Application of long non-coding RNA NEAT1 in regulating myofiber type conversion

Abstract

The application discloses application of long-chain non-coding RNA NEAT1 in regulation of muscle fiber type conversion, and is a new use of long-chain non-coding RNA NEAT1. The application studies the influence of NEAT1 on gene and protein expression related to muscle fiber type in muscle cells by interfering with NEAT1 expression or overexpressing NEAT1 in the muscle cells, finds that the long-chain non-coding RNA NEAT1 has important regulation on muscle fiber conversion of the muscle cells, the long-chain non-coding RNA NEAT1 can promote expression of slow muscle genes and proteins and inhibit expression of fast muscle genes and proteins. The expression of the fast and slow muscle genes and proteins in the muscle cells directly influences the proportion of muscle fiber types, so the proportion of muscle fiber types can be further influenced by regulating expression of the long-chain non-coding RNA NEAT1 in the muscle cells, and conversion of the muscle fiber types is realized. Thus, the conversion of the muscle fiber types can be regulated by inhibiting or overexpressing the long-chain non-coding RNA NEAT1, and meat quality of animals is further regulated, thereby providing a basis and new ideas for breeding lean or fattening animals.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of a long non-coding RNA NEAT1 in regulating myofiber type conversion. Background Technology

[0002] Skeletal muscle plays a crucial role in regulating metabolism and homeostasis in animals. The main component of skeletal muscle is muscle fibers. The type of muscle fibers is not fixed after birth; different types can transform under the influence of physiological or external factors. The relative abundance of muscle fibers in muscle tissue is related to animal performance and final meat quality development. Therefore, the composition of muscle fiber types is closely related to muscle yield and meat quality. The most abundant protein in muscle fibers is myosin. Currently, the most commonly used and accurate method for classifying muscle fiber types is based on the polymorphic expression of the myosin heavy chain gene in muscle fibers. Adult mammalian skeletal muscle expresses only four MyHC isoforms: type I, type IIA, type IIB, and type IIX. These can be distinguished by ATP histochemical staining to differentiate muscle quality. In fact, the type of muscle fibers depends on the dominant type, generally decreasing as the proportion of type II muscle fibers increases.

[0003] Long non-coding RNAs (lncRNAs) are a class of RNA molecules with little or no protein-coding ability and a length greater than 200 nt. They are characterized by low expression abundance, a small number of exons, and tissue-specific expression. lncRNAs have diverse functions, playing important roles in various biological processes such as cell proliferation and differentiation, gene imprinting, dose compensation, stem cell pluripotency, embryonic development, immune responses, and tumorigenesis. In recent years, several lncRNAs with important regulatory roles in muscle development have been discovered, but few lncRNAs regulating myofiber type conversion have been reported.

[0004] NEAT1 can maintain the stability of specific nucleosomes and nucleus spots in mammals and plays an important role in many gene regulation processes, but its effect on myofiber type has not been reported. Summary of the Invention

[0005] The purpose of this invention is to provide an application of long non-coding RNA NEAT1 in regulating myofiber type conversion, which can further provide a foundation and new ideas for breeding lean or fattening animals.

[0006] According to a first aspect of the present invention, the application of long non-coding RNA NEAT1 in regulating myofiber type conversion is provided. This reveals a novel use of long non-coding RNA NEAT1 in regulating myofiber type conversion, which can provide a foundation and ideas for its further application in improving animal meat quality.

[0007] In some embodiments, the application is an application that promotes the conversion of slow-twitch muscle fibers into fast-twitch muscle fibers.

[0008] In some embodiments, the application is to increase the expression of fast-twitch fiber-related genes and decrease the expression of slow-twitch fiber-related genes in muscles; the fast-twitch fiber-related genes are one or more of MyHC-IIA, MyHC-IIB, and MyHC-IIX; the slow-twitch fiber-related gene is MyHC-I.

[0009] In some implementations, the application is achieved by suppressing NEAT1 expression, which can be achieved by interfering with NEAT1 expression.

[0010] In some embodiments, the application is an application that promotes the conversion of fast-twitch muscle fibers into slow-twitch muscle fibers.

