Application of long-chain non-coding RNA NAV2-AS2 in promotion of Apelin protein expression

Abstract

The invention relates to the technical field of biomedicine, and provides an application of a long-chain non-coding RNANAV2-AS2 in promotion of Apelin protein expression. The invention further discloses application of the long-chain non-coding RNANAV2-AS2 in preparation of a medicine for promoting expression of Apelin mRNA (messenger ribonucleic acid) or Apelin protein. The expression of Apelin can be promoted by up-regulating the NAV2-AS2 in the human heart fibroblast, and the expression of Apelin can be inhibited by inhibiting the NAV2-AS2; the NAV2-AS2 has an inhibition effect on the expression of the miR-31, the miR-31 has an inhibition effect on the expression of the Apelin, and the effects are confirmed by luciferase reporter gene experiments; after miR-31 is over-expressed in human heart fibroblasts, the miR-31 is found to counteract the promotion effect of NAV2-AS2 on Apelin expression.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of long-chain non-coding RNA NAV2-AS2 in promoting the expression of Apelin protein. Background Art

[0002] Long non-coding RNAs (lncRNAs) are a type of non-coding RNA with a length greater than 200 nucleotides and no ability to encode proteins. Initially, they were considered "junk" in the transcription process. However, recent studies have shown that lncRNAs are involved in many important regulatory processes, including genomic imprinting, chromatin modification, transcriptional activation, transcriptional interference, nuclear transport, and other important regulatory processes, and are involved in a variety of disease processes, including cardiovascular disease, cancer, diabetes, and inflammatory diseases. The regulatory role of lncRNAs has increasingly attracted attention. Although research on lncRNAs has progressed rapidly in recent years, only a few lncRNAs have been fully understood in their functions and mechanisms in regulating disease and gene expression. Most lncRNAs have not yet been studied or even discovered.

[0003] MicroRNAs (miRNAs) are a type of non-coding RNA with about 20 nucleotides, relatively conservative in evolution, and no protein coding ability, but with strong protein regulation ability at the post-transcriptional level. The main way for miRNAs to regulate downstream target proteins is to bind to the 3' untranslated region (3'UTR) of the target protein mRNA according to the principle of base pairing, resulting in a decrease in the translation efficiency of the target protein, thereby regulating gene expression at the post-transcriptional level. At present, a large number of studies have confirmed that miRNAs can participate in the occurrence and development of various diseases such as various tumors, cardiovascular and cerebrovascular diseases, blood system diseases, mental system diseases and immune system diseases.

[0004] Apelin receptor (APJ) was initially identified as an orphan G protein-coupled receptor. With the discovery of its endogenous ligand Apelin in bovine stomach extracts in 1998, APJ was officially de-orphaned. The intracellular signals and organ responses mediated by Apelin and APJ are collectively referred to as the Apelin / APJ system. Studies have found that Apelin, also known as APLN, is a new type of adipose cytokine secreted by white fat. It is expressed in many tissues of the body, including the cardiovascular system, kidneys, lungs, central nervous system, skeletal muscle, breast, adrenal glands, ovaries, stomach, hepatocytes, placenta and breast milk, and is involved in regulating the pathophysiological processes of a variety of diseases.

[0005] However, we still know very little about the expression and regulation mechanism of endogenous Apelin in the heart. Based on this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide an application of a long non-coding RNA NAV2-AS2 in promoting the expression of Apelin protein, so as to clarify the regulatory relationship between NAV2-AS2 and Apelin.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides an application of a long-chain non-coding RNA NAV2-AS2 in preparing a medicine for promoting the expression of Apelin mRNA or Apelin protein.

[0009] Preferably, the nucleotide sequence of the long non-coding RNA NAV2-AS2 is as shown in SEQ ID No: 1.

[0010] The present invention analyzes the correlation between NAV2-AS2 and Apelin expression, and the results show that NAV2-AS2 is positively correlated with Apelin. In order to further verify the regulatory relationship of NAV2-AS2 to Apelin through laboratory methods, the expression of Apelin under the condition of NAV2-AS2 overexpression was verified by qRT-PCR, immunofluorescence and Western bolt experimental techniques.

