CN113999905B - Application of HNRNPA2B1 in cardiac fibroblasts - Google Patents

Abstract

The invention belongs to the technical field of cell engineering and genetic engineering, and relates to application of HNRNPA2B1 in cardiac fibroblasts. The sequence of HNRNPA2B1 is SEQ ID No.1 in a nucleotide sequence table. The invention researches key molecules and signal pathways of nicotine-induced myocardial fibers, and has great significance for explaining the internal mechanism of myocardial fibrosis generation. The invention verifies that after the interference of HNRNPA2B1, the expression of miR-125B-5p can be inhibited, the purpose of anti-fibrosis is achieved, the miR-125B-5p is an important functional target of HNRNPA2B1 in heart fibroblasts, and the function of the fibroblasts can be regulated by the HNRNPA2B1 through the miR-125B-5 p. Has good application value for researching the myocardial fibrosis mechanism induced by nicotine.

Description

Application of HNRNPA2B1 in cardiac fibroblasts

Technical Field

The invention belongs to the technical field of cell engineering and genetic engineering, and relates to application of HNRNPA2B1 in cardiac fibroblasts.

Background

Myocardial interstitial fibrosis is an important factor for inducing arrhythmia and causing heart failure. At present, an effective intervention strategy is lacking clinically, and the method is a great problem to be solved urgently at present. Myocardial fibrosis is an important pathological basis for the occurrence of heart failure, and is mainly characterized by myocardial fibroblast activation, abnormal deposition of collagen components, myocardial elasticity reduction to further influence cardiac output and finally cause cardiac insufficiency. If the excessive myocardial fibrosis can be inhibited in time, the overall function of the heart is expected to be improved. Inhibiting excessive myocardial fibrosis is an important entry point in the prevention and control of heart failure and myocardial fibrosis related diseases. The occurrence of myocardial interstitial fibrosis is associated with a number of factors. The main harmful component nicotine in tobacco is an important risk factor for cardiovascular diseases, but related researches are few at present, and the specific action mechanism of the nicotine is not clear. Chinese men always belong to one of the highest global smoking rate people. The adult tobacco survey in China in 2020 shows that the number of current smokers in China increased 1500 ten thousand years ago, and reaches 3.16 hundred million (15.2 per day per capita). Smoking remains a significant culprit in cardiovascular events. The discovery of drugs, specific molecules and signal pathways for regulating and controlling myocardial fibrosis has very important theoretical and clinical significance for clarifying the internal mechanism of the occurrence and development of myocardial fibrosis and searching for a potential new target for preventing and controlling heart failure and myocardial fibrosis related diseases.

The TGF- β pathway is one of the major signaling pathways regulating cell growth, differentiation and morphogenesis, and is a central regulatory factor of extracellular matrix deposition in fibrosis of many tissues, which can activate fibroblast proliferation and promote extracellular matrix production, playing an important role in the process of myocardial fibrosis. The TFG-beta/MAPK signaling pathway can promote cell proliferation and transformation, and is an important pathway for inducing myocardial fibrosis.

HNRNPA2B1 encodes 2 major proteins HNRNPA2 and HNRNPB1 by alternative splicing. Involved in packaging, alternative splicing, and trafficking, translation, and stabilization of cytoplasmic RNA of nascent mRNA. HNRNPA2 and HNRNPB1 also play a role in telomere maintenance, cell proliferation and differentiation, and glucose transport. It has recently been found that HNRNPA2B1 activates the MAPK/ERK signaling pathway to promote pancreatic cancer metastasis, and that serum antibodies raised against HNRNPA2B1 are found in the serum of patients with skin fibrotic diseases. The subfamilies HNRNPA1, K and E can promote the expression of collagen I and collagen III in myocardial fibroblasts, and TGF-beta can induce the expression of HNRNPA1, K and E and promote myocardial fibrosis.

Micrornas (mirnas) are a class of endogenous non-coding single-stranded small RNA of about 22 nucleotides in length, and can induce degradation of mRNA of a target gene or inhibit translation by complementary pairing with an untranslated region (UTR) of 3' of mRNA of Messenger Ribonucleic Acid (mRNA) to different degrees, thereby regulating expression of the target gene at a post-transcriptional level.

Disclosure of Invention

In order to research and control action targets and specific molecules and signal channels of myocardial fibrosis and clarify the internal mechanism of myocardial fibrosis generation and development, the invention provides the application of HNRNPA2B1 as a target in myocardial fibroblasts.