[0011] In some embodiments, the application is to increase the expression of slow-twitch muscle fiber-related genes and decrease the expression of fast-twitch muscle fiber-related genes in muscles; the fast-twitch muscle fiber-related genes are one or more of MyHC-IIA, MyHC-IIB, and MyHC-IIX; and the slow-twitch muscle fiber-related gene is MyHC-I.

[0012] In some implementations, the application is achieved by overexpressing NEAT1.

[0013] According to a second aspect of the present invention, a kit / nucleic acid molecule / recombinant protein / recombinant vector / product containing a reagent that inhibits the expression of the long non-coding RNA NEAT1 is provided for promoting the conversion of slow-twitch muscle fibers to fast-twitch muscle fibers in animal muscle types. This increases the expression ratio of fast-twitch muscle-related genes, thereby increasing the proportion of fast-twitch muscle fibers and further improving the meat quality of the animal.

[0014] According to a third aspect of the present invention, a kit / nucleic acid molecule / recombinant protein / recombinant vector / product containing a reagent that enables overexpression of the long non-coding RNA NEAT1 is provided for promoting the conversion of fast-twitch muscle fibers to slow-twitch muscle fibers in animal muscle types. This increases the expression ratio of slow-twitch muscle-related genes, thereby increasing the proportion of slow-twitch muscle fibers and further improving the meat quality of the animal.

[0015] In some implementations, the application achieves myofiber type conversion by regulating the glycolysis process in animal muscle tissue.

[0016] According to a fourth aspect of the present invention, there is an application of the long non-coding RNA NEAT1 in regulating glycolysis.

[0017] The beneficial effects of this application are:

[0018] This application investigates the effects of NEAT1 on the expression of genes and proteins related to myofiber type in muscle cells by interfering with or overexpressing NEAT1. The study reveals that lncRNA NEAT1 plays a crucial regulatory role in myofiber transformation: it promotes the expression of slow-twitch muscle genes and proteins while inhibiting the expression of fast-twitch muscle genes and proteins. Since the expression of fast and slow-twitch muscle genes and proteins directly affects the ratio of myofiber types, regulating the expression of lncRNA NEAT1 in muscle cells can further influence the ratio of myofiber types, thereby achieving myofiber type transformation. Therefore, by inhibiting or overexpressing lncRNA NEAT1, myofiber type transformation can be regulated, further controlling animal meat quality and providing a foundation and new ideas for breeding lean or fattening animals. Attached Figure Description

[0019] Figure 1 The figure shows the results of qPCR validation of the expression levels of myofiber type-related genes in porcine skeletal muscle satellite cells after NEAT1 gene overexpression: pcDNA3.1 represents the blank control plasmid, pcDNA3.1-pNEAT1 represents the porcine NEAT1 gene overexpression vector, MyHC-Ⅰ / slow represents the slow muscle gene expression results, and MyHC-ⅡA, MyHC-ⅡB, and MyHC-ⅡX represent the fast muscle gene expression results. ** and *** indicate highly significant differences.

[0020] Figure 2 The figure shows the protein expression results of porcine skeletal muscle satellite cell myofiber type-related genes after NEAT1 gene overexpression, validated by Western blot technology: pcDNA3.1 represents the blank control plasmid, pcDNA3.1-pNEAT1 represents the porcine NEAT1 gene overexpression vector, MyHC-Ⅰ / slow represents the slow muscle gene protein expression results, and MyHC-ⅡA, MyHC-ⅡB, and MyHC-ⅡX represent the fast muscle gene protein expression results. ** indicates extremely significant difference, and * indicates significant difference.

[0021] Figure 3 Figure showing the protein expression results of myofiber type-related genes in mouse C2C12 myoblasts after NEAT1 gene overexpression, validated by Western blot: pcDNA3.1 represents the blank control plasmid, pcDNA3.1-NEAT1 represents the mouse NEAT1 gene overexpression vector, MyHC-Ⅰ / slow represents the slow muscle gene protein expression results, and MyHC-ⅡA, MyHC-ⅡB, and MyHC-ⅡX represent the fast muscle gene protein expression results. ** indicates extremely significant difference, and * indicates significant difference.