[0011] The present invention uses the miRcode website to first predict the miRNAs that bind to NAV2-AS2 and Apelin respectively. Then, find out the miRNAs that can bind to both at the same time, and then verify whether these miRNAs can bind to both NAV2-AS2 animal and human sequences. Finally, after analysis by the NCBI website, it was found that only miR-31 met the experimental requirements. To verify this prediction result, it was found that the expression of miR-31 was significantly downregulated after overexpressing NAV2-AS2, from which it can be inferred that NAV2-AS2 has a certain inhibitory effect on miR-31 expression in human cardiac fibroblasts, and the binding relationship between the two was verified by a luciferase reporter gene experiment. At the same time, after overexpressing miR-31 in human cardiac fibroblasts, it was found that miR-31 offset the promoting effect of NAV2-AS2 on Apelin expression. It can be inferred that miR-31 has a certain inhibitory effect on Apelin expression in human cardiac fibroblasts, and the binding relationship between the two was verified by a luciferase reporter gene experiment. NAV2-AS2 and miR-31 were transfected simultaneously in human cardiac fibroblasts. The results showed that in the presence of TGF-β, miR-31 reversed the promoting effect of NAV2-AS2 on Apelin expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0013] Figure 1 is the correlation between NAV2-AS2 and Apelin expression;

[0014] Figure 2 The expression results of Apelin in human cardiac fibroblasts transfected with NAV2-AS248h in Example 1 (Note: *p<0.05, **p<0.01, ***p<0.001VS pcDNA-NC; #p<0.05, ##p<0.01VS pcDNA-NC+TGF-β);

[0015] Figure 3 This is a Venn diagram of differential miRNAs regulating Apelin and NAV2-AS2 in Example 2;

[0016] Figure 4 The luciferase reporter structures of NAV2-AS2 and Apelin in Example 2;

[0017] Figure 5 This is the experimental verification of the targeted binding of NAV2-AS2 to miR-31 in Example 2; (red letters indicate matching bases, n=3. Note: ****p<0.0001);

[0018] Figure 6 The level of miR-31 in human cardiac fibroblasts after transfection with NAV2-AS for 248h detected by qRT-PCR in Example 2; (n=5. Note: *p<0.05, ****p<0.0001VS pcDNA-NC; ####p<0.0001VS pcDNA-NC+TGF-β);

[0019] Figure 7 This is the experimental verification of Apelin as a target gene of miR-31 in Example 2; (Note: red letters indicate matching bases, n=3. ****p<0.0001);

[0020] Figure 8 The expression results of Apelin in human cardiac fibroblasts 48 hours after transfection with NAV2-AS2 and miR-31 in Example 2; (n=3. Scale bar: 50μm. Note: *p<0.05, **p<0.01, ****p<0.0001VS pcDNA-NC+TGF-β; ##p<0.01, ####p<0.0001VS pcDNA-NAV2-AS2+TGF-β; &p<0.05, &&p<0.01, &&&&p<0.0001VSpcDNA-NAV2-AS2+TGF-β+miR-31). DETAILED DESCRIPTION

[0021] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0022] Bioinformatics predicted the downstream genes of NAV2-AS2, and analysis showed that NAV2-AS2 was positively correlated with Apelin ( Figure 1 ), suggesting that Apelin may be a downstream target regulated by NAV2-AS2.

[0023] Example 1

[0024] In order to further clarify the regulatory relationship between NAV2-AS2 and Apelin, NAV2-AS2 overexpression plasmid was transfected into human cardiac fibroblasts to upregulate the expression level of NAV2-AS2 in human cardiac fibroblasts.

