CN113980985B - Application of LDHA (laser direct current) in cardiac fibroblasts - Google Patents

Abstract

The invention belongs to the technical field of cell engineering and genetic engineering, and relates to application of LDHA in heart fibroblast. The sequence of the LDHA is SEQ ID No.1 in a nucleotide sequence table. The proliferation, migration, differentiation and collagen synthesis capacity of fibroblasts are important mechanisms influencing myocardial fibrosis, and the invention proves that LDHA participates in the cardiac fibroblasts to influence the processes for the first time. Therefore, the invention provides a new target for treating myocardial fibrosis, and provides a new idea and method for diagnosing and preventing myocardial fibrosis in the aspect of biological gene means.

Description

Application of LDHA (laser direct current) in cardiac fibroblasts

Technical Field

The invention belongs to the technical field of cell engineering and genetic engineering, and relates to application of LDHA in heart fibroblast.

Background

Fibrosis is broadly defined as the accumulation of extracellular matrix and can be divided into repair fibrosis and reactive fibrosis. Tissue repair after myocardial infarction is replacement myocardial fibrosis, namely repair myocardial fibrosis; and the extracellular matrix accumulation and the metabolic process change caused by non-ischemic cardiomyopathy are reactive myocardial fibrosis. In alternative myocardial fibrosis formed following myocardial infarction, scarring of cross-linked collagen fibers provides strong structural support to the heart, and reactive fibrosis, resulting from prolonged maladaptive signals such as hypertensive heart disease, diabetic heart disease, hypertrophic heart disease, and the like, is manifested by perivascular and interstitial fibrosis. Myocardial fibrosis provides structural support when cells are lost, but myocardial structural changes caused by myocardial fibrosis can lead to further development of cardiac dysfunction, induce arrhythmia and worsen the condition of patients with heart failure. After the fibroblast differentiates into myofibroblast, the extracellular matrix is deposited in a large amount, and scar is formed at the infarction position to maintain the stable structure of the heart. In interstitial fibrosis, activation of fibrosis can occur without cardiomyocyte death, alpha-SMA expression is increased after fibroblast activation is converted into myofibroblasts, and extracellular matrix is diffusely deposited in the cardiac interstitium to influence the diastolic function of the heart.

Myocardial fibrosis refers to the hyperproliferation of fibroblasts in the heart and the transformation into alpha-SMA positive myofibroblasts under the stimulation of various pathological factors. Myofibroblasts are the major source of extracellular matrix, the major components of which are type I and type III collagen. Excessive deposition of extracellular matrix alters the original normal architecture of the heart and myocardial remodeling occurs. Myocardial fibrosis is the end-stage manifestation of a variety of cardiac diseases, leading to increased myocardial stiffness, enlargement of the heart chamber, systolic and diastolic dysfunction. Myocardial remodeling leads to the development of malignant cardiovascular events such as atrial fibrillation, malignant arrhythmias, heart failure and even sudden cardiac death.

Lactate Dehydrogenase (LDHA) is an enzyme that catalyzes the last step of the wobberg effect by converting pyruvate and NADH to lactate. In tumors, it contributes to epithelial-to-mesenchymal transition (EMT) and metastasis. It has been reported that the presence of up-regulated LDHA in many types of tumors is associated with poor prognosis. The LDHA reduction significantly inhibited cell transformation, significantly delayed tumor formation. Studies have shown that LDHA binds to NADH and promotes Reactive Oxygen Species (ROS) to induce catabolic changes by stabilizing the key pro-inflammatory mediator I κ B- ζ in chondrocytes. The increase of ROS is related to the effects of myocardial fibrosis and myocardial hypertrophy, and the effect of LDHA in the heart is not further researched.