[0022] Figure 4Figure showing the results of qPCR detection of the expression levels of myofiber type-related genes in porcine skeletal muscle satellite cells after siRNA interfered with lncRNA NEAT1: where si-NC represents blank control siRNA, si-pNEAT1 represents porcine interfering siRNA, ** indicates extremely significant difference, and * indicates significant difference;

[0023] Figure 5 Figure showing the results of qPCR detection of the expression levels of myofibril type-related genes in mouse C2C12 myoblasts after siRNA interfered with lncRNA NEAT1: where si-NC represents blank control siRNA, si-Neat1 represents mouse interfering siRNA, ** indicates extremely significant difference, and * indicates significant difference;

[0024] Figure 6 Figure showing the results of Western blot analysis of protein expression of porcine skeletal muscle satellite cell myofiber type-related genes after siRNA interference with lncRNA NEAT1: where si-NC represents blank control siRNA, si-pNEAT1 represents porcine interfering siRNA, ** indicates extremely significant difference, and * indicates significant difference;

[0025] Figure 7 Figure showing the results of Western blot analysis of protein expression of myofibril type-related genes in mouse C2C12 myoblasts after siRNA interference with lncRNA NEAT1: where si-NC represents blank control siRNA, si-Neat1 represents mouse interfering siRNA, ** indicates extremely significant difference, and * indicates significant difference;

[0026] Figure 8 This is a graph showing the protein detection results using RNA pulldown technology. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0028] Example 1: Obtaining lncRNA NEAT1

[0029] Primer design: Mouse and porcine lncRNA NEAT1 sequences were retrieved from the NCBI database (mouse NCBI gene sequence number: NR_003513.3, porcine NCBI gene sequence number: MN784916.1). Primers with restriction enzyme sites were designed based on the lncRNA NEAT1 sequence and vector plasmid map. The primer sequences are shown in Table 1 below (Neat1-FL represents the mouse primer, and pNEAT1-FL represents the porcine primer):

[0030] Table 1 Primer sequences for full-length lncRNA-NEAT1 amplification

[0031]

[0032] Note: F represents the upstream primer, and R represents the downstream primer; the underlined part is the enzyme cleavage site.

[0033] PCR amplification reaction: First, using qualified mouse and pig cell cDNA as templates, specific amplification was performed using full-length NEAT1 primers without restriction enzyme sites. After obtaining the target fragment, it was purified by gel extraction. Second, using the purified gel product as a PCR template, amplification was performed using designed full-length NEAT1 primers with restriction enzyme sites to obtain the full-length NEAT1 with restriction enzyme sites, which was then purified by gel extraction. The PCR amplification reaction system and reaction conditions are shown in Tables 2 and 3 below.

[0034] Table 2 PCR reaction system

[0035]

[0036]

[0037] Table 3 PCR reaction conditions

[0038]

[0039] Example 2: Construction of lncRNA NEAT1 overexpression vector

[0040] (1) The obtained full-length fragments of pNEAT1 (pig NEAT1) and NEAT1 (mouse NEAT1) with restriction sites and the pcDNA3.1(+) eukaryotic expression vector were digested with the same restriction endonucleases (BamHI and EcoRI). The reaction system is shown in Table 4 below. The cells were incubated at 37℃ for 2 h, and the digestion results were detected by 1.5% agarose gel electrophoresis and the target fragment was recovered.

[0041] Table 4. Double enzyme digestion reaction system

[0042]

[0043]

[0044] (2) Use the T4 DNA ligase kit to mix the recovered fragments according to the reaction system in Table 5 below, and react at 22℃ for 30 min.

[0045] Table 5 Connection Reaction System

[0046]

[0047] (3) Perform the conversion of the recombination product, the specific steps of which are as follows:

[0048] ① Take 50 μL of competent cells thawed on ice, add the ligation product, mix gently, and incubate on ice for 30 min.

[0049] ② Heat shock in a 42℃ water bath for 45 seconds, then quickly transfer to an ice bath for 2 minutes. Do not shake the centrifuge tube during this process.

[0050] ③ Add 500 μL of sterile LB medium (antibiotic-free) to the centrifuge tube, mix well, and incubate at 37°C and 220 rpm for 1 hour.