[0025] NAV2-AS2 plasmid (pcDNA-NAV2-AS2, designed and synthesized by Shanghai Gene Gene Co., Ltd.) was extracted according to the requirements of Tiangen Plasmid Extraction Kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). According to the manufacturer's instructions, the overexpression plasmid pcDNA-NAV2-AS2 was transfected into human cardiac fibroblasts using lipo8000 (purchased from Bio-Technology Co., Ltd.), and the specific steps are as follows:

[0026] Transfection overexpression plasmid pcDNA-NAV2-AS2

[0027] 24h before transfection, human cardiac fibroblasts were inoculated into six-well plates (purchased from Corning, USA) for culture, so that the cell density reached 70% on the second day of culture, and the old culture medium in the six-well plate was replaced with 2ml of fresh complete culture medium (containing serum and antibiotics). A sterilized centrifuge tube was used to prepare the DNA solution. 2.5μg of DNA plasmid was added to 125μl of DMEM culture medium without antibiotics and serum and gently mixed; 125μl of Lipo8000 transfection reagent-DNA mixture was evenly added to each well without incubation at room temperature, and culture was continued for 48h. After 48h, cells from each group were collected, and the cells were treated with TGF-β 6h before the cells were collected. The expression levels of Apelin mRNA and protein were verified by qRT-PCR, immunofluorescence and Westernbolt experiments.

[0028] The specific steps of TGF-β treatment are:

[0029] Before use, 10 μg of TGF-β recombinant protein (lyophilized powder) was placed in a refrigerated high-speed centrifuge and centrifuged at 12XG for 2 minutes. After centrifugation, 200 μl of sterile 0.1% BSA solution was taken into the TGF-β recombinant protein EP tube, gently mixed with a pipette, and placed in a mini centrifuge for centrifugation for 5 seconds. The TGF-β recombinant protein was then placed at 25°C for 5 minutes, configured to 50 μg / ml and placed in a -20°C refrigerator for storage. 6 hours before collecting the cells, the 50 μg / ml TGF-β recombinant protein was diluted to 5 ng / ml with distilled water, and the diluted TGF-β recombinant protein was added to human cardiac fibroblasts and cultured for 6 hours.

[0030] qRT-PCR experiments:

[0031] (1) Extract cellular RNA and determine its concentration.

[0032] Before the experiment, all relevant experimental consumables and instruments must be sterilized by high pressure and exposed to ultraviolet light for 30 minutes before the experiment begins. All operations are carried out in an ultra-clean workbench, the centrifuge is pre-cooled to 4°C in advance, and all steps are performed on ice.

[0033] After the old culture medium in the six-well plate was sucked out, it was washed twice with pre-cooled PBS, and 1 ml Trizol was added to each well. The cells were scraped vigorously with a cell scraper to fully lyse the cells. After standing for 5 minutes, the cells were transferred to a 1.5 ml EP tube; 200 μl (1 / 5 of Trizol) chloroform was added, and the cells were shaken vigorously for 5 minutes, and then stood for 5 minutes; 12XG, 4℃, centrifuged for 15 minutes. After centrifugation, the 1.5 ml EP tube was taken out, and the sample was divided into three layers (colorless supernatant aqueous phase, middle white layer, pink lower organic layer). RNA was distributed in the colorless supernatant aqueous phase. The colorless supernatant was carefully pipetted into another new 1.5 ml EP tube. 500 μl (1 / 2 of Trizol) isopropanol was added, and the cells were shaken up and down 7-8 times, and incubated on ice for 10 minutes; 12XG, 4℃, centrifuged for 10 minutes, and the supernatant was discarded. The EP tube was inverted on the filter paper to control the isopropanol. Add 1ml 75% ethanol (prepared with sterile water, the volume ratio of sterile water to ethanol is 1:3), and blow evenly with a pipette. Vortex and gently invert. Centrifuge at 7.5XG, 4℃ for 5min, pour off the supernatant, invert the EP tube on the filter paper, and dry the RNA at room temperature for 7min. Add 10μl of DEPC water to resuspend the RNA precipitate. After the white precipitate is completely dissolved, transfer it to a 0.2ml EP tube and place it in a -80℃ refrigerator for standby use. Use NanoDrop One ultra-micro spectrophotometer to measure the concentration of extracted RNA. First, add 2μl of distilled water to clean the probe up and down for 2min, then add 2μl of DEPC water to zero, then add 2μl of RNA for concentration measurement, and record the concentration and absorbance A260 / A280 ratio. A ratio of A260 / A280 between 1.8 and 2.0 indicates that the RNA purity is high.

