Substance for regulating expression of ATP7A gene in inhibition of adipogenic differentiation of porcine intramuscular precursor adipocyte

Abstract

The application discloses application of a substance for regulating expression of an ATP7A gene to inhibition of adipogenic differentiation of porcine intramuscular precursor adipocytes and belongs to the technical field of screening of genes related to adipogenic differentiation of precursor adipocytes. The application finds that the expression amount of ATP7A is significantly reduced after overexpression of miR-149-5p through RNA-seq, and finds that ATP7A is a target gene of miR-149-5p through online website prediction and double luciferase detection, and successfully identifies a porcine intramuscular fat (IMF) regulatory gene ATP7A (GeneID: 397159). The discovery of the gene provides a new theoretical basis for regulation of adipogenic differentiation of porcine intramuscular precursor adipocytes and provides an important reference for improvement of pork quality.

Description

Technical Field

[0001] This invention relates to the technical field of porcine intramuscular adipogenesis-related genes, and more specifically to the application of the ATP7A gene in inhibiting adipogenic differentiation of porcine intramuscular preadipocytes. Background Technology

[0002] Pork is the economic foundation of animal husbandry and a vital source of animal protein, energy, and iron for humans. Increasing research indicates that pork accounts for over 40% of global meat consumption. As living standards improve, people's demands for pork quality are also rising. Therefore, raising pork standards is of paramount importance to the pig farming industry. Essentially, pig breed influences fat and muscle growth, and IMF (intramuscular fat content) is highly correlated with pork quality. The sensory quality of meat is largely influenced by its IMF content, which is closely related to pork's flavor, tenderness, juiciness, water-holding capacity, and marbling.

[0003] IMF (Internal Fat Marrow) is a type of fat found in the perimuscular tunica albuginea, fasciculus, and endomuscular tunica albuginea. Compared to other adipose tissues in the body, IMF development is the most recent, and it contains more triglyceride phosphates, phospholipids, and cholesterol. Phospholipids are important compounds affecting the texture of meat products; they, along with triglycerides, constitute IMF, which is mainly composed of C:16 and C:18 fatty acids. IMF not only disrupts the cross-linking structure between muscle fiber bundles but also weakens the strength of connective tissue, thereby reducing muscle shear force and improving the tenderness of meat products. Simultaneously, as IMF content increases, the number of muscle fibers per unit area decreases, thus affecting the tenderness of meat products. The flavor of meat products is also significantly affected by fat decomposition. During processing, fat can break down into volatile substances such as aliphatic hydrocarbons, aldehydes, ketones, alcohols, esters, and carboxylic acids. Besides directly affecting the flavor of meat products through volatilization, these chemicals can also mix with other compounds to produce flavor compounds, further influencing the flavor of meat products. The quality of pork is determined by the types and percentages of fatty acids, which can serve as a benchmark for judging the quality of other meats. Monounsaturated and saturated fatty acids can improve meat quality, while polyunsaturated fatty acids have the opposite effect. Pork quality is a complex attribute, influenced by genetic factors as well as environmental, management, feeding, and slaughtering conditions. Therefore, understanding the mechanisms of intramuscular fat deposition in animals is crucial for improving meat quality.

[0004] miRNAs (microRNAs) are non-coding single-stranded RNAs approximately 22 nucleotides long that downregulate target gene expression primarily by acting on target mRNAs in two ways: (1) miRNAs typically bind to the 3'UTR (3' untranslated region) sequence of the mRNA target to degrade or inhibit translation; (2) they can also bind to unconventional targets in the 5'UTR (5' untranslated region) and CDS to block translation. miRNAs have been identified as important post-transcriptional regulators of gene expression, influencing many physiological and pathological processes, including development, proliferation, differentiation, and immune responses. Numerous studies have shown that many miRNAs, such as miR-122, miR-335, miR-27, miR-370, and miR-30, can act as important regulators in lipid metabolism and fat deposition.

[0005] MiR-149 is a novel miRNA discovered in recent years, located in intron 1 of the phosphatidylinositol glycan-1 (GPC1) gene. Its precursor produces two active strands, miR-149-5p and miR-149-3p, which exert their biological effects by acting on their respective target genes. Mohamed's research found that miR-149 inhibits PARP-2D expression in skeletal muscle through the SIRT-1 / PGC-1α network and promotes mitochondrial biogenesis. miR-149-3p regulates the transition between adipogenic and osteogenic differentiation of bone marrow mesenchymal stem cells by targeting FTO. MiR-149 regulates non-alcoholic fatty liver disease by targeting FGF-21 and reduces endoplasmic reticulum stress-induced inflammation and apoptosis in non-alcoholic fatty liver disease by negatively regulating ATF6. Studies by Liu et al. showed that miR-149-5p promotes the proliferation of porcine intramuscular preadipocytes and 3T3-L1 cells while inhibiting their differentiation.