[0051] ④ Centrifuge at 1500 rpm for 5 min at room temperature to remove most of the supernatant. Mix the remaining liquid by pipetting and add it to LA solid medium. Spread the cells evenly.

[0052] ⑤ Place the plate in a 37℃ incubator and incubate upright for 30 minutes, then invert the plate and incubate overnight.

[0053] ⑥ Sterilize the ultra-clean workbench with ultraviolet light in advance, pick single colonies from the resistance plate with the sterilization pipette tip, add them to 500μL of LA liquid medium, and expand the culture in a constant temperature shaker at 37℃ for 4-6 hours.

[0054] ⑦ After culture, the bacterial culture is sent for testing. The sequencing results are compared using SnapGene software to determine whether the sequence is correct.

[0055] ⑧ Transfer the successfully ligated and correctly sequenced positive clone bacterial solution into a 50mL centrifuge tube, mix the bacterial solution and LA medium at a ratio of 1:100, and incubate overnight at 37℃ and 220rpm.

[0056] ⑨ Use the Endo-free Plasmid Mini Kit II to extract the endotoxin-free plasmid, measure and record the concentration to obtain the porcine and mouse lncRNA-NEAT1 overexpression vector, and store at -20℃ for later use.

[0057] Example 3: Interference Fragment Synthesis

[0058] The mouse and porcine siRNA interference fragments used in this study were designed and synthesized by Suzhou Gemma Gene Co., Ltd., and their sequences are shown in Table 6 below (in Table 6, si-Neat1 represents the mouse siRNA interference fragment, si-pNEAT1 represents the porcine siRNA interference fragment, and siRNA NC represents the negative control fragment).

[0059] Table 6 siRNA interference fragment sequences

[0060]

[0061] Example 4: Effect of lncRNA NEAT1 overexpression vector on myofiber type conversion

[0062] The lncRNA NEAT1 overexpression vector (plasmid) obtained in Example 2 was used to transfect pig and mouse cells, respectively. The transfected cells were then validated by qPCR and protein expression, as detailed below:

[0063] 1. qPCR validation

[0064] (1) Cell RNA extraction

[0065] Total RNA was extracted using Trizol reagent purchased from Invitrogen. The specific steps are as follows:

[0066] ① Discard the culture medium and wash the cells twice with DPBS.

[0067] ② Discard DPBS, add 1 ml Trizol reagent to each well, pipette and detach the adherent cells several times, then transfer the liquid to a 1.5 ml nuclease-free centrifuge tube, vortex and let stand at room temperature for 10 min.

[0068] ③ Add 200 μL of chloroform, shake vigorously to mix, and let stand at room temperature for 2-3 minutes.

[0069] ④ Centrifuge at 12000 rpm and 4℃ for 15 min.

[0070] ⑤ Take the upper aqueous phase liquid into a new 1.5ml nuclease-free centrifuge tube, add an equal volume of pre-cooled isopropanol, mix by inverting, and let stand on ice for 10 minutes.

[0071] ⑥ Centrifuge at 12000 rpm and 4℃ for 10 min, observe the precipitate, and carefully discard the liquid.

[0072] ⑦ Add 1 ml of 75% ethanol to the precipitate and invert the container to suspend the precipitate.

[0073] ⑧ Centrifuge at 7500 rpm and 4℃ for 5 min, discard the liquid, repeat steps (7-8), and air dry at room temperature.

[0074] ⑨ Add 20 μL of enzyme-free DEPC water to dissolve the precipitate and measure the concentration.

[0075] (2) Preparation of cDNA

[0076] cDNA was synthesized using the EvoM-MLV reverse transcription kit according to the instructions.

[0077] (3) Real-time quantitative PCR

[0078] ① The obtained cDNA was used in RT-PCR experiments. The reaction system and reaction conditions are shown in Tables 7 and 8 below:

[0079] Table 7 qPCR reaction system

[0080]

[0081] Table 8 qPCR reaction conditions

[0082]

[0083] ② Data Analysis

[0084] The experiment was conducted using the Quant Studio™ 7 Flex real-time quantitative PCR system. Three sample replicates and three technical replicates were prepared for each experiment. After the reaction, the accuracy of the melting curve and amplification curve was verified. The detection results were applied using 2... -△△CT The method is used for analysis.

