Application of pig intestinal exosome miR-30b-5p in regulation of pig fat metabolism

Abstract

The application relates to the field of bioengineering technology, in particular to application of pig intestinal tract exosome miR-30b-5p in pig fat metabolism regulation, and discloses a nucleotide sequence of pig miR-30b-5p: 5'-TGTAAACATCCTACACTCAGCT-3', fat cell decomposition of pigs is promoted by improving pig intestinal tract exosome miR-30b-5p gene expression, or fat cell growth of pigs is promoted by inhibiting pig intestinal tract exosome miR-30b-5p gene expression, miR-30b-5p loaded by intestinal tract derived exosomes can be targeted and combined with pig fat FMO3 genes, pig fat cell decomposition is participated by mediating FMO3 signals, metabolism regulation of primary cells is realized, and pig fat deposition is effectively improved.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to the application of porcine intestinal exosomes miR-30b-5p in the regulation of porcine lipid metabolism. Background Technology

[0002] Fat deposition is closely related to carcass quality and feed conversion efficiency in pigs. Revealing the patterns and mechanisms of fat deposition and developing corresponding nutritional regulation strategies are effective ways to improve pig production efficiency. On the one hand, regulating regional fat deposition is key to pork carcass quality. Among the meat traits of pigs, intramuscular fat content is an important factor in pork quality and flavor. On the other hand, fat deposition is an inevitable result of the coordinated distribution of nutrients in the animal body. Adequate fat reserves are necessary to maintain normal life activities. However, excessive fat deposition is a significant indicator of energy homeostasis imbalance, leading to wasted feed resources and reduced farming efficiency. Therefore, regulating fat deposition is of great scientific significance for improving carcass quality and enhancing efficient pig production.

[0003] The interaction between the gut and adipose tissue is an effective means for the body to respond to exposures to the internal and external environments. The gut is the main organ for lipid sensing and regulation; intestinal secretory cells act as chemoreceptors, sensing substances within the intestinal lumen and secreting hormones to regulate lipid metabolism. Adipose tissue, as an immune and endocrine organ, secretes various adipokines and hormones to maintain intestinal homeostasis. Exosomes have been recognized as an important mechanism for mediating intertissue signaling and functional regulation; however, whether intestinal exosome signaling participates in lipid metabolism regulation and its potential targeting mechanisms remain unclear. Therefore, elucidating the targets of intestinal exosome-mediated lipid metabolism regulation is crucial for clarifying the mechanisms of lipid metabolism differences in pigs with different types of fat deposition. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of porcine intestinal exosomes miR-30b-5p in the regulation of porcine fat metabolism. The exosomes from the intestine loaded with miR-30b-5p can target and bind to the porcine fat FMO3 gene, participate in the decomposition of porcine adipocytes by mediating FMO3 signaling, realize the metabolic regulation of primary cells, and effectively improve porcine fat deposition.

[0005] To achieve the above objectives, the first aspect is:

[0006] This invention provides the application of porcine intestinal exosome miR-30b-5p in regulating porcine fat deposition, wherein the nucleotide sequence of porcine miR-30b-5p is: 5'-TGTAAACATCCTACACTCAGCT-3'.

[0007] Porcine adipocyte degradation can be promoted by increasing the expression of the miR-30b-5p gene in porcine intestinal exosomes, or porcine adipocyte growth can be promoted by inhibiting the expression of the miR-30b-5p gene in porcine intestinal exosomes.

[0008] In some implementations, the application includes any of the following:

[0009] Application of porcine intestinal exosomes miR-30b-5p in identifying the developmental potential of piglet adipocytes: The developmental potential of piglet adipocytes was determined by detecting the expression level of miR-30b-5p in porcine intestinal exosomes. Among them, the expression level of miR-30b-5p was inversely proportional to the developmental potential of piglet adipocytes.

[0010] Application of porcine intestinal exosome miR-30b-5p as an auxiliary molecular marker for the expression level of intramuscular fat in selected breeding pigs, wherein the expression level of miR-30b-5p is inversely proportional to the expression level of intramuscular fat in breeding pigs;

[0011] Application of porcine intestinal exosome miR-30b-5p in intestinal recognition of genetic molecular targets of lipid metabolism, wherein the genetic molecular target of lipid metabolism is the porcine fat FMO3 gene.

