature protein, and performing subsequent steps. Protein samples were subjected to polyacrylamide gel electrophoresis using 10% separation gel, electrophoresis voltage: 80V,30min;120V,60min; transferring the polyacrylamide gel after electrophoresis onto a PVDF membrane, and transferring the membrane current: 320mA; and after the film transfer is finished, taking out the PVDF film, and putting the PVDF film into 5% skimmed milk powder for sealing for 2 hours. After rinsing with a small amount of TBST, PVDF membranes were placed in an antibody dilution containing primary antibody (FAF 2/BNP/Tubulin) and placed in a refrigerator at 4 ℃ for overnight incubation. The next day, the PVDF membrane was taken out and washed 10min X4 times with TBST, and the PVDF membrane was placed in TBST solution containing the secondary antibody and incubated at room temperature for 2 hours. TBST was washed 10min X4 times. The PVDF membrane was incubated with ECL luminescence and developed in a dark room.

(2) Immunofluorescence detection FAF2 protein expression comprises the following specific steps: after stimulating H9c2 cells Ang II (10. Mu.M) in six well plates for 48H; discarding the culture medium, adding 4% paraformaldehyde solution, and fixing at room temperature for 15min; 1mL of 3% BSA was added to each well, and the wells were blocked at room temperature for 2 hours; FAF2 antibody (1:100) was diluted with PBS-formulated 3% BSA solution, 500. Mu.L of diluted antibody was added to each well, and incubated overnight at 4 ℃; the next day, PBS buffer was washed 3 times, 5min each time; alexa Fluor 594 goat anti-rabbit antibody (1:200) was diluted with 3% BSA solution, 500. Mu.L was added to each well and incubated for 2h at room temperature in the dark; washing with PBS buffer solution for 3 times, adding 500 mu LDAPI staining solution into each hole, and standing at 37 ℃ in dark place for 5min for incubation; the anti-fluorescence quenching chip sealing solution is diluted by PBS buffer solution (dilution ratio is 1:10), 1mL of the anti-fluorescence quenching chip sealing solution is added to each hole, and the anti-fluorescence quenching chip sealing solution is photographed under an inverted fluorescence microscope.

1.3 target RNA assay

Detection of Faf2 and myocardial cell hypertrophy marker Gene (Nppa, nppb, myh 7) expression using real-time fluorescent quantitative PCR (RT-qPCR): after H9c2 cells were treated with Ang II, the cells were collected in an enzyme-free sterile centrifuge tube; 1mL Trizol is added into each tube in a fume hood, and the mixture is cracked for 5min at room temperature; adding 200 mu L of chloroform, shaking for 30s, and standing at room temperature for 2min; centrifuging for 15min at 4 ℃ and 12000 g; the upper layer solution was taken and placed in a new 1.5mL EP tube, added with equal volume of isopropanol, mixed well and allowed to stand for 5min. Centrifuging for 10min at 4 ℃ with 12000 g; carefully remove the supernatant, filter paper to suck the residual liquid on the wall of the centrifugal tube, and reversely buckling and standing for 5min. 1mL of precooled 75% ethanol was added; the top and bottom of the centrifuge tube are turned upside down for several times, RNA at the bottom of the centrifuge tube is suspended, and the centrifuge tube is centrifuged for 10min at 4 ℃ and 12000 g; removing the supernatant again; standing at room temperature, and naturally air-drying for 5min; adding DEPC water, and denaturing at 99deg.C for 2min. RNA concentration and purity (OD 260/OD280 between 1.8 and 2.0) were measured using a Nano-300 micro-spectrophotometer. The extracted total RNA was then reverse transcribed into cDNA by following the reverse transcription kit procedure. The Gene of the target Primer was searched for on the Gene column of NCBI website to find the CDS region sequence on the mRNA sequence of the target Gene, and then the Primer-BLAST of NCBI was used to find the Primer sequence specific to the CDS region, with the Primer information as follows (synthesized by bioengineering).

TABLE 1PCR primer sequences

(1) Preparation of the reaction System

According to the reaction system recommended in the reagent specification of three compound holes of each group of samples, the following reaction system is prepared:

(2) After the system is prepared, the system is added into a PCR plate to carry out the next RT-qPCR reaction.

(3) The PCR plate was placed in a PCR instrument and the procedure was as follows.

(4) After the PCR process is completed, the result is imported into a computer, the data is processed by using a light 96System Software, and the data is quantified by using a Threshold cycle (Ct) method, wherein 18s is taken as an internal reference, and the expression quantity of a target gene is according to a formula 2 ^{-[(Ctofgene)-(Ctof18SrRNA)]} To calculate.