[0085] 2. Protein expression verification

[0086] The specific procedure for obtaining transfected cell samples from six-well plates is as follows:

[0087] (1) Cell protein extraction

[0088] ① Perform the entire experiment on ice. Discard the culture medium and wash the cells twice with PBS.

[0089] ② Add 200 μL of RIPA lysis buffer to each well (add PMSF at a ratio of 100:1 before use).

[0090] ③ Use a pipette to blow the liquid to ensure that the cells are in full contact with the lysis buffer. Use a pipette tip or cell scraper to scrape the cells off and transfer the liquid to a 1.5ml centrifuge tube.

[0091] ④ Shake vigorously and incubate on ice for 10 minutes.

[0092] ⑤ Centrifuge at 14000×g for 7 min at 4℃.

[0093] ⑥ Transfer the supernatant to a new 1.5ml centrifuge tube. If denaturation is not required, store the sample at -80℃.

[0094] ⑦ Denaturation: Dilute and mix the protein sample according to the ratio of supernatant to 6x protein loading buffer = 5:1, denature at 95-98℃ for 5-10 min, and store the sample at -20℃.

[0095] (2) SDS-PAGE gel preparation

[0096] ① Use dish soap to clean the long and short glass plates. After clamping them with clips, add pure water to check for leaks. Observe the liquid level after 10 minutes. If the liquid level does not drop, pour out the pure water and use filter paper to absorb the residual liquid.

[0097] ② Follow the instructions to prepare a 12% gel using the SDS-PAGE rapid gel preparation kit.

[0098] ③ The gel can be used after it solidifies. If it is not to be used immediately, soak it in pure water at 4℃ for later use. (3) SDS-PAGE gel electrophoresis

[0099] ④ Take out the comb from the prepared gel, assemble it into the electrophoresis tank, add pure water to check for leaks, and if the liquid level does not drop after 10 minutes, pour out the pure water, add the prepared electrophoresis solution, and prepare to load the sample.

[0100] ⑤ Add samples in sequence, adding 30 μg of denatured protein sample to each well, adding 5 μL of color pre-stained marker to both ends of the sample well, and filling the remaining wells with an equal amount of protein loading buffer.

[0101] ⑥ Connect the power supply and perform 200V constant voltage electrophoresis for 7 minutes to ensure the sample enters the separating gel. Then perform 120V constant voltage electrophoresis until the bromophenol blue indicator band reaches the bottom of the separating gel.

[0102] (4) Transfer membrane

[0103] ① Soak the clamps, filter paper and sponge used during membrane transfer in TBST in advance, prepare a 0.45μm PVDF membrane of appropriate size, and pre-cool and activate it with methanol for 10 min.

[0104] ② Remove the gel after electrophoresis and cut it off according to the marker bands and the molecular weight of the target protein.

[0105] ③ Assemble the sandwich clamp in the following order: negative electrode - sponge - 2 sheets of filter paper - gel - 2 sheets of filter paper - sponge - positive electrode, place it in the transfer tank, and fill it with transfer solution.

[0106] ④ Connect the power supply, transfer the membrane at a constant current of 200mA, and the time depends on the size of the target protein.

[0107] (5) Antigen-antibody immune reaction and imaging

[0108] ① After the transfer is complete, remove the PVDF membrane with tweezers and clean it twice with TBS for 5 minutes each time.

[0109] ② Seal with 6% skim milk powder prepared by TBST for more than 3 hours.

[0110] ③ Wash the membrane with TBS 3 times, 10 minutes each time.

[0111] ④ Incubate the primary antibody diluted with TBST at 4°C overnight.

[0112] ⑤ Primary antibody recovery: Wash the membrane 3 times with TBST and 1 time with TBS, each time for 10 minutes.

[0113] ⑥ The secondary antibody diluted with TBST was incubated on a shaker at 37°C for 1 hour.

[0114] ⑦ Wash the membrane 3 times with TBST and 1 time with TBS, each time for 10 minutes.