[0012] 3. The application according to claim 2, characterized in that, in the application of porcine intestinal exosome miR-30b-5p in identifying the developmental potential of piglet adipocytes, a dynamic individual development detection system is established based on porcine intestinal exosome miRNA signals.

[0013] The second aspect:

[0014] The application of porcine intestinal exosome miR-30b-5p in lipid metabolism is provided. The nucleotide sequence of porcine miR-30b-5p is 5'-TGTAAACATCCTACACTCAGCT-3'. The porcine intestinal exosome miR-30b-5p targets and binds to the porcine adipose FMO3 gene to initiate adipocyte catabolism.

[0015] Third aspect:

[0016] A porcine fat regulator is provided, the regulator containing a mimic of porcine intestinal exosome miR-30b-5p.

[0017] In some implementations...

[0018] The analogue sequence of miR-30b-5p: Positive sequence: 5'-UGUAAACAUCCUACACUCAGCU-3'.

[0019] Antonym sequence: 5'-CUGAGUGUAGGAUGUUUACAUU-3'.

[0020] Fourth aspect:

[0021] The applications of the above-mentioned swine fat regulators include any of the following applications:

[0022] Application of porcine fat miR-30b-5p in inhibiting fat deposition;

[0023] Application of porcine adipose miR-30b-5p as a signaling molecule to target and intervene in the FMO3 gene in porcine adipose tissue;

[0024] Application of genetically-assisted molecular markers for miR-30b-5p-mediated lipid metabolism regulation in porcine adipose tissue;

[0025] The application of porcine fat miR-30b-5p in the development of drugs and targets that regulate lipid metabolism.

[0026] The beneficial effects of this invention on the application of porcine intestinal exosome miR-30b-5p in the regulation of porcine lipid metabolism:

[0027] (1) The application of porcine intestinal exosomes miR-30b-5p in the regulation of porcine lipid metabolism. This invention is the first to discover that the expression of miR-30b-5p in porcine intestinal exosomes differs among different types of lipid deposition. It is also the first to elucidate that porcine intestinal exosomes miR-30b-5p has a significant association with lipid metabolism in primary porcine adipocytes. Specifically, high expression of miR-30b-5p in porcine intestinal exosomes promotes enhanced lipolysis in porcine adipocytes to inhibit lipogenesis and effectively regulates regional deposition of intramuscular fat. By transfecting primary porcine adipocytes with miR-30b-5p mimics, the expression level of miR-30b-5p in porcine adipocytes is enhanced, thereby promoting lipolysis in porcine adipocytes.

[0028] (2) The application of the porcine intestinal exosome miR-30b-5p in the regulation of porcine fat metabolism of the present invention is the first discovery that miR-30b-5p in porcine adipocytes participates in the metabolic regulation of porcine adipocytes by targeting and binding to the porcine adipocyte FMO3 gene. Among them, miR-30b-5p mediates FMO3 signaling to inhibit FMO3 gene expression. Since FMO3 gene can promote fat growth, fat growth can be inhibited by inhibiting FMO3 gene expression. Attached Figure Description

[0029] Figure 1 This is a diagram showing the results of porcine intestinal exosome isolation and identification in a specific embodiment of the present invention, including...

[0030] Figure A: Intestinal exosome particle size analysis of obese Lantang pigs and lean Landrace pigs;

[0031] Figure B: Analysis of the number of intestinal exosome particles per unit intestinal weight in obese Lantang pigs and lean Landrace pigs;

[0032] Figure C: Western blot results of intestinal exosome characterization proteins in obese Lantang pigs and lean Landrace pigs;

[0033] Figure D: Electron microscopy results of intestinal exosomes in obese Lantang pigs and lean Landrace pigs;

[0034] Langtang represents the Langtang pig; Landrace represents the Landrace pig.

[0035] Figure 2 This is a diagram showing the results of porcine primary adipocytes stimulated by porcine intestinal exosomes in a specific embodiment of the present invention, including...