2. Modeling of cardiac hypertrophy animal model (TAC method) and determination of target protein and RNA

2.1 moulding

After one week of adaptive feeding, 6C 57BL/6J experimental mice were randomized into two groups (Sham group and TAC group), sham group mice were operated with the same TAC group mice except that they did not constrict the aortic arch. The TAC model comprises the following specific steps: (1) Mice were anesthetized by intraperitoneal injection of 1% pentobarbital, dehaired in the chest area, placed on a constant temperature hot plate at 37 ℃, and rubbed with 75% medical alcohol to disinfect the skin. (2) Skin was cut at the midpoint of the two upper limbs of the mouse and soft tissues on both sides under the skin were released, the sternum was transected between the second ribs, and a chest support was placed. (3) The left and right thymus were gently separated along the superior sternal edge with a microtrap to cut the skin to the fourth rib, exposing the aortic arch. (4) The threading device belt 6-0 medical suture thread penetrates through the fat soft tissue of the upper edge of the aorta, penetrates out of the triangle area of the fat soft tissue of the lower edge of the aorta, the ophthalmic forceps pull out the suture thread, the original path exits the threading device belt 6-0 medical suture thread penetrates through the fat soft tissue of the upper edge of the aorta, penetrates out of the triangle area of the fat soft tissue of the lower edge of the aorta, the ophthalmic forceps pull out the suture thread, and the original path exits the threading device. (5) The 27G needle is padded at the aortic arch, the surgical knots are tied on the 6-0 suture, after the two knots are pulled to be solid, the superfluous suture is cut off, and the 27G needle is withdrawn. (6) 5-0 medical suture lines suture the left and right pectoral large muscles of the mouse. (7) Suturing skin, sterilizing with alcohol cotton ball, and placing the mice on a heating pad at 37deg.C until anesthesia is recovered. (8) Mice were sacrificed after TAC surgery for 4 weeks with routine feeding, and heart tissue was taken for protein and RNA extraction and detection.

2.2 protein assay methods

(1) Cardiac tissue protein extraction

After the experimental animal is finished, cervical vertebra is removed, the heart is taken down, left ventricular tissue is cut off, a certain amount of tissue is weighed and cut off, RIPAly buffer containing protease inhibitor is added according to the proportion of 1:10 (mg: mu L), and a tissue grinder is used for homogenizing. The parameters are as follows: run for 50s, stop for 10s, total 5 cycles. Centrifuging the homogenate at 4 ℃ and 12000rpm for 10min, collecting supernatant, and taking a small amount of supernatant to carry out protein quantification according to the BCA protein content determination kit. Adding 1/4 volume of 5 Xloading buffer solution into the quantified sample, mixing, decocting at 99deg.C for 15min, cooling, and storing in a refrigerator at-80deg.C.

(2) The Westernblot detection protein expression method is the same as experimental method 1.2 in example 1.

(3) Immunohistochemical detection of tissue protein expression

(a) Myocardial tissue was fixed with 4% paraformaldehyde and paraffin embedded. (b) Paraffin sections baked at 60℃overnight. (c) dewaxing: soaking in xylene for 10-15 min; soaking in xylene II for 10-15 min; soaking in absolute ethanol I for 10min; soaking in absolute ethanol II for 10min; soaking in 95% ethanol for 5min; soaking in 80% ethanol for 5min; washing with water for 1-2 min. (d) antigen retrieval: preparing 1X sodium citrate solution, performing high-temperature repair on the antigen, boiling in boiling water for 40min, sealing, naturally cooling, cooling to room temperature, dropwise adding 3% hydrogen peroxide solution on sliced tissues, and incubating at room temperature for 15min to block endogenous catalase. (e) closing: and (3) a proper size and closed range is defined around the tissue by using a grouping pen, 2 drops of goat serum sealing liquid are dripped, the goat serum sealing liquid is uniformly covered on the surface of the tissue, and the tissue is sealed for 1h at room temperature. (f) The tissue was rinsed 3X 5min with PBS, diluted primary antibody solution was added dropwise to the tissue to cover the tissue surface uniformly, and the wet cartridges were incubated overnight in a refrigerator at 4℃with specific reference to the respective instructions. (g) The solution was rinsed 3X 5min with PBS, and the polymer adjuvant was added dropwise and incubated at room temperature for 20min. (h) Washing with PBS for 3X 5min, adding diluted secondary antibody solution, and incubating for 1-2 h at room temperature. (i) Washing with PBS for 3X 5min, dripping freshly prepared DAB color development liquid, controlling the reaction time, and washing with tap water in time to terminate the color development reaction. (j) counterstaining: the Harris hematoxylin is counterstained for 30s to 1min, is differentiated by 1 percent hydrochloric acid alcohol after being washed by water, and is washed by tap water to turn blue. (k) dehydration and transparency: 90% alcohol for 5min;95% alcohol for 5min; absolute alcohol I for 10min; absolute alcohol II for 10min; xylene I for 10min; xylene II for 10min. And (l) dripping a neutral resin sealing piece. (m) observation: observing the expression condition of the protein in the tissue under an optical microscope, and photographing and storing.