[0115] ⑧ Under light-protected conditions, prepare the ECL luminescent solution according to the instructions and immerse it evenly on the PVDF membrane containing the protein sample.

[0116] ⑨ Image was taken using a chemiluminescence imager, and the grayscale values ​​were quantified and analyzed using ImageJ software.

[0117] 3. Results Analysis

[0118] The lncRNA NEAT1 overexpression vector was transfected into PSC porcine skeletal muscle satellite cells and C2C12 mouse myoblasts, respectively. After inducing differentiation for 3 and 5 days, respectively, the effect of the lncRNA NEAT1 overexpression vector on myofiber type conversion was detected by qPCR and Western blot. The results are as follows (in the results figure, MyHC-Ⅰ is the slow muscle gene, and MyHC-ⅡA, MyHC-ⅡB, and MyHC-ⅡX are the fast muscle genes):

[0119] like Figure 1 As shown, overexpression of pNEAT1 significantly increased the mRNA expression level of slow muscle genes in porcine skeletal muscle satellite cells, while the mRNA expression level of fast muscle genes was significantly reduced.

[0120] like Figure 2 As shown, overexpression of pNEAT1 significantly increased the expression of slow-twitch-related genes in porcine skeletal muscle satellite cells and inhibited the expression of fast-twitch-related genes.

[0121] like Figure 3 As shown, overexpression of Neat1 significantly increased the expression of C2C12 slow-twitch muscle-related gene proteins and inhibited the expression of fast-twitch muscle-related gene proteins.

[0122] The above results indicate that the overexpression vector can promote the expression of slow-twitch muscle fibers and inhibit the expression of fast-twitch muscle fibers, suggesting that it can promote the conversion of fast-twitch muscle fibers into slow-twitch muscle fibers.

[0123] Example 5: Effect of NEAT1 Interference on Myofiber Type Transformation

[0124] The qPCR and Western blot experiments in this embodiment are performed in the same manner as in Example 4.

[0125] The interfering fragment was transfected into PSC porcine skeletal muscle satellite cells and C2C12 mouse myoblasts, respectively. After inducing differentiation for 3 and 5 days, the effect of the interference on myofiber type conversion was detected by qPCR and Western blot. The results are as follows (in the results figure, MyHC-Ⅰ is the slow muscle gene, and MyHC-ⅡA, MyHC-ⅡB, and MyHC-ⅡX are the fast muscle genes):

[0126] like Figure 4 As shown, interfering with pNEAT1 significantly increased the mRNA expression of fast-twitch-related genes in porcine skeletal muscle satellite cells.

[0127] like Figure 5 As shown, interfering with Neat1 significantly increased the mRNA expression of C2C12 fast-twitch muscle-related genes and decreased the mRNA expression of slow-twitch muscle genes.

[0128] like Figure 6 As shown, interfering with pNEAT1 significantly promotes the protein expression of fast-twitch muscle-related genes in porcine skeletal muscle satellite cells and inhibits the protein expression of slow-twitch muscle-related genes.

[0129] like Figure 7 As shown, interfering with Neat1 significantly increased the protein expression of C2C12 fast-twitch muscle-related genes, while significantly inhibiting the protein expression of slow-twitch muscle-related genes.

[0130] The above results indicate that the interfering fragment can increase the expression of fast-twitch muscle-related genes and inhibit the expression of slow-twitch muscle-related genes, suggesting that it can promote the conversion of slow-twitch muscle fibers into fast-twitch muscle fibers.

[0131] Example 6: lncRNA-NEAT1 Interacting Protein Fishing

[0132] To further investigate the molecular mechanism by which lncRNA NEAT1 plays a role in myofibril type conversion, we first used RNA pulldown technology to extract proteins binding to Neat1 and pNEAT1 from the myotubes of differentiated PSC and C2C12 cells. We then excised the protein bands specifically bound to Neat1 and pNEAT1 and identified the extracted proteins using proteomic profiling. The steps are as follows:

[0133] 1. RNA pulldown protein extraction

[0134] ①Use one 10cm culture dish as a sample, add 10mL of pre-cooled DPBS to wash the cells 3 times.