In order to achieve the purpose, the invention is realized by adopting the following technical scheme:

the application of DARTS experiments, WB experiments, Coomassie brilliant blue staining, mass spectrometry and further analysis of later-stage experiments proves that HNRNPA2B1 plays a role in the process of inducing myocardial fibrosis by nicotine. The nucleic acid sequence of HNRNPA2B1 is SEQ ID No.1 in the nucleotide sequence table.

Another objective of the invention is to provide siRNA of small interfering RNA fragment for inhibiting HNRNPA2B1 gene.

The siRNA sequences are as follows:

si-r-HNRNPA2B1_001 ：5’-GGATTTGGCTTTGTAACTT-3’,

si-r-HNRNPA2B1_002： 5’-GGAAGATACTGAGGAGCAT-3’，

si-r-HNRNPA2B1_003 ：5’-GGAAATTATGGAAGTGGAA-3’。

the invention verifies that HNRNPA2B1 is combined with miR-125B-5p and the expression of the miR-125B-5p is regulated and controlled through qRT-PCR and RIP experiments, and the miR-125B-5p sequence is as follows: 5'-UCCCUGAGACCCUAACUUGUGA-3' are provided. HNRNPA2B1 increases miR-125B-5p expression and accelerates proliferation and differentiation of cardiac fibroblasts and accumulation of collagen.

The qRT-PCR primer sequences used were:

β-actin：

in the forward direction 5'-CGTTGACATCCGTAAAGACC-3' of the direction,

a reverse direction 5'-TAGAGCCACCAATCCACACA-3';

Col1：

in the forward direction 5'-CCAGCGGTGGTTATGACTTCA-3' of the direction,

a reverse direction 5'-TGCTGGCTCAGGCTCTTGA-3';

Col3：

in the forward direction 5'-GGTCACTTTCACTGGTTGACGA-3' of the direction,

a reverse direction 5'-TTGAATATCAAACACGCAAGGC-3';

a-SMA：

in the forward direction 5'-CATCCGACCTTGCTAACGGA-3' of the direction,

a reverse direction 5'-GTCCAGAGCGACATAGCACA-3';

Fn：

in the forward direction 5'-GGATCCCCTCCCAGAGAAGT-3' of the direction,

a reverse direction 5'-GGGTGTGGAAGGGTAACCAG-3';

TGFb1：

in the forward direction 5'-GCCGCCCGAAGGGTAGAT-3' of the direction,

a reverse direction 5'-GAGGCAAGACTGGTGTCTCC-3';

TGFbR1：

in the forward direction 5'-TGCCTGCTTCTCATCGTGTT-3' of the direction,

a reverse direction 5'-TGCTTTTCTGTAGTTGGGAGTTC-3';

HNRNPA2B1：

in the forward direction 5'-CAGACTGTGTGGTTATGCGG-3' of the direction,

and reverse direction 5'-TCAACCACCCTGCCATCAAT-3'.

Compared with the prior art, the invention has the advantages and positive effects that:

the invention researches key molecules and signal paths of nicotine-induced myocardial fibrosis and has great significance for explaining the internal mechanism of myocardial fibrosis generation. The invention provides the effect of HNRNPA2B1 on cardiac fibroblasts, and HNRNPA2B1 is taken as a research object, HNRNPA2B1 interference small-fragment siRNA is synthesized, the cardiac fibroblasts are transfected, and HNRNPA2B1 is found to promote the proliferation, migration, differentiation and collagen synthesis of the cardiac fibroblasts. Studies were also conducted by controlling the function of miR-125b-5p, whose expression is in cardiac fibroblasts; the invention verifies that after HNRNPA2B1 is interfered, the expression of miR-125B-5p can be inhibited, and the aim of resisting fibrosis is fulfilled. The miR-125B-5p is shown to be an important functional target of HNRNPA2B1 in the cardiac fibroblasts, and HNRNPA2B1 can regulate the functions of the fibroblasts through the miR-125B-5 p. Has good application value for researching the myocardial fibrosis mechanism induced by nicotine.

Drawings

Figure 1 is nicotine-induced cell proliferation.

FIG. 2 shows the interference gene knockdown of HNRNPA2B1 gene.

FIG. 3 shows DARTS and CETSA experimental results.

Fig. 4 is a graph of cardiac fibroblast activation and associated signaling pathways following nicotine addition.

FIG. 5 shows the results of the RIP experiment. SNP70 is a positive control.

In FIG. 2, ctr is the negative control of small interfering RNA, s1 is si-r-HNRNPA2B1_001, s2 is si-r-HNRNPA2B1_002, and s3 is si-r-HNRNPA2B1_ 003.