[0036] Figure A: Western blot results of primary porcine adipose cells treated with intestinal exosomes from obese Lantang pigs and lean Landrace pigs; Figure B: Oil Red O results of primary porcine adipose cells treated with intestinal exosomes from obese Lantang pigs and lean Landrace pigs;

[0037] Figure C: Results of triglyceride content detection in intestinal exosomes of obese Lantang pigs and lean Landrace pigs;

[0038] NC represents physiological saline; LT-EXO represents intestinal exosomes of Blue Tang pigs; LD-EXO represents intestinal exosomes of Landrace pigs; TG represents triglycerides.

[0039] Figure 3 This is a diagram showing the miRNA-seq sequencing results of porcine intestinal exosomes with different lipid deposition types in a specific embodiment of the present invention, including...

[0040] Figure A: Venn plot results of differential miRNAs in intestinal exosomes between obese Lantang pigs and lean Landrace pigs;

[0041] Figure B: Volcano plot results of differential miRNAs in intestinal exosomes between obese Lantang pigs and lean Landrace pigs;

[0042] Figure 4 This is a functional verification of the porcine adipose miR-30b-5p gene in a specific embodiment of the present invention, including...

[0043] Figure A: Results of miR-30b-5p transfection efficiency of porcine primary adipocytes after transfection with miR-30b-5p mimics;

[0044] Figure B: Results of triglyceride content in porcine primary adipocytes after transfection with miR-30b-5p mimic;

[0045] Figure C: Oil Red O results of porcine primary adipocytes transfected with miR-30b-5p mimic;

[0046] Figure D: Changes in FMO3 and FASN gene expression in porcine primary adipocytes after transfection with miR-30b-5p mimic. Detailed Implementation

[0047] Preferred embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While preferred embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0048] Example 1

[0049] This embodiment focuses on pigs with different fat deposition types and their isolated intestinal exosomes. A method for isolating porcine intestinal exosomes is disclosed, including the identification and structural observation of intestinal exosomes isolated from obese Blue Tang pigs and lean Landrace pigs, respectively.

[0050] Specifically, the identification and observation of intestinal exosomes in pigs with different fat deposition types (obese Blue Tang pigs and lean Landrace pigs) included the following steps:

[0051] Three 3-day-old pigs of different fat deposition types (obese Lantang pigs and lean Landrace pigs) were selected. After euthanasia, intestinal tissue was collected. Under aseptic conditions, the intestinal contents were removed by washing with pre-cooled PBS. The cleaned intestines were divided into 4-5 cm segments. The mesenteric fat was bluntly peeled off and the intestinal epithelium was completely exposed in PBS solution after washing with pre-cooled PBS. The intestinal tissue was rinsed three times and then treated with 10 mmol / L dithiothreitol for 5 min to remove intestinal mucus. The intestines were then digested in 8 mM EDTA solution for 30 min. The epithelium was removed and added to pre-cooled PBS with shaking. The intestines were then centrifuged at 600×g for 5 min at 4°C to remove the precipitate. The intestines were centrifuged again at 600×g for 5 min at 4°C to remove cellular impurities. The supernatant was filtered through a 0.22 μm filter and centrifuged at 120,000×g for 2 h to collect the precipitate. The precipitate was then washed with pre-cooled PBS. Finally, the exosomes produced from the intestinal tissue were harvested.

[0052] Porcine intestinal exosome particle size analysis: 10 μL of harvested exosome solution was added to 1 mL of PBS solution, and nanoparticle tracking analysis was performed using a Zetaview instrument to obtain... Figure 1 The results in Figure A indicate that there are differences in the particle size of intestinal exosomes between obese Lantang pigs and lean Landrace pigs.

[0053] Analysis of exosome particle count in pig intestines: The intestines of the slaughtered piglets were weighed, and the number of exosome particles in the intestines of pigs with different fat deposition types was determined by Zetaview. The number of exosome particles per unit weight of intestine was calculated for different fat deposition types of pigs. Figure 1 The results in Figure B indicate a difference in the number of exosome particles in the intestines between obese Lantang pigs and lean Landrace pigs.