2.3RNA determination method

(1) Cardiac tissue RNA extraction

After cervical vertebra removal and sacrifice, the heart is taken down, left ventricular tissue is cut off, a certain amount of tissue is weighed and cut off, grinding beads and 1ml of Trizol lysate are added, and the mixture is put into a tissue grinder for grinding until the mixture is homogenized. The parameters are as follows: run for 45s, stop for 10s, 6 cycles total. The subsequent procedure is the same as in experimental procedure 1.3 of example 1. The primer sequences used are shown in the following table:

TABLE 2PCR primer sequences

3. Data processing

The GraphPadPrism statistical mapping software was used for the analysis and all values were expressed as mean ± standard deviation (mean ± SD). Statistics were examined using unpaired Student's t, with p <0.05 considered statistically significant differences. Specific p values are noted in the figure.

3. Experimental results

1. Target protein and RNA expression levels in myocardial hypertrophy cell models

And (3) after stimulating H9c2 myocardial cells by Ang II for 48 hours, collecting cells, and detecting the expression level of target proteins and RNA by western blot, RT-qPCR and cellular immunofluorescence. FAF2, BNP protein expression levels are shown as A, B in fig. 1, FAF2, nppa, nppb, myh7mRNA expression is shown as C in fig. 1, and FAF2 fluorescence intensity is shown as D in fig. 1.

The results show that compared with the control group, the expression levels of BNP protein and Nppa, nppb, myh mRNA which are markers of myocardial hypertrophy in the model group are obviously increased, which indicates that the modeling of the myocardial hypertrophy cell model is successful; compared with the control group, the expression level of the FAF2 protein and mRNA in the model group is obviously increased, which indicates that the FAF2 protein is highly expressed in a myocardial hypertrophy cell model.

2. Target protein and RNA expression levels in animal models of cardiac hypertrophy

And detecting the expression level of target protein and RNA in heart tissue of the cardiac hypertrophy animal model by Western blot, immunohistochemistry and RT-qPCR. FAF2, BNP protein expression levels are shown as A, B, D in fig. 2, FAF2, nppa, nppb, myh7mRNA expression levels are shown as C in fig. 2.

The results show that compared with a control group, the expression levels of BNP protein and Nppa, nppb, myh7mRNA of a cardiac hypertrophy marker in heart tissues of a model group are obviously increased, which indicates that the modeling of a cardiac hypertrophy animal model is successful; compared with the control group, the expression level of the FAF2 protein and mRNA in the model group is obviously increased, which indicates that the FAF2 protein is highly expressed in the animal model of myocardial hypertrophy.

This example demonstrates the high expression of FAF2 protein in cardiac hypertrophy cells/animal models.

Example 2: FAF2 overexpression leads to upregulation of myocardial cell center muscle hypertrophy marker expression levels

1. Experimental materials

pcDNA3.1 (Vector); the pcDNA3.1-Flag-FAF2 (OE-FAF 2) plasmid is constructed and synthesized by PPL company; lipofectamine 3000 is purchased from Thermo Scientific; opti-MEM medium was purchased from Gibco corporation.

2. Experimental method

1. Cell culture and grouping

The culture conditions of H9c2 cells were the same as in experimental method 1.1 of example 1. H9c2 was divided into Vector and OE-FAF2 groups, vector was transfected with empty plasmid (pcDNA3.1), OE-FAF2 was transfected with full-length FAF2 plasmid (pcDNA3.1-Flag-FAF 2), and three duplicate wells were placed in each group.

2. Transfection plasmid

After the H9c2 cell density reaches 80% -90%, the cells are digested by trypsin-EDTA digestion solution, resuspended and spread in six-well plates, each well containing about 1×10 ⁶ Individual cells. Divided into two groups of three duplicate wells.

The transfection procedure was as follows: (1) the medium in the 6-well plate was aspirated and washed once with PBS. The wells were replaced with 1ml of serum-free medium per well. (2) preparing A solution: mu.g of plasmid and 5. Mu. l P3000 were diluted with 200. Mu.l of Opti-MEM. (3) preparing solution B: mu.l lipo3000 was diluted with 200. Mu.l Opti-MEM. And (4) respectively and gently mixing the solution A and the solution B, and standing for 5min. (5) And (3) sucking the solution B, adding the solution B into the solution A, gently mixing, and standing at room temperature for 20min. (6) The A+B mixture was added to the medium in each well, and after 5 hours, the medium was replaced with DMEM complete medium containing 10% FBS, and the culture was continued for 48 hours.