Detailed Description

In order that the above objects, features and advantages of the present invention may be more clearly understood, the present invention will be further described with reference to specific embodiments. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments of the present disclosure.

Example 1

1. And (4) pretreatment.

Subject: 2-3d Wistar rat born rat heart fibroblasts, supplied by Beijing Wittingle Inc., in DMEM high-sugar medium from Gibco, USA.

(1) Preparation before experiment: sterilizing with ultraviolet for 30 min.

(2) Taking heart fibroblasts of suckling mice, digesting the heart fibroblasts by type II collagenase, and carrying out differential adherence in a culture bottle to remove non-fibroblasts.

(3) Discarding supernatant, adding culture medium again, and adding 5% CO at 37 deg.C₂And (5) standing and culturing for 24 hours in an incubator.

2. Seeding and transfection of cardiac fibroblasts.

(1) When the confluency of cardiac fibroblasts reached 90% under microscopic observation, the medium was discarded and the cells were washed twice with pre-warmed PBS containing 1% diabody (penicillin and streptomycin).

(2) Adding preheated 0.25% pancreatin into culture flask, placing into incubator, standing for 2min, and adding stop solution (10% FBS DMEM high sugar medium) to stop digestion when most of cells float under microscope.

(3) The cells were gently pipetted with a pipette tip to prepare a cell suspension, which was transferred to a 15 mL centrifuge tube and centrifuged at 800 rpm for 5 min.

(4) Suspending the cell sediment by proper amount of complete culture medium, counting the cells, uniformly distributing the counted cells in each hole, and putting the cells into an incubator for continuous culture.

(5) The cell morphology and the confluence degree are observed for about 24 hours, and the cell morphology is good and the confluence degree reaches 70% -90%, so that the cell can be used for transfection. The 3 rd passage cardiac fibroblasts were transfected and the transfection procedure was performed according to the 2000 kit instructions of Invitrogen.

(6) After transfection, cells were at 37 ℃ and 5% CO₂And continuing culturing in the incubator.

(7) Cells were harvested 48h after transfection.

3. Cardiac fibroblast proliferation assay.

Reference is made to the Cell-light EdU Apollo 567 In vitro Kit instructions from Ruibo Biotech, Inc., Guangzhou, as follows (96-well plate).

The cells were cultured in a cell culture medium at 1000: edu solution was diluted at a ratio of 1 to give Edu medium at a concentration of 50. mu.M.

Each well was incubated for 2h with 100. mu.L of Edu medium at a concentration of 50. mu.M, and the medium was discarded.

The cells were washed with PBS 1-2 times, 5min each time.

Add 50. mu.L of cell fixative (4% paraformaldehyde in PBS) to each well and incubate for 30min at room temperature, discard the fixative.

Add 100. mu.L PBS to each well, wash on a decolorization shaker for 5min, discard PBS.

Add 100. mu.L of 1 XApollo staining reaction solution into each well, incubate for 30min in dark, room temperature, decolorizing and shaking table, discard the staining reaction solution.

Adding 100 mu L of penetrating agent (PBS containing 0.3% Triton x-100) to decolorize and wash for 2-3 times, each time for 5min, and discarding the penetrating agent.

Deionized water was added according to a 100: 1 to obtain 1 Xhoechst 3342 reaction solution, and storing in dark.

Add 100. mu.L of 1 × Hoechst3342 reaction solution into each well, incubate for 30min in dark at room temperature in a decolorized shaker, and then discard the staining reaction solution.

150 μ L of PBS was added to each well and washed 1-3 times.

Add 100. mu.L of PBS to each well and store until use.

Cardiac fibroblasts were treated with a range of different concentrations of nicotine in the range of 1-1000 nM, and photograph collection and proliferation rate analysis were performed using a fluorescence microscope. The results are shown in fig. 1, and it can be seen that the proliferation of cardiac fibroblasts can be significantly induced after the addition of nicotine, and the cell proliferation amount varies with the nicotine concentration.

Example 2

qRT-PCR experiments.

(1) And (3) cleaning adherent cells for 2-3 times by using cold PBS, inclining a cell culture dish, completely sucking the PBS, adding 1mL of Trizol, standing for 5min until the cells are completely cracked under a microscope, uniformly blowing and beating the cells by using a gun head, and transferring the cells to a 1.5mL RNase-free centrifuge tube.

