Application of pig intestinal exosome miR-320 in regulation and control of lipid deposition of pig liver

Abstract

The invention relates to the technical field of bioengineering, in particular to application of a pig intestinal exosome miR-320 in regulation and control of lipid deposition of pig livers. The nucleotide sequence of the miR-320 is 5 '-AAAAGCTGGGGTTGAGAGGGCGA-3'. It is found and proved for the first time that by improving the expression level of the miR-320 in pig intestinal exosomes, the miR-320 can act on pig liver cells in a targeted mode and remarkably promote lipid deposition of the pig liver cells, a new key target is provided for molecular breeding, precise nutrition regulation and liver lipid metabolism disease research of pigs, and the application has the advantages that the miR-320 can be applied to the pig intestinal exosomes, and the application of the miR-320 in the pig intestinal exosomes is developed. On the basis, the invention further provides a reinforcing agent containing the miR-320 simulant, the reinforcing agent can be used for promoting liver lipid accumulation, optimizing feed energy distribution and developing related metabolism regulation drugs, and the reinforcing agent has important application value for improving carcass quality and increasing production benefits of live pigs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to the application of pig intestinal exosome miR-320 in regulating pig liver lipid deposition. BACKGROUND

[0002] The liver is the hub of lipid synthesis, oxidation and transport, moderate deposition is conducive to energy turnover and growth, while excessive accumulation is easy to cause metabolic diseases and reduce production performance. Therefore, optimizing the regulation mode of pig liver lipid metabolism is of great significance for improving carcass and meat quality, regional coordination of pig energy supply, and promoting the healthy and efficient development of pig industry.

[0003] The energy metabolism signal cross-talk between the intestine and the liver is the key to lipid regulation. The intestine and the liver, as important organs of digestion and absorption, are both independent and connected. In the ecological niche, the distal regulation of the intestine based on the portal vein and bile circulation has been proven to be an effective way to achieve liver energy integration. In fact, intestinal-derived nutrient signals, intestinal microbial signals, and hormone signals are important participants in the regulation of liver lipid metabolism. Exosome signals, as a new unit of tissue cross-talk metabolic regulation, have gradually attracted attention. Recent studies have revealed that intestinal exosomes are a new way to participate in the regulation of liver metabolism. However, whether intestinal exosomes mediate the regulation of liver lipid deposition and its potential mechanisms, especially in pigs, are still unknown. Therefore, analyzing the key target of pig intestinal exosome-mediated regulation of liver lipid deposition has far-reaching significance for regional regulation of pig energy utilization and improving pig production efficiency.

[0004] Non-coding RNA, as a common functional small molecule in exosomes, widely participates in the metabolic regulation process in vivo. In particular, miRNA composed of 20-22 nucleotide fragments is considered an important hub for glucose and lipid metabolism regulation. The cross-tissue delivery of exosome miRNA is a new way to target lipid regulation and optimize energy supply. However, it is still unclear which specific miRNA in pig intestinal exosomes mediates its regulation of liver lipid deposition, the identity of this key molecule and its mechanism of action, which has seriously restricted the development of precise regulation of pig energy metabolism through this pathway. Therefore, analyzing this key miRNA and elucidating its regulation mechanism is crucial for developing new strategies for lipid metabolism regulation and improving pig breeding efficiency. SUMMARY

[0005] The present application aims to avoid the shortcomings in the prior art and provide the application of miR-320 gene in pig intestinal exosomes in regulating pig liver lipid metabolism. Based on the enhancement of miR-320 gene expression in pig liver tissue, the lipid deposition in pig liver can be effectively promoted.

[0006] To achieve the above-mentioned purpose, the first aspect:

[0007] Provided is an application of pig intestinal exosome miR-320 in regulating lipid deposition in pig liver, wherein the nucleotide sequence of the pig intestinal exosome miR-320 is 5'-AAAAGCTGGGTTGAGAGGGCGA-3',

[0008] By increasing the expression of pig intestinal exosome miR-320 gene, the lipid deposition in pig liver cells is promoted.