Autophagy is a highly conserved metabolic process specific to eukaryotic cells, and mainly means that lysosomes degrade intracellular structures (biological macromolecules such as organelles, long-life proteins or nucleic acids and the like) and provide raw materials for cell reconstruction, regeneration and repair, so that the cells are recycled and reused. The main role of autophagy is to clear, degrade damaged cellular structures, senescent organelles and macromolecules no longer needed, etc. within the cell and to selectively remove damaged mitochondria. Thus, autophagy is closely indistinguishable from a balance of cell growth, development and cellular homeostasis. Autophagy, which contributes to the synthesis of cellular products and maintains a metabolic balance, is the primary mechanism by which cells acquire energy in a state of deficiency in nutrients. Autophagy plays an important role in cardiomyocytes, and the main reasons include: since cardiomyocytes are long-lived, late-dividing cells and have little ability to differentiate and regenerate, autophagy in cardiomyocytes plays an important role in maintaining cardiac function and activity. Secondly, as mentioned above, autophagy can selectively remove damaged mitochondria, and cardiomyocytes are rich in mitochondria, so autophagy has important significance on myocardial pathophysiological states and has myocardial protection effect. ③ the level of apoptosis of the cardiomyocytes in the patients with the heart disease is low, and the death of the cardiomyocytes in the patients with the heart disease is related to the type II programmed cell death, namely the autophagic cell death.

Disclosure of Invention

The invention provides application of LDHA gene as a target in preparation of anti-myocardial fibrosis drugs aiming at the problems in the traditional myocardial fibrosis research process.

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

nicotine induces myocardial fibrosis and exacerbates the development of myocardial fibrosis in a concentration-dependent manner. The invention provides that LDHA protein plays a role in the process of inducing myocardial fibrosis by nicotine.

The invention provides a method for changing the expression quantity of an LDHA gene in a fibroblast by a genetic engineering technology, which can influence the proliferation, migration, collagen synthesis and differentiation of the fibroblast, wherein the sequence of the LDHA gene is SEQ ID No.1 in a nucleotide sequence table.

The invention also provides siRNA for inhibiting LDHA gene, which has the following sequence:

si-r-LDHA_001 ：5’-GCTTGTGCCATCAGTATCT-3’；

si-r-LDHA_002 ：5’-GGGAGAGATGATGGATCTT-3’；

si-r-LDHA_003 ：5’-TCCCATTTCCACCATGATT-3’。

after siRNA segments for inhibiting LDHA genes are supplemented in cardiac fibroblasts, the proliferation and differentiation of the cardiac fibroblasts can be inhibited, collagen deposition is inhibited, and an anti-fibrosis effect is achieved.

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

the proliferation, migration, differentiation and collagen synthesis capacity of fibroblasts are important mechanisms influencing myocardial fibrosis, and the invention firstly proposes and proves that LDHA participates in the cardiac fibroblasts to influence the processes. Therefore, the invention provides a new target for treating myocardial fibrosis, and provides a new idea and method for diagnosing and preventing myocardial fibrosis in the aspect of biological gene means.

Drawings

FIG. 1 shows DARTS experimental results.

FIG. 2 shows the result of knocking-out efficiency of siRNA to LDHA by qRT-PCR detection.

Fig. 3 shows the effect of LDHA knockdown on nicotine-induced cell proliferation activity.

FIG. 4 shows the result of Western blot detection of the effect of LDHA knock-down and nicotine stimulation on the activation index alpha-SMA of cardiac fibroblasts and the proliferating cell nuclear antigen PCNA.

FIG. 5 shows the effect of knocking down LDHA on autophagy as detected by Western blot.

FIG. 6 shows expression conditions of PCNA and alpha-SMA, which are related indexes of fibroblast proliferation and activation, detected by Western blot after inhibitor FX11 of LDHA is pretreated.

FIG. 7 shows a Western blot to examine the effect of inhibitor FX11 of LDHA on blocking of autophagy induced by nicotine.

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) preparing materials.

(1) Preparation before experiment: the culture flask was sterilized by UV light for 30 min.

(2) Taking heart of suckling mouse, and cutting into 1-3 mm³The small fragments were digested 3 times with collagenase type II and differentially attached in culture flasks for 1.5 hours to remove cardiomyocytes.