(2) 200 mul of precooling liquid (Trizol tissue fluid: chloroform: 5: 1) is added into each tube, the tube is vigorously shaken for 15-30 s, and after being placed on ice for 5min, the tube is centrifuged for 15 min at 12000 rpm and 4 ℃. The liquid is divided into three layers: the upper layer is colorless RNA, the middle layer is a white water phase containing phenol-chloroform, and the lower layer is light red. The upper colourless liquid phase was carefully aspirated (avoiding touching the intermediate phase). The upper phase (about 300. mu.L) was transferred to a new 1.5mL centrifuge tube, an equal amount of isopropanol solution was added, shaken gently upside down for 10 times, and allowed to stand on ice for 10 min. Then centrifuged at 12000 rpm, 4 ℃ for 10 min.

(3) The supernatant was discarded, the pellet was retained, and 1mL of pre-cooled, 75% ethanol-DEPC was added along the tube wall to wash the RNA, followed by centrifugation at 12000 rpm at 4 ℃ for 5min and then the supernatant was removed.

(4) And (4) sucking the liquid, and naturally drying at room temperature for 5-10 min, and simultaneously avoiding excessive drying of the RNA precipitate.

(5) Depending on the amount of precipitate, 20. mu.L of DEPC water was added to dissolve the RNA precipitate. The RNA concentration and purity were determined spectrophotometrically.

As the qRT-PCR, SYBR Green qRT-PCR Master Mix (2X), ROX Solution rendered Mix (2X) from Thermo was used. By 2^-△△CtThe method calculates the relative expression quantity of mRNA and miRNA.

The qRT-PCR primers used were as follows:

β-actin：

in the forward direction 5'-CGTTGACATCCGTAAAGACC-3' of the direction,

a reverse direction 5'-TAGAGCCACCAATCCACACA-3';

Col1：

in the forward direction 5'-CCAGCGGTGGTTATGACTTCA-3' of the direction,

a reverse direction 5'-TGCTGGCTCAGGCTCTTGA-3';

Col3：

in the forward direction 5'-GGTCACTTTCACTGGTTGACGA-3' of the direction,

a reverse direction 5'-TTGAATATCAAACACGCAAGGC-3';

a-SMA：

in the forward direction 5'-CATCCGACCTTGCTAACGGA-3' of the direction,

a reverse direction 5'-GTCCAGAGCGACATAGCACA-3';

Fn：

in the forward direction 5'-GGATCCCCTCCCAGAGAAGT-3' of the direction,

a reverse direction 5'-GGGTGTGGAAGGGTAACCAG-3';

TGFb1：

in the forward direction 5'-GCCGCCCGAAGGGTAGAT-3' of the direction,

a reverse direction 5'-GAGGCAAGACTGGTGTCTCC-3';

TGFbR1：

in the forward direction 5'-TGCCTGCTTCTCATCGTGTT-3' of the direction,

a reverse direction 5'-TGCTTTTCTGTAGTTGGGAGTTC-3';

HNRNPA2B1：

in the forward direction 5'-CAGACTGTGTGGTTATGCGG-3' of the direction,

and reverse direction 5'-TCAACCACCCTGCCATCAAT-3'.

As shown in FIG. 2, after cardiac fibroblasts were transfected with different small interfering siRNA fragments (transfection concentration of 50 nM), qRT-PCR detected the knockdown efficiency for HNRNPA2B1, and the results showed that fragments si-r-HNRNPA2B1_001(s1), si-r-HNRNPA2B1_002(s2), and si-r-HNRNPA2B1_003(s3) were all able to significantly knock down the expression of HNRNPA2B1, with the highest knockdown efficiency of si-r-HNRNPA2B1_ 001. Ctr (scarmble siRNA) is a negative control of siRNA, and the sequence of small interfering siRNA fragments is as follows:

si-r-HNRNPA2B1_001：5’-GGATTTGGCTTTGTAACTT-3’，

si-r-HNRNPA2B1_002：5 ’-GGAAGATACTGAGGAGCAT-3’，

si-r-HNRNPA2B1_003：5’-GGAAATTATGGAAGTGGAA-3’。

the small interference siRNA fragment by Guangzhou city Ruibo biological science and technology limited synthesis.

Example 3

Target point stability experiment of drug affinity reaction (DARTS for short).

The specific operation steps are as follows:

(1) preparation of M-PER lysate (1 mL).

mu.L of protease inhibitor cocktail, 10. mu.L of 200mM phosphatase inhibitor, 50. mu.L of 1M sodium fluoride, 100. mu.L of 100mM disodium beta-glycerophosphate, 100. mu.L of 50mM sodium pyrophosphate, and 730. mu.L of M-PER were mixed to obtain M-PER lysate.