[0009] In some embodiments, the application comprises any one of the following:

[0010] The application of pig intestinal exosome miR-320 as a biomarker in regulating the potential of lipid deposition in pig liver cells, the expression level of miR-320 in pig intestinal exosome can be detected, combined with molecular biological verification of liver lipid deposition and correlation with lipid deposition related phenotypes, and early warning and grading evaluation indicators for pig liver lipid deposition are constructed by using bioinformatics means;

[0011] The application of pig intestinal exosome miR-320 in the function of regulating liver lipid, the expression abundance of miR-320 in pig intestinal exosome can be changed by gene intervention or nutrient regulation, so as to realize the targeted regulation of pig intestinal tract on liver lipid deposition;

[0012] The application of pig intestinal exosome miR-320 in pig precise nutrition supply and healthy feeding management, the application optimizes the regional supply mode of feed energy by coordinating the glycolipid metabolism mode of liver and improving pig feeding strategy through precise regulation of intestinal exosome miR-320;

[0013] The application of pig intestinal exosome miR-320 in assisting breeding molecular marker selection, the application assists the selection of metabolic stable type or better fat deposition line by constructing an innovative breeding evaluation system of pig intestinal exosome miR-320 expression and production traits, carcass quality and slaughter performance parameters;

[0014] The application of pig miR-320 in the research of human and mammalian liver lipid metabolism diseases, the application provides new insights and intervention target exploration for human or mammalian obesity, insulin resistance and liver lipid metabolism disorder research through the research of liver lipid deposition regulation participated by pig intestinal miR-320 in piglet model.

[0015] The second aspect:

[0016] An enhancer of pig liver miR-320 is provided, and the enhancer contains a mimic of pig liver cell miR-320.

[0017] In some embodiments, the sequence of the miR-320 mimic is as follows:

[0018] Sense sequence: 5'-AAAAGCTGGGTTGAGAGGGCGA-3',

[0019] Antisense sequence: 5'-GCCCUCUCAACCCAGCUUUUUU-3'.

[0020] The miR-320 mimic is an artificially synthesized double-stranded small RNA molecule, the sequence of which is designed to mimic the mature structure of endogenous miR-320 in cells and its function. The sequence of the sense strand is exactly the same as that of the natural mature miR-320 (5'-AAAAGCTGGGTTGAGAGGGCGA-3'), and the antisense strand is partially complementary to it. After enzymatic digestion in cells, it can be efficiently integrated into the RNA-induced silencing complex (RISC), thereby specifically recognizing and inhibiting the translation of target genes.

[0021] The miR-320 enhancer is an umbrella concept, referring to any substance or composition that can directly or indirectly increase the level or biological activity of functional miR-320 in cells.

[0022] The third aspect:

[0023] The application of the pig liver miR-320 enhancer described above is provided, including the application of any one of the following:

[0024] Application of pig liver miR-320 in promoting liver lipid accumulation;

[0025] Application of pig liver miR-320 as a molecular target for lipid deposition regulation in assisted molecular breeding markers;

[0026] Application of pig liver miR-320 in the development of drugs and targets for the regulation of pig liver lipid metabolism;

[0027] Application of pig liver miR-320 in the evaluation of the nutritional value of system animals and the optimization of energy allocation patterns of daily rations;

[0028] Application of pig liver miR-320 in the treatment of liver lipid metabolism diseases in model animals using piglets as models.

[0029] Compared with the prior art, the present application has the following advantages:

[0030] The application mainly illustrates the application of pig intestinal exosome miR-320 in regulating pig liver lipid deposition, for the first time, it is found that the expression of intestinal exosome miR-320 of pigs with different fat deposition types is different, and it is first clarified that the pig intestinal exosome miR-320 has a direct regulating effect on the lipid deposition of pig liver primary cells, wherein the high expression of miR-320 in pig intestinal exosome can promote the lipid deposition of pig liver cells, by transfecting the mimic of miR-320 into pig liver primary cells to enhance the expression level of miR-320 in pig liver cells, so as to confirm that it can directly promote the lipid deposition of pig liver cells. Based on the regulation of pig liver miR-320, especially pig intestinal exosome miR-320, it is a key means to realize the regulation of liver lipid deposition and the control of energy area deposition. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is the pig miR-320 expression result graph in different fat deposition types in the embodiment of the application, including