(3) Removing supernatant from the flask, adding DMEM medium, and culturing at 37 deg.C with 5% CO₂Culturing in an incubator for 24 h.

Wherein, the suckling mouse is a Wistar rat born for 2-3 days and purchased from Jinnanpunyue animal breeding company Limited. The medium was gibco's high-glucose DMEM medium in the united states.

2. Seeding and transfection of cardiac fibroblasts.

(1) When the confluency of the cardiac fibroblasts reached 90% under an inverted microscope, the medium was removed and the cells were washed 2 times with 1% double antibody (penicillin and streptomycin) in PBS.

(2) Digestion was carried out for 2 min with 0.25% trypsin, and when most of the cells were microscopically rounded, digestion was terminated by adding 10% FBS in DMEM.

(3) Cells were gently pipetted using a Pasteur pipette to make a cell suspension and transferred to a 15mL centrifuge tube and centrifuged at 800 rpm for 5 min.

(4) And (4) resuspending the cell sediment by using a complete culture medium, uniformly seeding the cell sediment into each hole after the cell counting is completed, and placing the cell sediment into an incubator to continue culturing.

(5) After 24 hours, the morphology and confluence of the cells are observed, and the cells can be used for transfection when the cell state is good and the confluence reaches 70% -90%. The 3 rd generation cardiac fibroblasts were transfected according to the instructions of the Lipofectamine 2000 transfection kit from sefmei corporation.

(6) After transfection, the cells were further cultured at 37 ℃ in a 5% carbon dioxide incubator.

(7) Cells were harvested 48h after transfection.

3. Target stability experiments for Drug Affinity Reactions (DARTS).

(1) Preparation of M-PER lysate (1 mL): 10 μ L protease inhibitor cocktail, 50 μ L1M sodium fluoride, 100 μ L100 mM sodium beta-glycerophosphate, 100 μ L50 mM sodium pyrophosphate, 10 μ L200 mM sodium orthovanadate, 730 μ L M-PER.

(2) After precooling PBS to wash cells for 2 times, adding 200 mu L PBS into each hole, scraping the cells in 6 holes to a 1.5mL EP tube by using a cell scraper, centrifuging for 5min at the temperature of 1000 rpm and 4 ℃, removing the PBS, adding 500 mu LM-PER lysate, blowing and beating by using a gun head, placing on ice for 30min, and blowing and beating 2-3 times by using the gun head in the middle.

(3) 13000rpm, 4 ℃ for 10 min.

(4) After the centrifugation, the supernatant was collected and the concentration was measured.

(5) And (3) subpackaging 500 mu L of protein into two 1.5mL centrifuge tubes on average, adding 2 mu L of ultrapure water into one tube, adding 2 mu L of the gulf-pudding stock solution into the other tube, and incubating for 1-1.5h at room temperature by a light-shielding shaking table.

(6) Preparation of TNC: mu.L of 1M Tris-HCl 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 a TNC stock solution, which was diluted 10-fold with ultrapure water when used.

(7) Each tube of protein was divided into 4 tubes of 50. mu.L each. Four empty centrifuge tubes were prepared and different concentrations of protease were prepared. Following protease (stock concentration 10 mg/mL, 2. mu.L per tube): protein =0, 1: 800, 1: 400, 1:200 were added to the corresponding protein tubes, respectively, and 2 μ L of diluted TNC stock solution was added to the control group. After incubation at room temperature for 15 min, digestion was stopped by adding 1 μ L of cocktail per tube and incubating for 5min on ice.

(8) Adding protein sample buffer solution, boiling in water bath for 5-10 min, and storing at-80 deg.C.

After digestion is completed, the gel is run, silver stained or coomassie brilliant blue stained, and the difference bands are cut off and sent to mass spectrometry. And (4) screening out target proteins related to functions according to the molecular weight and the functions. The results are shown in FIG. 1. As can be seen in figure 1, nicotine was able to bind to LDHA inhibiting its degradation by proteolytic enzymes.