[0054] Western blot analysis of porcine intestinal exosomes: Total protein in exosomes was detected by BCA assay and then calibrated. 15 μg of protein was loaded per well. Exosome marker proteins Alix, TSG101, CD63, CD9, and the intestinal-specific protein GPA33 were detected. Figure 1 The results in Figure C indicate that the intestinal exosomes isolated from the intestines of obese Lantang pigs and lean Landrace pigs contain exosome marker proteins and intestinal-specific expressed proteins.

[0055] Transmission electron microscopy (TEM) analysis: 10 μL of the harvested exosome solution was placed on a polyvinyl acetate-coated copper grid and incubated at room temperature for 5 min. Excess exosome solution at the edge of the copper grid was aspirated. Uranyl acetate was added to the copper grid for negative staining for 1 min. The prepared copper grid was then placed on a TEM and the morphology of the exosomes was observed at 100 kV. Figure 1 The results of the D diagram indicate that the substances separated from the intestines of obese Lantang pigs and lean Landrace pigs by high-speed centrifugation are intestinal exosomes.

[0056] The experimental results above show that the above separation and identification methods can effectively harvest porcine intestinal exosomes with different types of fat deposition, providing practical materials for studying the lipid metabolism regulation function of porcine intestinal exosomes.

[0057] Example 2

[0058] To further illustrate the regulatory function of porcine intestinal exosomes on porcine primary adipocytes, Example 2 was conducted based on Example 1:

[0059] This embodiment studies the regulatory function of porcine intestinal exosomes with different lipid deposition types on porcine primary adipocytes. It discloses that porcine intestinal exosomes with different lipid deposition types can effectively promote the growth of porcine adipocytes, and the lipid-promoting effect of intestinal exosomes from obese Lantang pigs is stronger, indicating that porcine intestinal exosomes can effectively regulate the lipid differentiation of porcine primary adipocytes, including:

[0060] Regulation of lipid metabolism mediated by porcine intestinal exosomes: On the first day of differentiation induction, 2×10⁻⁶ mmol / L was added to the culture system. 9Different doses of intestinal exosomes from pigs with different types of fat deposition were used to supplement the culture system with an equal dose of exosomes every two days, and the entire induction period was stimulated with exosomes for 8 days.

[0061] Primary porcine adipocytes stimulated by porcine intestinal exosomes with different types of fat deposition were analyzed using Western blotting to detect the expression levels of adipogenesis-related proteins in these cells. Figure 2 Figure A in the middle, from Figure 2 Figure A shows that the lipid deposition-related proteins FASN, CD36, PPAR-γ, and FABP4 in porcine primary adipose cells were differentially expressed after stimulation by porcine intestinal exosomes in different lipid deposition types, suggesting that porcine intestinal exosomes play an important role in regulating porcine lipid deposition function.

[0062] Subsequently, the content and distribution of lipid droplets were observed by Oil Red O staining. Figure 2 Figure B shows the determination of esterification in primary porcine adipocytes stimulated by intestinal exosomes in obese Lantang pigs and lean Landrace pigs, using reagents from the triglyceride assay. Figure 2 Figure C in the diagram.

[0063] In this embodiment, porcine intestinal exosomes with different fat deposition types were used to stimulate porcine primary adipocytes to explore the function of porcine intestinal exosomes in the regulation of lipid metabolism in porcine adipocytes. The isolated porcine intestinal exosomes were used to stimulate porcine primary adipocytes, and the changes in lipid metabolism phenotype of porcine primary adipocytes were confirmed by Western blot, Oil Red O, and triglyceride detection, demonstrating that porcine intestinal exosomes with different fat deposition types participate in the metabolic regulation of porcine primary adipocytes.

[0064] Example 3

[0065] To further illustrate how porcine intestinal exosomes participate in the metabolic regulation of porcine primary adipocytes, Example 3 was conducted based on Example 2:

[0066] This embodiment studies the differences in miRNA components of porcine intestinal exosomals with different types of fat deposition. The miRNA-seq sequences of 3-day-old obese Lantang pigs and lean Landrace pigs intestinal exosomals were disclosed. Analysis and screening of differentially expressed miRNAs revealed differential expression of porcine intestinal exosomal miR-30b-5p in porcine intestinal exosomals with different fat deposition types. Porcine intestinal exosomal miR-30b-5p was identified as a key extracellular genetic signaling molecule for differential regulation of metabolic function in porcine primary adipocytes by porcine intestinal exosomals. The steps included:

[0067] Porcine intestinal exosome miRNA-seq transcriptome data mining: The experimental procedure was performed according to the standard steps provided by Illumina, including library preparation and sequencing experiments. Small RNA sequencing libraries were prepared using the TruSeq SmallRNA Sample Prep Kits (Illumina, San Diego, USA). After library preparation, the constructed libraries were sequenced using Illumina HiSeq 2000 / 2500, with single-end read lengths of 1 x 50 bp. miRNA data analysis was performed using the ACGT101-miR software (LCSciences, Houston, Texas, USA). The analysis process is as follows: (1) Remove 3' adapters and junk sequences: obtain clean data; (2) Length screening: retain sequences with a base length of 18-26 nt; (3) Comparison analysis with various RNA databases: align the remaining sequences (excluding miRNAs) with mRNA, RFam, and Repbase databases and filter them; (4) miRNA identification: obtain valid data and identify miRNAs by comparing precursors and genomes.

[0068] Bioinformatics comparative analysis was performed on the intestinal exosomal miRNAs of different lipid deposition types obtained by transcriptome sequencing. Based on the expression abundance of miRNAs in the intestinal exosomals of obese Lantang pigs and lean Landrace pigs, the following results were obtained: Figure 3 Figure A illustrates that there are some common miRNAs and some uniquely expressed miRNAs in porcine intestinal exosomal miRNAs across different lipid deposition types. By comparing the differentially expressed miRNAs in porcine intestinal exosomal miRNAs from different lipid deposition types, we obtained... Figure 3 Figure B illustrates the differential expression of miR-30b-5p in the intestinal exosomes of obese Lantang pigs and lean Landrace pigs.

[0069] In this embodiment, transcriptomic sequencing was performed on miRNAs in porcine intestinal exosomes with different lipid deposition types to explore the relationship between the regulation of porcine primary adipocyte metabolic function by porcine intestinal exosomes and the differential expression of porcine intestinal exosome miRNAs. Through transcriptomic analysis of porcine intestinal exosome miRNA-seq, combined with visualization comparison using Venn plots and volcano plots, it was found that porcine intestinal exosomes with different lipid deposition types showed differential expression of miR-30b-5p, revealing the expression pattern of porcine intestinal exosome miRNAs in different lipid deposition types.

[0070] Example 4

[0071] To further illustrate the expression patterns of porcine intestinal exosome miRNAs in different types of lipid deposition, Example 4 was conducted based on Example 3:

[0072] This study investigated the lipid metabolism function of miR-30b-5p in porcine primary adipocytes. Using miRNA transfection technology, miR-30b-5p mimics were used to overexpress miR-30b-5p in porcine primary adipocytes to verify its role in regulating metabolic function. This demonstrates that miR-30b-5p delivered by porcine intestinal exosomes can achieve differential regulation of lipid metabolism phenotypes, including:

[0073] Transfection of porcine primary adipocytes: After porcine primary adipocytes were grown and fused, they were transfected using RfectPMV2siRNA primary cell transfection reagent.

[0074] Transfection sequence of the control group:

[0075] Justice Sequence 5'-UUCUCCGAACGUGUCACGUTT-3';

[0076] Antonym sequence 5'-ACGUGACACGUUCGGAGAATT),

[0077] miR-30b-5p analog sequence:

[0078] The sequence of justice is: 5'-UGUAAACAUCCUACACUCAGCU-3';

[0079] Antisense sequence: 5'-CUGAGUGUAGGAUGUUUACAUU-3', transfected 3 times during the entire experimental period, with each transfection performed every 2 days.

[0080] The expression level of miR-30b-5p in porcine primary adipocytes transfected with miR-30b-5p was verified by RT-qPCR to determine the transfection efficiency. Figure 4 Figure A in the figure illustrates that the transfection reagents and methods used in this example can achieve overexpression of miR-30b-5p in porcine primary adipocytes.