[0032] A graph: the expression abundance result of intestinal exosome miR-320 of obese blue-dang pigs and lean long-white pigs;

[0033] B graph: the expression abundance result of intestinal tissue miR-320 of obese blue-dang pigs and lean long-white pigs;

[0034] C graph: the expression abundance result of liver tissue miR-320 of obese blue-dang pigs and lean long-white pigs;

[0035] Lantang represents blue-dang pigs; Landrace represents long-white pigs; Lantang-EXO represents blue-dang pig intestinal exosome; Landrace-EXO represents long-white pig intestinal exosome; Relative miR-320 expression represents the relative expression level of miR-320; intestine represents the intestinal tract; liver represents the liver.

[0036] Figure 2 is the pig miR-320 target gene enrichment analysis result graph in the embodiment of the application, including A graph: the GO enrichment analysis result graph of pig miR-320 target genes;

[0037] B graph: the KEGG enrichment analysis result graph of pig miR-320 target genes.

[0038] Figure 3 is the pig intestinal exosome regulating function verification of pig liver primary cell lipid deposition in the embodiment of the application, including

[0039] Figure A: The results of lipid droplet oil red O staining of porcine primary hepatocytes after treatment with intestinal exosomes of obese Lantang pigs and lean Landrace pigs;

[0040] Figure B: The results of Western blot of porcine primary hepatocytes after treatment with intestinal exosomes of obese Lantang pigs and lean Landrace pigs;

[0041] Figure C: The results of miR-320 expression level of porcine primary hepatocytes after treatment with intestinal exosomes of obese Lantang pigs and lean Landrace pigs;

[0042] Figure D: The results of triglyceride content of porcine primary hepatocytes after treatment with intestinal exosomes of obese Lantang pigs and lean Landrace pigs;

[0043] OA / PA represents oleic acid / palmitic acid induction; LD-EVs represents Landrace pig intestinal exosomes; LT-EVs represents Lantang pig intestinal exosomes; TG content (mmol / g protion) represents triglyceride content (millimole / gram of protein).

[0044] Figure 4 Figure A: The results of lipid droplet oil red O staining of porcine primary hepatocytes after treatment with intestinal exosomes of obese Lantang pigs and lean Landrace pigs;

[0045] Figure B: The results of Western blot of porcine primary hepatocytes after treatment with intestinal exosomes of obese Lantang pigs and lean Landrace pigs;

[0046] Figure C: The results of miR-320 expression level of porcine primary hepatocytes after treatment with intestinal exosomes of obese Lantang pigs and lean Landrace pigs;

[0047] Figure D: The results of triglyceride content of porcine primary hepatocytes after treatment with intestinal exosomes of obese Lantang pigs and lean Landrace pigs;

[0048] OA / PA represents oleic acid / palmitic acid induction; NC represents the control group transfection group. DETAILED DESCRIPTION

[0049] Preferred embodiments of the present application will be described in more detail with reference to the drawings. Although the preferred embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present application is more thoroughly and completely conveyed to those skilled in the art, and the scope of the present application is fully conveyed to those skilled in the art.

[0050] Example 1

[0051] The research object of the embodiment is a differential analysis and comparison of miR-320 expression of pigs with different fat deposition types. The present example first discloses the change of miR-320 expression abundance in intestinal tissue, intestinal exosome and liver tissue of 3-day-old obese blue pond pigs and lean long white pigs, finds that the expression of pig miR-320 is related to the lipid deposition phenotype, and deduces that miR-320 may be a key signal molecule for mediating intestinal-liver lipid deposition regulation through molecular biology detection, and the steps include:

[0052] The present application verifies the expression of miR-320 in each tissue of pigs with different fat deposition types by RT-qPCR technology. First, the intestinal exosome of pigs with different fat deposition types separated by ultracentrifugation is harvested, and the expression abundance of miR-320 is determined, and the figure A in Figure 1 indicates that there is a significant difference in the expression of miR-320 in the intestinal exosome of pigs with different fat deposition types, and the abundance of miR-320 in the intestinal exosome of blue pond pigs is higher than that of long white pigs. The donor tissue of the intestinal exosome is then detected to evaluate the loading performance of miR-320 in the intestinal exosome, and the figure B in Figure 1 indicates that the expression level of miR-320 in the intestinal tissue of obese blue pond pigs is significantly higher than that of lean long white pigs, which is consistent with the change of miR-320 expression in the intestinal exosome, suggesting that the differential expression of miR-320 in the intestinal exosome may be related to the difference of miR-320 at the intestinal level. Finally, the expression abundance of miR-320 in the liver is detected, and the figure C in Figure 1 indicates that there is a difference in the expression of miR-320 in the liver of pigs with different fat deposition types, and the expression level of miR-320 in the liver of obese blue pond pigs is significantly higher than that of lean long white pigs.

[0053] The present embodiment detects the expression abundance of miR-320 in the intestinal exosome, intestinal tissue and liver of pigs with different fat deposition types, and points out that miR-320 may be a key signal molecule for intestinal exosome-mediated intestinal-liver signal transmission.

[0054] Example 2

[0055] To further explore the biological function that the differentially expressed miR-320 found in example 1 may be involved in, the present embodiment is based on example 1:

[0056] The research object of the present embodiment is the prediction of the metabolic regulation function of pig miR-320. The target genes of miR-320 in Example 1 are screened (TargetScan_stcore>50 and Miranda-Energy<-10) by TargetScan and Miranda databases and GO and KEGG enrichment analysis is performed to explore the lipid metabolism function of pig miR-320 involved in different fat deposition types. Through GO enrichment analysis of differentially expressed miR-320, it is found that these genes that may have a target relationship with miR-320 are related to lipid metabolism regulation, as shown in FIG. A of Figure 2 Through KEGG enrichment analysis of differentially expressed miR-320, it is found that these genes that may have a target relationship with miR-320 are involved in the regulation of insulin signaling and AMPK signaling pathways, indicating that miR-320 is a key element of lipid metabolism regulation, as shown in FIG. B of Figure 2

[0057] Example 3

[0058] To further illustrate the regulation function of pig intestinal exosomes on lipid deposition of pig primary liver cells, Example 3 is carried out on the basis of Example 2:

[0059] The research object of the present embodiment is the regulation function of pig intestinal exosomes of different fat deposition types on lipid deposition of pig primary liver cells. In the present embodiment, OA (250 μM) / PA (125 μM) mixed treatment is used to induce fatty degeneration of pig liver cells, and 10 μg / mL of LT-EVs or LD-EVs is added to the culture system, respectively, and treated for 48 h. Through oil red O staining of lipid droplets in pig primary liver cells stimulated by intestinal exosomes of different fat deposition types, it is found that the intestinal exosomes of lean Landrace pigs have no regulation function on lipid deposition of pig primary liver cells induced by OA / PA, but the intestinal exosomes of obese Lantang pigs significantly promote lipid accumulation in pig primary liver cells, as shown in FIG. A of Figure 3 Through detection of lipid transport key protein CD36 in pig primary liver cells, it is found that the intestinal exosomes of obese Lantang pigs significantly promote the expression of liver lipid transport key protein CD36, while the intestinal exosomes of lean Landrace pigs have no function, as shown in FIG. B of Figure 3 To further verify whether the intestinal exosome miR-320 in Example 1 and Example 2 is an intermediate medium for intestinal-liver signal transmission, the expression of miR-320 in pig primary liver cells treated by intestinal exosomes of different fat deposition types is detected, and Figure 3 ​Figure C in the drawings of the present application, it is found that the intestinal exosomes of the obese Landrace pigs significantly promote the expression of miR-320 in the pig liver cells. Finally, the effect of the intestinal exosomes of pigs with different fat deposition types on the synthesis of triglycerides after the treatment of the primary liver cells is detected, and it is obtained that Figure 3 Figure D in the drawings of the present application, consistent with the changes of Figures A and B, the intestinal exosomes of the obese Landrace pigs significantly promote the accumulation of triglycerides in the primary pig liver cells. In combination with Example 1 and Example 2, it is illustrated that the signal transmission of miR-320 mediated by the pig intestinal exosomes between the intestine and the liver can be the key molecule for the regulation of lipids.