Example 2

qRT-PCR experiments.

Washing adherent cardiac fibroblasts for 2-3 times by using precooled PBS, inclining a cell culture dish, completely sucking the PBS, adding 1mL of Trizol tissue fluid, cracking the cells 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.

Adding 200 mu L of chloroform into each tube, shaking vigorously for 15-30 s, standing for 5min, and then centrifuging at 12000rpm and 4 ℃ for 10 min. The upper colorless liquid phase was carefully aspirated. Transferring the upper liquid phase into a new 1.5mL centrifuge tube, adding an equal volume of isopropanol solution, shaking up and down gently for 10 times, and standing on ice for 10 min. Then, the mixture was centrifuged at 12000rpm at 4 ℃ for 15 min. The supernatant was discarded and 1mL of pre-cooled, 75% ethanol-DEPC was added along the tube wall to wash the RNA pellet, centrifuged at 12000rpm for 4 ℃ for 5min and then discarded. And (4) sucking the liquid, and naturally drying at room temperature for 5-10 min to avoid excessive drying of the RNA precipitate. Depending on the amount of precipitate, about 20. mu.L of DEPC water was added to dissolve the RNA precipitate. The RNA concentration and purity were determined spectrophotometrically.

The reverse transcription reaction was performed using the PrimeScriptTM RT Master Mix (Perfect Real Time) Kit from TAKARA. As the qRT-PCR, SYBR Green qRT-PCR Master Mix (2X) and ROX Solution rendered Mix (2X) from Thermo were used, respectively. By 2^-△△CtThe method calculates the relative expression quantity of mRNA and miRNA.

The qRT-PCR primer sequences 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';

LDHA：

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

and reverse direction 5'-AACACAACTGGACCAACTGGA-3'.

As shown in FIG. 2, when cells were transfected with 3 siRNA fragments si-r-LDHA _001 (s 1), si-r-LDHA _002(s2), si-r-LDHA _003(s3) targeting LDHA and control scramble siRNA (Ctr), it was found that all three fragments could inhibit LDHA expression to different extents. The sequences of 3 siRNA fragments are respectively as follows:

si-r-LDHA_001：5’-GCTTGTGCCATCAGTATCT-3’，

si-r-LDHA_002：5’-GGGAGAGATGATGGATCTT-3’，

si-r-LDHA_003：5’-TCCCATTTCCACCATGATT-3’。

the 3 siRNA fragments were synthesized by Ruibo Biotech, Inc., Guangzhou.

The qRT-PCR result shows that si-r-LDHA-001 in the three siRNA fragments can obviously inhibit the expression of CTGF, alpha-SMA, Fn, Col1 and Col3 in mRNA, namely LDHA can promote the expression of the mRNA of the alpha-SMA in heart fibroblasts.

Example 3

Cardiac fibroblast proliferation assay.

In this example, Cell proliferation experiments were performed using the Cell-light EdU Apollo 567 In vitro Kit from Ruibo Biotech, Inc., Guangzhou.

The method comprises the following specific steps:

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. 50 μ L of 4% paraformaldehyde in PBS was added to each well, and after incubation at room temperature for 30min, the fixative was removed.

Adding 100 μ L PBS fixing solution with concentration of 4% paraformaldehyde into each well, washing with decolorizing shaker for 5min, and removing the fixing solution.

Add 100. mu.L of 1 XApollo staining reaction solution into each well, decolorize and shake-bed incubate for 30min in dark at room temperature, and then discard the staining reaction solution.

Adding 100 mu L of penetrant (PBS of 0.3% Triton X-100) into each hole, decoloring and shaking the mixture for 2-3 times, washing the mixture for 5min each time, and discarding the penetrant.

The reaction solution (1: 100) of Hoechst3342 was diluted with deionized water and stored in the dark.

Adding 100 mu L of diluted Hoechst3342 reaction solution into each hole, incubating for 30min in a light-proof, room temperature and decolorizing shaker, and then discarding the staining reaction solution.