[0034] (2) PCR amplification

[0035] Removal of genomic DNA: Based on PrimeScript TM RT reagent Kit with gDNA Eraser (PerfectRealTime) kit (purchased from Takara, Japan) instructions, prepare the reaction mixture (as shown in Table 1), and operate on ice throughout the process. In order to ensure the accuracy of the reaction solution preparation, the Master Mix should be prepared first when performing each reaction, and 9μl of Master Mix should be added to each reaction tube. Finally, the RNA sample should be added, mixed and placed in the PCR instrument, and the following program should be set: 42℃, 2min; 4℃, ∞.

[0036] Table 1 Reagents and dosages used in the reaction mixture

[0037] Reagents Usage 5×gDNAEraserBuffer 2.0μl gDNAEraser 1.0μl Total RNA 500ng <![CDATA[RNaseFreedH2O]]> up to 10 μl

[0038] (3) Reverse transcription

[0039] Obtain cDNA solution by reverse transcription for Real Time PCR. Prepare the reaction mixture (as shown in Table 2) to ensure the accuracy of the reaction solution. When performing each reaction, prepare MasterMix first, add 10μl of Master Mix and 10μl of reaction solution to remove genomic DNA to each reaction tube, mix gently and immediately perform reverse transcription reaction, put into PCR instrument after mixing, and set the following program: 37℃, 15min; 85℃, 5s; 4℃, ∞.

[0040] Table 2 Reagents and dosages used in the reaction mixture

[0041] Reagents Usage Reaction solution for removing genomic DNA 10.0μl PrimeScriptRTEnzymeMixI 1.0μl RTPrimerMix 1.0μl 5×PrimeScriptBuffer2(forRealTime) 4.0μl <![CDATA[RNaseFreedH2O]]> 4.0μl Total 20μl

[0042] (4) Real Time PCR

[0043] Prepare the reaction solution according to Table 3, add the prepared reaction solution to the PCR reaction plate, and test it on the machine. The standard PCR amplification procedure is shown in Table 4, and the real-time PCR primer sequence design is shown in Table 6. According to the threshold cycle (threshold, cycle, Ct) values ​​of each gene obtained, GAPDH was used as the internal reference gene, and 2 -△△Ct The expression level of Apelin in different samples was calculated by this method.

[0044] Table 3 Reagents and dosages used in the reaction mixture

[0045] Reagents Usage TBGreenPremixExTaqII(TliRNaseHPlus)(2×) 5μl PCRForwardPrimer (10 μM) 0.5μl PCR Reverse Primer (10 μM) 0.5μl RT reaction solution (cDNA solution) 1μl Sterilized water 3μl Total 10μl

[0046] Table 4 PCR amplification standard procedure

[0047]

[0048] Western blot experiments

[0049] Prepare the protease inhibitor and RIPA lysis buffer at a ratio of 1:50, that is, add 20 μl of protease inhibitor to 1 ml of RIPA lysis buffer, mix and shake before use.