(2) The cells were washed 2 times with pre-cooled PBS, the PBS was aspirated, 500. mu.L of M-PER lysate (2/3 dishes in one according to cell density) was added to each dish, the cells were scraped with a cell scraper and transferred to a 1.5mL centrifuge tube, and incubated for 30min at 4 ℃ with a rotary shaker.

(3) Centrifuging at 13000 rpm and 4 ℃ for 10min, taking the supernatant, and measuring the concentration. Storage at-80 ℃ or subsequent next step.

(4) Two 1.5mL centrifuge tubes were each filled with 225. mu.L of protein, one tube was added with 2. mu.L of LDMSO, the other tube was added with 2. mu.L of nicotine, and incubated on a rotary shaker at room temperature in the dark for 1-1.5 h.

(5) Preparation of TNC buffer solution: 1mL of 10 XTNC, 500. mu.L of 1M Tris-HCl buffer (pH 8.0), 100. mu.L of 5M sodium chloride, 100. mu.L of 1M calcium chloride, and 300. mu.L of ultrapure water were mixed to obtain TNC. When in use, TNC was diluted 10 times with ultrapure water.

(6) Each tube of protein was divided into 4 tubes (total 8 tubes), 50. mu.L per tube. Four empty centrifuge tubes were prepared, formulating different concentrations of protease: the protease was added at a ratio of protease/protein =0, 1:800, 1:400, 1: 200. Adding diluted protease into corresponding protein tubes (grouped as-0, +0, -1:800, +1:800, -1:400, +1:400, -1:200, +1:200, -as control group and + as medicine group, and adding gel sample according to the order), respectively, and adding 2 μ L into each tube; in the-0 and +0 groups, 2. mu.L of 1 XTNC was added and incubated at room temperature for 15 min, 1. mu.L of the protease inhibitor cocktail was added to each tube and incubated on ice for 5min to stop digestion.

(7) According to the protein: adding the sample buffer solution at a ratio of 4:1, boiling in water bath for 10min, and storing in refrigerator at-80 deg.C.

(8) Adding sample according to the sequence, directly dyeing the sample with Coomassie blue for 1 h without membrane conversion after electrophoresis, replacing destaining solution, eluting until a clear strip (generally 24 h) appears, selecting mass spectrometry, and performing membrane conversion, color development and verification according to the steps at the later stage.

Wherein the Coomassie brilliant blue dye solution consists of 0.05 g of Coomassie brilliant blue R-250, 91mL of 50% methanol aqueous solution and 9mL of glacial acetic acid.

The decolorized solution is prepared by mixing 10mL of glacial acetic acid, 80mL of distilled water and 10mL of methanol.

The results are shown in fig. 3A, and it can be seen from fig. 3A that nicotine can bind to HNRNPA2B1 to protect its proteins from degradation, suggesting that HNRNPA2B1 is a potential binding target of nicotine.

Example 4

CETSA experiments.

The experimental procedure was as follows:

1. a melting curve of the protein of interest is obtained.

(1) Cell expansion to 2X 10⁶Four melting curves required 1.2X 10⁸And (4) cells.

(2) The cells were cultured in a suitable cell culture dish (control group with water as solvent control, experimental group with nicotine) and the heating step was performed after cell isolation.

(3) The petri dish was removed from the incubator and placed on ice, washed 3 times with PBS, scraped off with cells from the control and experimental groups, respectively, placed in a 1.5mL centrifuge tube, and placed on ice.

(4) The number of cells was counted and the activity of the cells was evaluated.

(5) Centrifuge at 1000rpm in 4 ℃ centrifuge for 5 min.

(6) 15 mL of PBS resuspended cells, centrifuged again and the supernatant discarded (this step can be repeated).

(7) The cell suspension was distributed into 10 0.2 mL qRT-PCR vials with 100 μ Ι _ of cell suspension per vial, each vial was labeled with a specified temperature, 40 vials were divided into 10, 4 each, and left at room temperature.

(8) The tubes were heated at the first six temperatures (40-67 ℃) for 3 min using a 96-well plate thermocycler, immediately removed, incubated at room temperature for 3 min, placed on ice, and when all the vials were heated, the samples were immediately frozen as per step 12.

(9) The cell suspension after heat treatment was rapidly frozen in liquid nitrogen (or frozen for storage in a freezer at-80 ℃ C.), and the samples were stored at-80 ℃ for one night.

(10) The cells were frozen-thawed twice using liquid nitrogen (-80 ℃ freezer) and a thermocycler set at 25 ℃ (or 37 ℃ dry box) to ensure uniform temperature between tubes, with a shaker shaking briefly after each thaw, and the lysate was placed on ice.