Adding 150 mu L of PBS into each hole, and washing for 1-3 times; then adding 100 mu L of PBS into each hole for storage for later use; after the staining was completed, a photograph was taken with a fluorescence microscope.

Immunofluorescence detection of a-SMA and PCNA: first, fibroblasts were fixed with 4% paraformaldehyde for 15 minutes; the wells were punched with 0.3% Triton X-100 for 3 minutes; wash 3 times with Phosphate Buffered Saline (PBS) for 5 minutes each. After blocking the membrane with 10% donkey serum, the membrane was kept at room temperature for 40 minutes. Primary antibody plating cells were incubated with primary culture a-SMA and PCNA antibody (1: 200, diluted) overnight at 4 ℃. PBS was washed 3 times for 5 minutes each. 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.

The results are shown in fig. 3, 500 nM nicotine stimulates heart fibroblasts, and EDU detection, CCK8 experiment and immunofluorescence cell nuclear proliferation antigen (PCNA) all verify that nicotine causes cell proliferation, but is obviously inhibited. Figure 3 demonstrates that nicotine can induce myocardial fibrosis through LDHA. The nicotine pretreatment can obviously promote the proliferation of cardiac fibroblasts, and the knocking-down of LDHA can inhibit the action of nicotine.

Example 4

Western Blotting experiment.

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

After 48h after cell transfection, the cells were washed 3 times with PBS, and 80. mu.L of RIPA lysate was added to each well of 6-well plate cells for cell lysis, and the cell lysate was transferred to a 1.5mL centrifuge tube. Centrifuging at 12000rpm 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 buffer and blocked with 5% skimmed milk powder for 2h at room temperature. The membrane was washed with 1 XPBST buffer, diluted with the desired antibody dilution and incubated overnight at 4 ℃. The primary antibody (LDHA primary antibody; alpha-SMA primary antibody; all of which are conventional commercial products) was recovered as a 1 XPBST buffer wash membrane. A secondary antibody (secondary antibody: horseradish peroxidase-labeled goat anti-rabbit IgG-HRP, purchased from China fir gold bridge) was incubated at room temperature for 2 h. 1 x PBST buffer washing membrane. 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 it can be seen from fig. 4 that nicotine pretreatment can significantly induce the expression of a-SMA and PCNA, suggesting that cardiac fibroblast activation and proliferation capacity is significantly increased. After the LDHA is knocked down, the activation and proliferation of the heart fibroblast induced by nicotine can be obviously improved.

In order to further detect whether the regulation effect of LDHA on the myocardial fibrosis by nicotine is realized by regulating autophagy, the autophagy condition after LDHA knock-down is further detected by Western blot. The results are shown in figure 5, which induces increased expression of the autophagy marker protein LC3-II and the autophagy-specific substrate P62 after nicotine stimulation, suggesting a blockade of autophagy flow. Knocking down LDHA can inhibit the blocking of nicotine to autophagy flow.

Further, FX11 (model HY-16214, available from MCE) which is an inhibitor of LDHA was added, and western blot was used to detect the expression of PCNA and alpha-SMA in fibroblasts, and to detect the autophagy blocking condition. The results are shown in FIGS. 6 and 7. As can be seen in figure 6, nicotine-induced increases in PCNA and α -SMA were significantly improved upon pretreatment with inhibitor FX11 added to LDHA, suggesting that FX11 is capable of improving nicotine-induced fibroblast proliferation and activation. As can be seen from fig. 7, after adding FX11 inhibitor of LDHA for pretreatment, it can improve the nicotine-induced autophagy block and reduce the intracellular aggregation of LC3 and p 62.

Where not specifically stated in the examples, conventional procedures in the art are employed, and materials not specifically stated are all commercially available reagents.