[0135] ② Add 1ml of RIP Buffer, scrape the cells off with a cell scraper, and transfer them to a 1.5ml centrifuge tube.

[0136] ③ Add 15 μL PMSF to each tube, mix well, and remove air bubbles.

[0137] ④ Use an ultrasonic cell disruptor to break down cells: power 25%, on for 9.9 seconds, off for 9.9 seconds, time 4 minutes.

[0138] ⑤ Centrifuge at 14000 rpm and 4℃ for 10 min, discard the precipitate, and determine the protein concentration using the BCA method according to the instructions. Store at -80℃.

[0139] 2. RNA pulldown

[0140] ① Take 3 μg of biotin-labeled RNA, add 2 μL of RNase Inhibitor, mix well, incubate at 90℃ for 2 min, and immediately place on ice for 2 min.

[0141] ② Add RNA Structure Buffer to 100 μL and incubate at room temperature for 20 min to allow the RNA to automatically form a secondary structure.

[0142] ③ Dissolve the protein prepared in 2.3.11.3 on ice. Add 1 μL PMSF and 5 μL RNase Inhibitor to every 1 mg of protein, and add RIP Buffer to make up to 1 mL. Mix well.

[0143] ④ Mix 3 μg of biotin-labeled RNA with 1 mg of protein and incubate at room temperature for 1 h by rotation.

[0144] ⑤ Prepare 30 μL of biotin-labeled magnetic beads in advance, and wash them 3 times with 1 mL of RIP Buffer for 5 min each time.

[0145] ⑥ Mix the magnetic beads with the RNA-protein complex and incubate at room temperature by rotation for 1 hour.

[0146] ⑦ After incubation, rinse with RIP Buffer 5 times, 5 minutes each time.

[0147] ⑧ Discard the RIP buffer, add 20 μL LEPC water and 10 μL protein loading buffer, mix well, denature at 98℃ for 10 min, incubate on ice for 5 min, and store at -20℃.

[0148] ⑨ Western blot analysis: After electrophoresis, the entire gel was stained using a silver staining kit according to the instructions. The results are as follows: Figure 8 As shown.

[0149] 3. Sequencing results

[0150] Protein strip sequencing was performed by Beijing Novogene Technology Co., Ltd., and some sequencing results are shown in Tables 9 and 10 below:

[0151] Table 9. List of protein samples obtained from Neat1 pulldown (pig)

[0152]

[0153]

[0154] Table 10 List of pNeat1 pulldown proteins (mice)

[0155]

[0156]

[0157] Analysis of the proteins retrieved by Neat1 pulldown in Tables 9 and 10 revealed that proteins encoded by genes such as LDHB and PKM interact with lncRNA NEAT1 in pigs, while proteins encoded by genes such as Ldha and Pkm interact with lncRNA NEAT1 in mice. These genes are key proteins involved in glycolysis, which is an important step affecting the conversion of muscle fibers between fast and slow muscle types. Therefore, it is inferred that lncRNA NEAT1 participates in regulating the conversion of muscle fiber types in pigs and mice by participating in the glycolysis process.

[0158] The above descriptions are merely some embodiments of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the inventive concept of the present invention, and all such modifications and improvements fall within the scope of protection of the invention.

Claims

1. The application of long non-coding RNA NEAT1 in regulating myofiber type conversion, characterized in that, The application promotes the conversion of fast-twitch muscle fibers to slow-twitch muscle fibers by overexpressing NEAT1; or promotes the conversion of slow-twitch muscle fibers to fast-twitch muscle fibers by inhibiting NEAT1 expression.

2. The application of kits or nucleic acid molecules that can inhibit the expression of long non-coding RNA NEAT1 in promoting the conversion of slow muscle fibers to fast muscle fibers in animal muscle types.

3. Kits or nucleic acid molecules that can overexpress long non-coding RNA NEAT1 can be used to promote the conversion of fast-twitch muscle fibers to slow-twitch muscle fibers in animal muscle types.

4. The application according to any one of claims 1-3, wherein, The application achieves muscle fiber type conversion by regulating the glycolysis process in animal muscle tissue.

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