[0081] The lipid metabolism regulation capacity of miR-30b-5p in porcine primary adipocytes was determined by triglyceride content. Figure 4 Figure B illustrates that overexpression of miR-30b-5p in porcine primary adipocytes inhibits polyester differentiation and promotes adipocyte catabolism. Subsequently, the lipid droplet content and distribution of porcine primary adipocytes before and after transfection were observed using Oil Red O staining. Figure 4Figure C shows that transfection of porcine adipocytes with miR-30b-5p inhibited lipid droplet production in porcine adipocytes. Subsequently, Western blot analysis was performed to determine the miR-30b-5p-mediated metabolic pathway in porcine adipocytes. Figure 4 Figure D illustrates that overexpression of miR-30b-5p in porcine primary adipose cells can mediate lipid metabolism regulation in porcine primary adipose cells by targeting and binding to the porcine adipose FMO3 gene.

[0082] This embodiment measures the lipid metabolism regulatory function of miR-30b-5p in porcine primary adipocytes, exploring the relationship between miR-30b-5p and adipocyte differentiation in porcine adipocytes. By transfecting porcine primary adipocytes with miR-30b-5p mimics, overexpression of miR-30b-5p was achieved in porcine primary adipocytes. Combined with RT-qPCR, triglyceride content, Oil Red O, and Western blot, it was confirmed that miR-30b-5p in porcine primary adipocytes has the function of inhibiting adipogenic differentiation and promoting adipocyte catabolism.

[0083] Based on Examples 1 to 4, this invention obtained intestinal exosomes from 3-day-old obese Lantang pigs and lean Landrace pigs by particle size analysis, electron microscopy, and Western blot analysis. To verify the lipid metabolism function of porcine intestinal exosomes, primary adipocytes were stimulated with intestinal exosomes from both obese Lantang and lean Landrace pigs. Western blot analysis, triglyceride content, and Oil Red O concentration analysis revealed that intestinal exosomes from pigs with different lipid deposition types could promote adipocyte differentiation, and the lipid deposition regulation ability of Lantang pig intestinal exosomes was superior to that of Landrace pig intestinal exosomes. To further explore the reasons for the differences in lipid regulation ability of intestinal exosomes from pigs with different lipid deposition types, miRNA-seq transcriptome sequencing was performed on porcine intestinal exosomes. Based on Venn diagrams and differential volcano diagrams, differential expression of miR-30b-5p was found in intestinal exosomes from pigs with different lipid deposition types. Next, we investigated the function of miR-30b-5p in porcine primary adipocytes. By transfecting porcine primary adipocytes with a miR-30b-5p mimic, we confirmed that the mimic effectively overexpressed miR-30b-5p in porcine primary adipocytes using RT-qPCR. Subsequently, we confirmed that miR-30b-5p in porcine primary adipocytes could inhibit adipocyte differentiation by measuring triglycerides and observing Oil Red O lipid droplets. We then used Western blot to detect pathway changes of miR-30b-5p in regulating the metabolic function of porcine primary adipocytes and found that miR-30b-5p regulates the metabolic function of porcine adipocytes by targeting and binding to the porcine adipocyte FMO3 gene, thereby promoting adipocyte degradation. This invention is the first to discover that porcine intestinal exosomes can promote polyester differentiation in porcine adipocytes; the first to demonstrate that differential expression of miR-30b-5p in intestinal exosomes is the main reason for the differences in lipid metabolism regulation function of intestinal exosomes in different types of porcine fat deposition; and the first to discover that porcine adipose tissue miR-30b-5p targets and binds to the FMO3 gene to participate in the regulation of lipid metabolism in porcine adipocytes. These findings have significant scientific implications for improving carcass quality and enhancing efficient pig production.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of mimics of porcine intestinal exosomes miR-30b-5p in regulating porcine fat deposition, characterized in that, The nucleotide sequence of the porcine intestinal exosome miR-30b-5p is: 5'-TGTAAACATCCTACACTCAGCT-3'. The expression of the miR-30b-5p gene in porcine intestinal exosomes was increased to promote the breakdown of porcine adipocytes. The breeds of pigs mentioned were obese Lantang pigs and lean Landrace pigs. miR-30b-5p analog sequence: The sequence of justice is: 5'-UGUAAACAUCCUACACUCAGCU-3'. Antsense sequence: 5'-CUGAGUGUAGGAUGUUUACAUU-3', the application is an in vitro application.

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