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 LDHA in cardiac fibroblasts

<130> 1

<160> 10

<170> PatentIn version 3.5

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gtgctggagc cactgtcgcc gatctcgcgc acgctactgc tgctgctcgc ccgtcgtccc 60

ccatcgtgca ctaagcggtc ccaaaagatt caaagtccaa gatggcagcc ctcaaggacc 120

agctgattgt gaatcttctt aaggaagaac aggtccccca gaacaagatt acagttgttg 180

gggttggtgc tgttggcatg gcttgtgcca tcagtatctt aatgaaggac ttggctgatg 240

agcttgccct tgttgatgtc atagaagata agctaaaggg agagatgatg gatcttcagc 300

atggcagcct tttccttaag acaccaaaaa ttgtctccag caaagattat agtgtgactg 360

caaactccaa gctggtcatt atcaccgcgg gggcccgtca gcaagaggga gagagccggc 420

tcaatttggt ccagcgaaac gtgaacatct tcaagttcat cattccaaat gttgtgaaat 480

acagtccaca gtgcaaactg ctcatcgtct caaacccagt ggatatcttg acctacgtgg 540

cttggaagat cagcggcttc cccaaaaaca gagttattgg aagtggttgc aatctggatt 600

cggctcggtt ccgttacctg atgggagaaa ggctgggagt tcatccactg agctgtcacg 660

ggtgggtcct gggagagcat ggcgactcca gtgtgcctgt gtggagtggt gtgaatgtcg 720

ccggcgtctc cctgaagtct ctgaacccgc agctgggcac ggatgcagac aaggagcagt 780

ggaaggatgt gcacaagcag gtggttgaca gtgcatacga agtgatcaag ctgaaaggtt 840

acacatcctg ggccattggc ctctccgtgg cagacttggc cgagagcata atgaagaacc 900

ttaggcgggt gcatcccatt tccaccatga ttaagggtct ctatggaatc aaggaggatg 960

tcttcctcag cgtcccatgt atcctgggac aaaatggaat ctcagatgtt gtgaaggtga 1020

cactgactcc tgacgaggag gcccgcctga agaagagtgc agataccctc tggggaatcc 1080

agaaggagct gcagttctaa agtcttccca gtgtcctagc acttcactgt ccaggctgca 1140

gcagggtttc tatggagacc acgcacttct catctgagct gtggttggtc cagttgtgtt 1200

gaggtggtct gggggaaatc tcagttccac agctctaccc tgctaagtgg tacttgtgta 1260

gtggtaacct ggttagtgtg acaatcccac tgtctccaag acacactgcc aactgcatgc 1320

aggctttgat taccctgtga gcctgctgca ttgctgtgct acgcaccctc accaaacatg 1380

cctaggccat gagttcccag ttagttataa gctggctcca gtgtgtaagt ccatcgtgca 1440

tatcttgtgc ataaatgttc tacaggatat tttctgtatt atatgtgtct gtagtgtaca 1500

ttgcaatatt atgtgaaatg taagatctgc atatggatga tggaaccaac cactcaagtg 1560

tcatgccaag gaaaacacca aataaacctt gaacagtg 1598

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gcttgtgcca tcagtatct 19

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gggagagatg atggatctt 19

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tcccatttcc accatgatt 19

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ggatcccctc ccagagaagt gggtgtggaa gggtaaccag 40

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atctggattc ggctcggttc aacacaactg gaccaactgg a 41

Claims (2)

1. The application of siRNA for inhibiting LDHA gene in preparing medicine for regulating nicotine-induced cardiac fibroblast proliferation is disclosed, wherein the sequence of LDHA gene is SEQ ID No.1 in a nucleotide sequence table; the siRNA sequence is as follows:

si-r-LDHA_001：5’-GCTTGTGCCATCAGTATCT-3’；

si-r-LDHA_002：5’-GGGAGAGATGATGGATCTT-3’；

si-r-LDHA_003：5’-TCCCATTTCCACCATGATT-3’。

2. the use of claim 1, wherein qRT-PCR is used to detect the expression level of LDHA gene, and the primer sequences used in qRT-PCR detection are 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';

LDHA：

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

and reverse direction 5'-AACACAACTGGACCAACTGGA-3'.

Images