[0050] After the cell culture fluid in the human cardiac fibroblast culture bottle is cleaned, it is rinsed with PBS three times, 2 ml each time, and dried with filter paper. 300 μl of RIPA lysis solution (containing protease inhibitors) is added to each cell culture bottle, and it is allowed to stand for 5 seconds. After blowing with a pipette, the lysis solution is fully contacted with the human cardiac fibroblasts for 2 minutes, and the adherent cells are scraped off with a cell scraper and sucked into the EP tube. After lysis on ice for 10 minutes, the vortex is shaken for 30 seconds, once for 3 minutes, and shaken three times. The lysed cells are placed in a 1.5 ml EP tube, centrifuged at 4°C, 13XG, and the supernatant is sucked into the EP tube, and the protein concentration is determined (BCA method determination, wherein the BCA protein concentration determination kit is purchased from Biyuntian Biotechnology Co., Ltd.): According to the instructions of the BCA protein concentration determination kit, a 25 mg / ml protein standard solution is prepared, and after preparation, it is stored at -20°C for a long time. The protein standard solution with a concentration of 25 mg / ml is diluted to a protein standard working solution with a concentration of 0.5 mg / ml using ddH2O or PBS. According to the number of experimental wells, prepare BCA working solution in the ratio of A solution: B solution = 50:1. Prepare a 96-well plate, refer to Table 5 for sample addition, and use ddH2O or PBS to make up for less than 20μl. Each protein concentration determination is set to 3 replicate wells, add 1μl, 2μl and 4μl of the protein solution to be tested, add ddH2O or PBS to make up to 20μl, and then add 200μl of A solution + B solution mixture (50:1) to each sample well. Place the 96-well plate on a microplate fast oscillator for 30s, and then incubate it in a 37℃ incubator for 30min. Use an enzyme marker to measure the absorbance at 562nm and draw a standard curve. Calculate the protein concentration of the sample based on the standard curve and the volume of the sample used.

[0051] Table 5 96-well plate loading reagents and concentrations

[0052] serial number 1 2 3 4 5 6 7 8 Standard BSA / μl 0 1 2 4 8 12 16 20 <![CDATA[ddH2O / PBS / μl]]> 20 19 18 16 12 8 4 0 Concentration (mg / ml) 0 0.025 0.05 0.1 0.2 0.3 0.4 0.5

[0053] According to the protein expression level, the concentration of the primary antibody (Table 7) was determined, and the antibody was diluted to an appropriate concentration using 1×PBST (configuration: 95.7 g of ready-to-use PBS powder was added to ddH2O to a total volume of 1 L, and stirred evenly with a stirring rod to fully dissolve it to prepare a 10×PBS solution. 100 ml of 10×PBS solution was added to ddH2O to 1 L, and then 500 μl of Tween-20 was added to mix well to prepare a 1×PBST solution). The PVDF membrane was completely placed in the antibody, incubated at 4°C overnight, the primary antibody was recovered for labeling and stored at 4°C, and the PVDF membrane was washed 3 times with 1×PBST, each time for 10 min.

[0054] The secondary antibody (Table 8) was diluted to an appropriate concentration using 1×PBST according to the instructions of the SDS-PAGE gel rapid preparation kit (purchased from Bio-Technology Co., Ltd.), incubated at 25°C for 2h, and labeled after recovering the secondary antibody and storing at 4°C. The PVDF membrane was washed 3 times with 1×PBST for 10min each time.

[0055] The ECL kit (purchased from Bio-Tech Biotech) was used to mix solution A and solution B in a ratio of 1:1 to prepare the developer in the dark. The developer was placed on a plastic wrap, and the film was placed on it so that the developer and the strip were in full contact. The light was turned off and the strip was incubated for 2 minutes. Then, after the strip was developed and fixed, the gray value of the strip was measured using Image J software.

[0056] Immunofluorescence

[0057] Human cardiac fibroblasts were plated in 24-well plates with glass slides and cultured. When the growth density reached 75%, the cells were treated: fixed with 4% paraformaldehyde for 15 minutes, and the cell surface was washed with PBS. 0.1% Triton X-100 was penetrated for 30 minutes, the cell surface was washed with PBS, and goat serum was blocked at room temperature for 1 hour. Apelin primary antibody (1:100) was incubated overnight; the next day, AlexaFluor488-labeled goat anti-rabbit IgG (H+L) fluorescent secondary antibody was added and incubated at room temperature for 1 hour, 1×DAPI stain (purchased from Beyotime Biotechnology) was incubated at room temperature for 10 minutes to color the cell nucleus, and the images were collected by laser confocal microscopy.