[0060] Example 4

[0061] In order to finally confirm that the lipid deposition phenotype observed in Example 3 is directly mediated by the miR-320 carried by the exosomes, the present example is carried out on the basis of Example 3:

[0062] The research object of the present example is the application of the miR-320 mimic in the regulation of lipid deposition of the OA / PA-induced pig primary liver cells. In combination with Example 3, the present example realizes the overexpression of miR-320 in the pig primary liver cells by using the miR-320 mimic under the condition of OA / PA induction, so as to verify the role of miR-320 in the regulation of lipid deposition of the pig primary liver cells, and it is illustrated that the change of miR-320 in the pig liver tissue can realize the differential regulation of liver lipid deposition, including:

[0063] Transfection of pig primary liver cells: after the growth and fusion of the pig primary liver cells, the pig primary liver cells are transfected by using the RfectPMV2 siRNA primary cell transfection reagent.

[0064] The transfection sequence of the control group is:

[0065] The sense sequence is 5'-UUCUCCGAACGUGUCACGUTT-3';

[0066] The antisense sequence is 5'-ACGUGACACGUUCGGAGAATT-3',

[0067] The mimic sequence of miR-320 is:

[0068] The sense sequence is 5'-AAAAGCTGGGTTGAGAGGGCGA-3';

[0069] The antisense sequence is 5'-GCCCUCUCAACCCAGCUUUUUU-3'.

[0070] In the present example, the miR-320 mimic is transfected into the pig primary liver cells after the induction of OA / PA for the first time, and the lipid droplets of the transfected pig primary liver cells are subjected to oil red O staining, and it is obtained thatFigure 4 Figure A in the figure, it was found that the number of lipid droplets in the pig primary liver cells treated by pig miR-320 mimics increased significantly. In combination with the change of CD36 protein in Example 3, the high-fat environment provided by OA / PA may promote the lipid transport of liver cells, and then we detected the expression of CD36 in the transfected pig primary liver cells, and obtained Figure 4 Figure B in the figure, it was found that pig miR-320 mimics significantly promoted the expression of CD36 in pig primary liver cells, indicating that pig miR-320 mimics promoted the lipid transport of liver cells. Finally, the level of triglyceride in pig primary liver cells treated by pig miR-320 mimics was detected, and obtained Figure 4 Figure C in the figure, it was found that pig miR-320 mimics significantly increased the level of triglyceride in pig primary liver cells.

[0071] In this embodiment, the lipid metabolism regulation function of miR-320 in pig liver primary cells was determined, the relationship between the expression of miR-320 and lipid deposition in pig liver cells was explored, the overexpression of miR-320 in pig liver primary cells was realized by transfecting the mimics of miR-320 into pig liver primary cells, and it was confirmed that miR-320 in pig primary liver cells had the function of promoting the lipid deposition of liver cells by combining oil red O, triglyceride content and Western blot.