After culturing H9c2 cells for 48 hours, using RIPA cell lysate to lyse cells on ice to extract cell proteins, and adopting western blot to detect the expression of FAF2 and BNP protein of a myocardial cell hypertrophy marker, wherein the detailed steps are the same as those of the experimental method 1.2 of the example 1; the total RNA of the cells was extracted using Trizol and then subjected to subsequent RT-qPCR detection, and the RNA assay was performed in the same manner as in example 1, experimental method 1.3.

3. Data processing

3. Experimental results

To explore the function of FAF2 in cardiomyocytes, we overexpressed the FAF2 protein in cardiomyocytes. The FAF2 overexpression efficiency is detected by western blot and RT-PCR. The results showed that FAF2 protein and mRNA expression levels were significantly elevated in FAF 2-overexpressed OE-FAF2 cells compared to Vector cells, indicating that we successfully achieved FAF2 overexpression in H9c2 cells (A, B in fig. 3). The RT-PCR test results showed that the expression level of the central muscle hypertrophy marker Nppa, nppb, myh7 of the OE-FAF2 group overexpressing FAF2 was significantly increased compared to the Vector group (C in fig. 3), indicating that overexpression of FAF2 protein induced cardiomyocyte hypertrophy, exhibiting a phenomenon similar to that of the cardiomyocyte model.

Example 3: FAF2 knock-down regulates expression level of myocardial hypertrophy marker in myocardial hypertrophy cell model

1. Experimental materials

pGPU6-GFP-Neo vector (NC); pGPU6-GFP-Neo-shFAF2 (shFAF 2- #1, shFAF2- #2shFAF2- #3, shFAF2- # 4) (shFAF 2) plasmid was purchased from Shanghai Ji Ma company; lipofectamine 2000 was purchased from Thermo Scientific; opti-MEM medium was purchased from Gibco, 35% hydrogen peroxide solution (cat. H433859-250 ml) was purchased from Alatine; phalloidin (cat No. 40734ES 75) was purchased from shanghai next san biosciences.

2. Experimental method

1. Build H ₂ O ₂ Induced myocardial hypertrophy cell model

1.1 cell culture and grouping

The culture conditions of H9c2 cells were the same as in experimental method 1.1 of example 1. H9c2 is divided into NC group and shFAF2 group, NC group transfects empty plasmid (pGPU 6-GFP-Neo), shFAF2 group transfects FAF2 knockdown plasmid (pGPU 6-GFP-Neo-shFAF 2), each group is provided with three compound holes.

1.2 method for establishing model

Cell cycle was synchronized by starving the cells with DMEM medium containing 1% fbs for 12h prior to cell experiments. Subsequent addition of H ₂ O ₂ (100. Mu. Mol/L) for 24h. After 24h, the cellular proteins and RNA were collected for subsequent detection.

2. Transfection of FAF2 knockdown plasmids

After the H9c2 cell density reaches 80% -90%, the cells are digested by trypsin-EDTA digestion solution, resuspended and spread in six-well plates, each well containing about 1×10 ⁶ Individual cells. The method is divided into NC groups and shFAF2 groups, and each group is provided with three compound holes.

The transfection procedure was as follows: (1) the medium in the 6-well plate was aspirated and washed once with PBS. The wells were replaced with 1ml of serum-free medium per well. (2) preparing A solution: mu.g of plasmid was diluted with 200. Mu.l of Opti-MEM. (3) preparing solution B: mu.l lipo2000 was diluted with 200. Mu.l Opti-MEM. And (4) respectively and gently mixing the solution A and the solution B, and standing for 5min. (5) And (3) sucking the solution B, adding the solution B into the solution A, gently mixing, and standing at room temperature for 20min. (6) After 5 hours, the A+B mixture was added to the medium in each well and replaced with DMEM complete medium containing 10% FBS, and the culture was continued for 24 hours.

After transfection of H9c2 cells to 24 hours, the cell culture medium was replaced with DMEM medium containing 1% FBS for 12 hours, followed by addition of H ₂ O ₂ (100. Mu. Mol/L) stimulation for 24h.