[0058] like Figure 2 As shown, qRT-PCR ( Figure 2 A: qRT-PCR detection of Apelin mRNA expression in human cardiac fibroblasts transfected with NAV2-AS for 248 hours, n=4), Western blot ( Figure 2 B: Western blot detection of Apelin protein expression in human cardiac fibroblasts transfected with NAV2-AS248h. n=4) and immunofluorescence ( Figure 2 C: Immunofluorescence detection of Apelin protein expression in human cardiac fibroblasts transfected with NAV2-AS2 for 48 hours. n=3. Scale bar: 50μm) All confirmed that Apelin expression was upregulated after NAV2-AS2 overexpression for 48 hours. At the same time, the expression of Apelin was also inhibited after NAV2-AS2 was inhibited by TGF-β. The above data show that upregulating NAV2-AS2 can promote the expression of Apelin, and inhibiting NAV2-AS2 can inhibit the expression of Apelin, suggesting that Apelin is a potential downstream target regulated by NAV2-AS2.

[0059] Example 2

[0060] The prediction analysis of the miRcode website showed that there are 37 miRNAs that can bind to Apelin, 16 miRNAs that can bind to NAV2-AS2, and 5 miRNAs that can bind to both Apelin and NAV2-AS2 (e.g. Figure 3 However, after comparing the nucleotide sequences on the NCBI website, it was found that only miR-31 could bind to both human and mouse NAV2-AS2 sequences, so miR-31 was selected as the research target.

[0061] Construction of dual luciferase reporter gene vector.

[0062] The binding sites of NAV2-AS2, Apelin and miR-31 were predicted by Targetscan website. Wild-type and mutant sequences of NAV2-AS2 and Apelin were constructed based on the predicted binding sites and cloned into the dual-luciferase vector psi-CHECK2 (purchased from Promega Biotechnology Co., Ltd., USA). The vector map is shown in Figure 4 (The sequence shown in the figure is located from position 459 to position 483 of sequence 1), Figure 4 A is a luciferase reporter structure containing NAV2-AS2 wild type (wt) or NAV2-AS2 mutant (mut): the bases marked in red are the only difference between NAV2-AS2 wild type (wt) and NAV2-AS2 mutant (mut). The wild type (wt) refers to the sequence of human NAV2-AS2, and the mutant (mut) refers to the sequence of NAV2-AS2 with the red mutation site. The same is true for the following Apelin, Figure 4 B is a luciferase reporter structure containing Apelin 3'UTR or Apelin mutant 3'UTR. The design and construction of the vector was completed by Guangzhou Aizhe Biotechnology Co., Ltd.

[0063] The Targetscan website predicts that NAV2-AS2 has binding sites with miR-31. Figure 5 As shown in A, NAV2-AS2-wt or NAV2-AS2-mut dual luciferase reporter vectors and miR-31mimics, miR-31NC and miR-31inhibitor, miR-31inhibitorNC (purchased from Guangdong Ruibo Biotechnology Co., Ltd.) were co-transfected into HEK-293T cells, and dual luciferase reporter gene experiments were performed to verify whether NAV2-AS2 directly targets miR-31. The results showed that compared with NAV2-AS2-mut, the luciferase activity of the NAV2-AS2-wt group was greatly inhibited, and the difference was statistically significant (such as Figure 5 In addition, qRT-PCR (same steps as in Example 1, primers refer to Table 6) confirmed again that miR-31 levels were upregulated in human cardiac fibroblasts after TGF-β treatment, and miR-31 levels were reduced after overexpression of NAV2-AS2 (as shown in Figure 6 The above experimental data show that NAV2-AS2 has a targeted binding relationship with miR-31.

[0064] The Targetscan website predicts that Apelin has binding sites with miR-31. Figure 7 As shown in A. When the Apelin wild-type dual-luciferase reporter vector was co-transfected with miR-31mimics in HEK-293T cells, the luciferase activity was greatly inhibited, but miR-31mimics had no effect on the luciferase activity of the 3'-UTR of the Apelin mutant (as shown in Figure 7 Therefore, the dual luciferase reporter gene experiment proved that Apelin is a direct target gene of miR-31.