[0072] In combination with examples 1 to 4, the present application determines the expression of miR-320 in the intestinal exosomes, intestines and liver tissues of obese blue and lean Landrace pigs, and performs GO and KEGG function enrichment analysis on the target genes by bioinformatics analysis, determines that miR-320 is involved in lipid metabolism regulation, and the high expression of miR-320 in the intestinal exosomes of blue pigs may be a new way of lipid deposition regulation. To explore whether the intestinal exosomes are involved in the regulation of liver lipid deposition and the relationship between the intestinal exosomes and miR-320, the high-fat environment of porcine primary hepatocytes is induced by OA / PA, and different fat deposition type pig intestinal exosomes are added, combined with oil red O staining, CD36 Western blot analysis and triglyceride content determination, it is confirmed that the pig intestinal exosomes promote the lipid deposition of porcine primary hepatocytes. In addition, according to the RT-qPCR, the expression of miR-320 in the porcine primary hepatocytes treated by the exosomes is detected, which confirms that the exosome treatment can effectively improve the expression level of miR-320 in the porcine primary hepatocytes, which indicates that the intestinal exosome-mediated miR-320 transmission between the intestine and the liver may be a new way of lipid deposition regulation. To further reveal the function of pig miR-320 in the regulation of liver lipid deposition, the miR-320 mimic is transfected into the porcine primary hepatocytes, combined with oil red O staining, CD36 Western blot analysis and triglyceride content determination, it is confirmed that miR-320 is a key miRNA for regulating pig liver lipid deposition. The finding has important scientific significance for regulating liver lipid deposition, optimizing feed energy supply, improving carcass quality and improving pig production efficiency.

[0073] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Application of porcine intestinal exosome miR-320 in regulating lipid deposition in pig liver, characterized in that, The nucleotide sequence of the pig intestinal exosome miR-320 is 5'-AAAAGCTGGGTTGAGAGGGCGA-3', The pig intestinal exosome miR-320 promotes the lipid deposition of pig liver cells by increasing the expression of the pig intestinal exosome miR-320 gene.

2. Use according to claim 1, characterized in that, The application includes any of the following: The pig intestinal exosome miR-320 is used as a biomarker for regulating the potential of piglet liver cell lipid deposition, and the expression profile of pig intestinal miR-320 is detected to construct an early warning and grading evaluation index for piglet liver lipid deposition; The pig intestinal exosome miR-320 is used in the application of liver lipid regulation function, and the loading level of miR-320 in the exosome is regulated by gene / nutrition intervention means to realize the targeted regulation of piglet intestinal tract on liver lipid deposition; The pig intestinal exosome miR-320 is used in the application of pig precise nutrition supply and healthy feeding management, the expression abundance of intestinal exosome miR-320 is regulated to realize the production target individualized lipid deposition regulation, optimize the feed energy supply mode, and assist in improving pig feeding strategy; The pig intestinal exosome miR-320 is used in the application of auxiliary breeding molecular marker selection, and a trinity innovation breeding evaluation system of pig intestinal exosome miR-320, production traits, carcass quality and slaughter performance is constructed to assist in breeding metabolic robust type or better fat deposition line; The pig miR-320 is used in the application of human and mammalian liver lipid metabolism disease research, the regulation of liver lipid deposition involving intestinal miR-320 in piglet model is researched to provide new insights and intervention target exploration for human or mammalian obesity, insulin resistance and liver lipid metabolism disorder regulation research.

3. An enhancer of porcine liver miR-320, characterized in that, The enhancer contains a mimic of pig liver cell miR-320.

4. The enhancer of pig liver miR-320 according to claim 3, characterized in that, The sequence of the mimic of miR-320 is as follows: The positive sequence is 5'-AAAAGCTGGGTTGAGAGGGCGA-3', The negative sequence is 5'-GCCCUCUCAACCCAGCUUUUUU-3'.

5. Use of porcine liver miR-320, characterized in that, The pig liver miR-320 uses the enhancer of the pig liver miR-320 as claimed in claim 3 or 4, and the application includes any of the following applications: The pig liver miR-320 is used in the application of promoting liver lipid accumulation; The pig liver miR-320 is used as a molecular target for lipid deposition regulation in the application of auxiliary molecular breeding markers; The pig liver miR-320 is used in the development of drugs and targets for pig liver lipid metabolism regulation; the pig liver miR-320 is used in the evaluation of system animal nutrition value and the optimization of dietary energy allocation mode; the pig liver miR-320 is used in the treatment of piglet model animal liver lipid metabolism disease targets.

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