(11) After the final thawing, the tube was briefly shaken and centrifuged at 12000 rpm for 10min at 4 ℃ to pellet the cell debris together with the aggregated proteins, and the tube was carefully removed and placed on ice.

(12) And (3) taking 90 mu L of the supernatant into a new test tube, and adding a loading buffer solution to boil the protein for 10 min.

(13) Load 20. mu.L per well and WB detect the target protein.

(14) Protein gray values were measured at different temperatures and melting curves were plotted.

2. A dose-response curve at a fixed temperature was obtained.

The temperature of this step is determined by the analysis of the previous step and is illustrated by way of example at 50 ℃.

(1) The cells were uniformly planted in six-well plates and treated according to different concentrations of the drug.

(2) The six-well plate was removed from the incubator and placed on ice, washed 3 times with PBS, and the treatment groups of different concentrations were scraped off with cells, placed in 1.5mL centrifuge tubes, and placed on ice.

(3) The number of cells was counted and the activity of the cells was evaluated.

(4) Centrifuge at 1000rpm in 4 ℃ centrifuge for 5 min.

(5) Centrifuge again and discard the supernatant.

(6) Preparing cell lysate containing 10%, respectively adding 100 μ L of cell lysate into centrifuge tubes with different concentrations, blowing to resuspend cells, respectively transferring to corresponding 200 μ L centrifuge tubes, and labeling with different concentrations.

(7) Place 200. mu.L of the centrifuge tube in a 96-well plate thermal cycler, heat the tube at 50 ℃ for 3 min, immediately remove the tube, place on ice, and immediately place in liquid nitrogen or-80 ℃ freezer.

(8) The cells were frozen-thawed twice using liquid nitrogen (-80 ℃ freezer) and a thermocycler set at 25 ℃ (or 37 ℃ dry box) to ensure uniform temperature between tubes, with a shaker shaking briefly after each thaw, and the lysate was placed on ice.

(9) After the final thaw, the tube was briefly shaken and centrifuged at 12000g for 10min at 4 ℃ to pellet the cell debris along with the aggregated proteins, and the tube was carefully removed and placed on ice.

(10) Take 90. mu.L or 80. mu.L of supernatant in a new tube (avoid touching the tube wall and precipitating), BCA kit for protein concentration determination, add loading buffer to boil the protein for 10 min.

(11) Load 20. mu.L per well and WB detect the target protein.

Protein gray values were measured at different concentrations and melting curves were plotted.

The results are shown in fig. 3B, where HNRNPA2B1 increased thermal stability compared to the group without nicotine, suggesting that nicotine may increase its thermal stability by direct binding to HNRNPA2B 1.

Example 5

Western Blot experiment.

(1) And (3) extracting the total protein of the heart fibroblast.

48h after cell transfection, the cells were washed 3 times with PBS, protein lysis was performed by adding 80. mu.L of protein lysate to each well of 6-well plate cells, and the cell lysate was transferred to a 1.5mL centrifuge tube. Centrifuging at 12000 rpm and 4 deg.C for 15 min, collecting supernatant, transferring into new 1.5mL centrifuge tube, and storing at low temperature to prevent protein degradation.

SDS-PAGE electrophoresis: after initial quantification of protein samples by BCA method, 20 μ g total protein per group and 5 × loading buffer were run at 5: 1 mixing and boiling for 10 min. SDS-PAGE electrophoresis is carried out until the bromophenol blue just comes out of the gel bottom.

(2) And (5) transferring the film.

Soaking a polyvinylidene fluoride membrane (PVDF membrane) for 15 s by formaldehyde, washing by clear water, and soaking the PVDF membrane, the adsorption filter paper and the formaldehyde in a membrane transferring solution. Stacking the adsorption filter paper (1 sheet), the gel, the PVDF membrane and the adsorption filter paper (1 sheet) in sequence from the anode, and closing the cathode. Loading into an electrophoresis tank. The membrane conversion process is carried out on ice, the constant pressure is 80V, and the membrane conversion time is selected according to different target proteins.

(3) And (4) detecting protein and analyzing results.

After the transfer of the membrane was complete, the membrane was rinsed in 1 XPBST and blocked with 5% skimmed milk powder for 2h at room temperature. The membrane was washed with 1 XPBST, diluted with the desired antibody dilution and incubated overnight at 4 ℃. Primary antibodies (HNRNPA2B1 primary antibody; alpha-SMA primary antibody; all of which are conventional commercial products) were recovered, 1 XPBST washed membranes. And incubating a secondary antibody (secondary antibody: horseradish peroxidase-labeled goat anti-rabbit IgG-HRP, China fir gold bridge) for 2h at room temperature. 1 × PBST membrane washing. Detection was performed by ECL chemiluminescence, exposure was performed using a Bio-Rad exposure system and pictures were taken. The electrophoretic band intensity value was measured using Image J.