[0065] In order to further verify the regulatory relationship among NAV2-AS2, miR-31 and Apelin, qRT-PCR, western blot and immunofluorescence experiments were used to detect the expression levels of Apelin mRNA and protein in the pcDNA-NC + TGF-β group, pcDNA-NAV2-AS2 + TGF-β group, pcDNA-NAV2-AS2 + TGF-β + miR-31 group and pcDNA-NAV2-AS2 + TGF-β + miR-NC group, respectively (the detection method was the same as in Example 1).

[0066] The results showed that compared with pcDNA-NC+TGF-β, the expression levels of Apelin mRNA and protein in the pcDNA-NAV2-AS2+TGF-β group were significantly increased; and the expression levels of Apelin mRNA and protein in the pcDNA-NAV2-AS2+TGF-β+miR-31 group were significantly decreased ( Figure 8 A-8C). The above results indicate that up-regulated NAV2-AS2 can promote the expression of Apelin mRNA and protein in human cardiac fibroblasts, while overexpression of miR-31 can reverse the effect of up-regulated NAV2-AS2 on Apelin expression.

[0067] The primers used in the RT PCR process in the above examples are shown in Table 6 below.

[0068] Table 6 Real-time PCR primer sequence design

[0069]

[0070] The primary antibodies and secondary antibodies used in the Western blot experiments in the above examples are shown in Tables 7 and 8 below.

[0071] Table 7 Related information of primary antibody

[0072] Primary Antibody Species Manufacturer Product Number Dilution ratio Apelin Rabbit Abcam (Cambridge, USA) ab230536 1:1000 GAPDH Mouse BioSky Biotech AG019 1:1000

[0073] Table 8 Related information of secondary antibodies

[0074] Secondary Antibodies Manufacturer Product Number Dilution ratio HRP-labeled Goat Anti-Rabbit BioSky Biotech A0208 1:1000 HRP-labeledGoatAnti-Mouse BioSky Biotech A0216 1:1000

[0075] It can be seen from the above embodiments that the present invention clarifies the downstream regulatory targets of NAV2-AS2. In the present invention, the known genomic data are first screened and analyzed in the heart failure group by bioinformatics. We speculate that Apelin may be a potential downstream target of NAV2-AS2, so the correlation between NAV2-AS2 and Apelin expression was analyzed. The results showed that NAV2-AS2 was positively correlated with Apelin. In order to further verify the regulatory relationship of NAV2-AS2 to Apelin through laboratory methods, we verified the expression of Apelin when NAV2-AS2 was overexpressed by qRT-PCR, immunofluorescence and Westernbolt experimental techniques.

[0076] We used the miRcode website to predict the miRNAs that bind to NAV2-AS2 and Apelin respectively. Then, we looked for miRNAs that can bind to both at the same time. Finally, after analysis on the NCBI website, we found that only miR-31 met our experimental requirements. To verify this prediction, we found that after overexpressing NAV2-AS2, the expression of miR-31 was significantly downregulated, from which we can infer that NAV2-AS2 has a certain inhibitory effect on miR-31 expression in human cardiac fibroblasts, and the binding relationship between the two was verified by luciferase reporter gene experiments. At the same time, after overexpressing miR-31 in human cardiac fibroblasts, it was found that miR-31 offset the promoting effect of NAV2-AS2 on Apelin expression. From this, we can infer that miR-31 has a certain inhibitory effect on Apelin expression in human cardiac fibroblasts, and the binding relationship between the two was verified by luciferase reporter gene experiments.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of long non-coding RNA NAV2-AS2 in the preparation of drugs for promoting the expression of Apelin mRNA or Apelin protein.

2. The use according to claim 1, characterized in that The nucleotide sequence of the long non-coding RNA NAV2-AS2 is shown in SEQ ID No:

1.

3. The use according to claim 1 or 2, characterized in that: The long non-coding RNA NAV2-AS2 acts as a competitive inhibitor of miR-31 to promote the expression of Apelin protein.

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