Cell protein is extracted by using RIPA cell lysate to lyse cells on ice, and the expression of FAF2 protein is detected by using western blot, and the detailed steps are the same as those of the experimental method 1.2 of the example 1; the total RNA of the cells was extracted using Trizol and then subjected to subsequent RT-qPCR detection, and the RNA assay was performed in the same manner as in example 1, experimental method 1.3.

3. Cell size detection by cell phalloidin staining

The method comprises the following specific steps: (1) The H9c2 cell slide grows for 24 hours to ensure that the density reaches 50 percent of confluence, and FAF2 knockdown plasmid transfection and H are carried out ₂ O ₂ And (5) stimulating treatment. (2) Wait for H ₂ O ₂ After 24h of stimulation (100. Mu. Mol/L), the culture medium was aspirated and the cells were washed 2 times with 1 XPBS (pH 7.4) pre-warmed at 37 ℃. (3) Cell fixation was performed using 4% formaldehyde solution in PBS, for 10min at room temperature. (4) Cells were washed 3 times with PBS at room temperature for 10min each. (5) Permeabilization with 0.5% Triton X-100 solution was performed for 5min at room temperature. (6) Cells were washed 3 times with PBS at room temperature for 10min each. (7) 200. Mu.L of the prepared TRITC-labeled phalloidin working solution (200 nM) was used to cover the cells on the coverslip and incubated at room temperature for 30min in the dark. (8) the coverslip was washed 3 times with PBS for 5min each. (9) Nuclei were counterstained with 200 μl DAPI solution (100 nM) for about 5min. (10) The coverslip was washed with PBS and then inverted onto a slide onto which a drop of water-soluble coverslip had been dropped. Excess caplets were gently rubbed off using a paper towel, and permanently capped with nail polish. (11) Fluorescence observation photographing under a fluorescence microscope was performed, and TRITC excitation/emission filter (Ex/em=545 was selected570 nm) and DAPI excitation/emission filter (Ex/em=364/454 nm).

4. Data processing

The GraphPadPrism statistical mapping software was used for the analysis and all values were expressed as mean ± standard deviation (mean ± SD). The differences between groups were examined using one-way anova with p <0.05 being considered statistically significant differences. Specific p values are noted in the figure.

3. Experimental results

To further explore the function of FAF2 in cardiomyocytes, we constructed 4-site knockdown plasmids (shFAF 2- #1, shFAF2- #2, shFAF2- #3, shFAF2- # 4) for knockdown of FAF 2. Western blot detection results show that compared with NC groups, shFAF2- #3 and shFAF2- #4 groups of cells have significantly reduced FAF2 protein, which indicates that we successfully realize the knockout of FAF2 protein in H9c2 cells (A in FIG. 4). RT-PCR detection results show that compared with NC groups, the expression of Faf2 mRNA in shFAF2- #3 and shFAF2- #4 groups of cells is obviously reduced, and further prove that shFAF2- #3 and shFAF2- #4 plasmids can realize the knock-down inhibition effect of FAF2 at protein and mRNA levels (B in FIG. 4). The test result of phalloidin staining shows that the knockdown FAF2 protein has no obvious effect on the size of normal myocardial cells, and the knockdown FAF2 can obviously reduce the cell size in a myocardial hypertrophy cell model (C in fig. 4).

The above examples demonstrate for the first time that FAF2 protein is highly expressed in cardiac hypertrophy cells/animal models; the over-expression experiment proves that the over-expression of the FAF2 protein can cause the up-regulation of the expression level of the myocardial cell center muscle hypertrophy marker, and the phenomenon similar to a myocardial hypertrophy cell model is presented; the knock-down experiment proves that inhibiting FAF2 protein can relieve myocardial cell hypertrophy in a myocardial hypertrophy cell model, and has no obvious influence on the size of normal myocardial cells. Therefore, the FAF2 protein can be used as a target spot for screening and preparing medicaments for treating myocardial hypertrophy, and the FAF2 protein inhibitor has the prospect of being developed into medicaments for treating myocardial hypertrophy.

The above-described embodiments serve to describe the substance of the present invention in detail, but those skilled in the art should understand that the scope of the present invention should not be limited to this specific embodiment.

Claims

The FAF2 protein is used as a target spot in screening and preparing medicaments for treating myocardial hypertrophy.
Application of FAF2 protein inhibitor in preparing medicine for treating myocardial hypertrophy.
3. The use according to claim 2, wherein the medicament is prepared into a pharmaceutically acceptable dosage form by taking the FAF2 protein inhibitor as an active ingredient and using pharmaceutically acceptable auxiliary materials.
4. Use according to claim 3, wherein the adjuvant is a solid, liquid or semi-solid adjuvant.
5. The use according to claim 3, wherein the dosage forms comprise tablets, capsules, injections and oral liquids.