The results are shown in fig. 4, and the addition of nicotine can significantly induce the activation of cardiac fibroblasts, which is shown by the significant increase of a-SMA, and activate fibrosis-related signaling pathways such as TGF β, JNK, p38 and the like. Compared with a control group, the activation of alpha-SMA and the above-mentioned pathway protein is obviously inhibited after the knock-down of HNRNPA2B 1.

Example 6

Immunofluorescence detects a-SMA and PCNA.

The method comprises the following steps:

(1) fibroblasts were fixed with 4% paraformaldehyde for 15 minutes.

(2) The wells were perforated using 0.3% Triton X-100 for 3 minutes.

(3) Wash 3 times with Phosphate Buffered Saline (PBS) for 5 minutes each.

(4) Blocking the membrane with 10% donkey serum and keeping at room temperature for 40 min.

(5) Applying a first antibody: primary cultured cells were incubated with a-SMA and PCNA antibody (1: 200, diluted) overnight at 4 ℃.

(6) Applying a second antibody: cells were incubated with fluorescent secondary antibodies, Alexa Fluor 488 (donkey anti-rabbit) IgG and Alexa Fluor 568 (donkey anti-mouse), diluted 1:200, respectively, purchased from Invitrogen, usa for 40 min.

(7) Photographing and result analysis.

The experimental result shows that the expression of a-SMA and PCNA is obviously induced after nicotine treatment, and the trend is obviously improved after HNRNPA2B1 is knocked down.

Example 7

RNA co-immunoprecipitation assay (RIP).

This example was performed according to the instructions of the Millipop RIP kit (cat # 17-701).

The result is shown in figure 5, and it can be seen from the figure that HNRNPA2B1 can bind to miR-125B-5p and affect the expression of miR-125B-5p, and the expression of miR-125B-5p is reduced after HNRNPA2B1 is knocked out.

Among them, FIG. 5A shows that HNRNPA2B1 is able to bind to miR-125B-5p, where SNP70 is a positive control. FIG. 5B shows that miR-125B expression is significantly increased after nicotine addition, whereas miR-125B expression is inhibited after knock-down of HNRNPA2B 1. The miR-125b-5p inhibitor and the miR-125b-5p analogue are co-transfected at the same time of the cardiac fibroblasts, and the miR-125b-5p analogue can remarkably induce the expression of the myocardial fibrosis marker protein, but the adding of the inhibitor results are opposite.

The above description is only a preferred embodiment of the present invention, and not intended to limit the present invention in other forms, and any person skilled in the art may apply the above modifications or changes to the equivalent embodiments with equivalent changes, without departing from the technical spirit of the present invention, and any simple modification, equivalent change and change made to the above embodiments according to the technical spirit of the present invention still belong to the protection scope of the technical spirit of the present invention.

SEQUENCE LISTING

<110> Jinan City central hospital

<120> application of HNRNPA2B1 in cardiac fibroblasts

<130> 1

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 1777

<212> DNA

<213> Artificial sequence

<400> 1

aaggttctag aaaagcggcg gtctcggctc cagcggcagt agcagcggcg ccggtcccgt 60

gtgcaggtgc tccgtgcggc gcagtttctt ggccatcggc tgctctcacc acagcgtcag 120

gacgagtccc gtgcgtgtcc gtccgcggag gtctttctct tctcgctcgg ctgcgggaaa 180

tcgagctgaa gcgactgagt ccgcgatgga gaaaacttta gaaactgttc ctttggagag 240

gaaaaagaga gaaaaggaac agttccgtaa gctctttatt ggtggcttaa gctttgaaac 300

cacagaagaa agtttgagaa actactacga gcaatgggga aagctcacag actgtgtggt 360

tatgcgggat cctgcaagca aaagatcaag aggatttggc tttgtaactt tctcatctat 420

ggctgaggtt gatgctgcca tggctgcaag gcctcattcc attgatggca gggtggttga 480

gccaaaacgt gctgtggcaa gagaggagtc tgggaaacca ggagcccatg tgactgtgaa 540

gaaactgttc gttggtggaa ttaaggaaga tactgaggag catcacctta gagattactt 600

tgaagaatat ggaaaaattg atactattga aataattact gataggcagt ctggaaaaaa 660

aagaggcttt ggctttgtta cctttgatga ccatgatcct gtggataaga ttgtcttgca 720

gaaatatcac accataaatg gtcacaatgc agaagttaga aaggcattgt ctagacaaga 780

aatgcaggaa gtccaaagtt ctaggagtgg aagaggagga aactttggtt ttggagattc 840

tcgaggtggc ggtggcaatt ttggaccagg accaggaagc aactttaggg ggggttctga 900

tggatatgga agtggacgtg gatttgggga tggctataat gggtatggag gaggacctgg 960

aggtggcaat tttggaggta gccctggtta tggaggagga agaggaggat atggtggtgg 1020

aggacctgga tatggcaacc agggtggggg ctacggaggt ggttatgaca actatggagg 1080

aggaaattat ggaagtggaa attacaatga ttttggaaat tataaccagc aaccttctaa 1140

ctacggtcca atgaagagtg gaaactttgg tggtagcagg aacatgggag gaccatatgg 1200

tggagggaac tatggtcctg gaggaagtgg aggaagtggg ggctatggtg gaaggagccg 1260

atattgagct tcttcctact taccatgggc ttcactgtat aaataggaga ggatgagagc 1320

ccagaggtaa cagaacagct tcaggttatc gaaataacaa tgttaaggaa actcttatct 1380

cagtcatgca taaatatgca gtgatatggc agaagacacc agagcagatg cagagagcca 1440

ttttgtgaat ggattggatt atttaatacc ttactgtgga ggaaggattg taaaaaaaaa 1500

tgcctttgag acagtttctt agctttttaa ttgttgtttc tttctagtgg tctttgtaag 1560

agtgtagaag cattccttct ttgataatgt taaatttgta agtttcaggt gacatgtgaa 1620

acctttttta agatttttct caaaaagttt tgaaaagcta ttagccagga tcatggtgta 1680

ataagacata acgtttttcc tttaaaaatt taagtgcgtg tgtagagtta agaagctgtt 1740

gtacatttat gatttaataa aataattcta aaggaaa 1777

<210> 2

<211> 57

<212> DNA

<213> Artificial sequence

<400> 2

ggatttggct ttgtaacttg gaagatactg aggagcatgg aaattatgga agtggaa 57

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence

<400> 3

ucccugagac ccuaacuugu ga 22

<210> 4

<211> 40

<212> DNA

<213> Artificial sequence

<400> 4

cgttgacatc cgtaaagacc tagagccacc aatccacaca 40

<210> 5

<211> 40

<212> DNA

<213> Artificial sequence

<400> 5

ccagcggtgg ttatgacttc atgctggctc aggctcttga 40

<210> 6

<211> 44

<212> DNA

<213> Artificial sequence

<400> 6

ggtcactttc actggttgac gattgaatat caaacacgca aggc 44

<210> 7

<211> 40

<212> DNA

<213> Artificial sequence

<400> 7

catccgacct tgctaacgga gtccagagcg acatagcaca 40

<210> 8

<211> 40

<212> DNA

<213> Artificial sequence

<400> 8

ggatcccctc ccagagaagt gggtgtggaa gggtaaccag 40

<210> 9

<211> 38

<212> DNA

<213> Artificial sequence

<400> 9

gccgcccgaa gggtagatga ggcaagactg gtgtctcc 38

<210> 10

<211> 43

<212> DNA

<213> Artificial sequence

<400> 10

tgcctgcttc tcatcgtgtt tgcttttctg tagttgggag ttc 43

<210> 11

<211> 40

<212> DNA

<213> Artificial sequence

<400> 11

cagactgtgt ggttatgcgg tcaaccaccc tgccatcaat 40

Claims (2)

1. The application of the interfering RNA segment siRNA of HNRNPA2B1 gene in preparing the medicine for regulating and controlling the nicotine-induced cardiac fibroblast proliferation is disclosed, wherein the sequence of the HNRNPA2B1 gene is SEQ ID No.1 in a nucleotide sequence table, and the siRNA sequence is as follows:

   si-r-HNRNPA2B1_001：5’-GGATTTGGCTTTGTAACTT-3’，

   si-r-HNRNPA2B1_ 002: 5'-GGAAGATACTGAGGAGCAT-3' or

   si-r-HNRNPA2B1_003：5’-GGAAATTATGGAAGTGGAA-3’。
2. 2. The use according to claim 1, wherein the expression level of HNRNPA2B1 gene in the regulation process is detected by qRT-PCR, the primer sequences used in the qRT-PCR detection process are as follows,

   in the forward direction 5'-CAGACTGTGTGGTTATGCGG-3' of the direction,

   and reverse direction 5'-TCAACCACCCTGCCATCAAT-3'.

Images