[0006] Therefore, how to discover the target gene of miR-149-5p and apply it to inhibit intramuscular fat formation in pigs is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention discovered the downstream target gene ATP7A of miR-149-5p and found the role of this gene in inhibiting the adipogenic differentiation of porcine intramuscular preadipocytes.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] Application of substances that regulate ATP7A gene expression in inhibiting adipogenic differentiation of porcine intramuscular preadipocytes.

[0010] Preferably, the substance regulating ATP7A gene expression includes miR-149-5p, which inhibits ATP7A gene expression.

[0011] As with the same inventive concept as the above-mentioned technical solution, the present invention also seeks protection for the application of the ATP7A gene in inhibiting the adipogenic differentiation of porcine intramuscular preadipocytes, wherein the ATP7A gene sequence is shown in SEQ ID NO.1 and the accession number is Gene ID:397159.

[0012] ATP7A gene sequence:

[0013]

[0014] Preferably, the adipogenic differentiation of porcine intramuscular preadipocytes is inhibited by suppressing or knocking out the expression of the ATP7A gene.

[0015] As having the same inventive concept as the above-mentioned technical solution, the present invention also seeks protection for the use of miR-149-5p in regulating ATP7A gene expression.

[0016] Preferably, miR-149-5p inhibits the expression of the ATP7A gene by targeting and binding to the 3'UTR of the ATP7A gene.

[0017] As an inventive concept with the same technical solution as above, this invention also claims protection for the application of miR-149-5p targeting and binding to the ATP7A gene in inhibiting adipogenic differentiation of porcine intramuscular preadipocytes.

[0018] Preferably, overexpression of miR-149-5p decreases ATP7A expression, thereby inhibiting adipogenic differentiation of porcine intramuscular preadipocytes.

[0019] As described above, compared with existing technologies, this invention utilizes RNA-seq technology to screen key genes affecting intramuscular fat formation in porcine intramuscular preadipocytes after overexpression of miR-149-5p. Previous studies have shown that overexpression of miR-149-5p significantly inhibits adipogenic differentiation of porcine intramuscular preadipocytes. RNA-seq revealed that the expression level of ATP7A was significantly reduced after overexpression of miR-149-5p, and online prediction and dual-luciferase assays confirmed that ATP7A is a target gene of miR-149-5p, successfully identifying the porcine intramuscular fat regulatory gene ATP7A (GeneID: 397159). The discovery of this gene provides a new theoretical basis for the regulation of adipogenic differentiation of porcine intramuscular preadipocytes and offers important reference for improving pork quality.

[0020] Meanwhile, this invention demonstrated through overexpression and interference of miR-149-5p in porcine intramuscular preadipocytes that ATP7A is a target gene of miR-149-5p, and that ATP7A is a Cu 2+ Transporter proteins, which may affect Cu 2+ The transport of ATP7A affects the formation of intramuscular fat in pigs. Meanwhile, the expression level of ATP7A in the longissimus dorsi and leg muscles of local pigs is significantly higher than that in foreign pigs. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 The attached figure shows the relative expression level of ATP7A in porcine intramuscular preadipocytes after adipogenic differentiation following overexpression of miR-149-5p, as detected by qRT-PCR.

[0023] Figure 2 The attached figure shows the differences in Oil Red O staining during adipogenic differentiation of porcine intramuscular preadipocytes after overexpression and interference with miR-149-5p; where A and B: Oil Red O staining of porcine intramuscular preadipocytes 8 days after induction of differentiation; C and D: Quantitative analysis of Oil Red O staining results (* represents P<0.05 and ** represents P<0.01).

[0024] Figure 3 The attached figures show the results of verifying that miR-149-5p regulates ATP7A to inhibit adipogenic differentiation of porcine intramuscular preadipocytes; A: Binding site of miR-149-5p to ATP7A 3'UTR; B: Dual-luciferase reporter assay results; C: Relative expression level of miR-149-5p after overexpression in porcine intramuscular preadipocytes; D: Relative expression level of ATP7A in porcine intramuscular preadipocytes on day 8 after induction of differentiation; E: Relative expression level of miR-149-5p after interference in porcine intramuscular preadipocytes; F: Relative expression level of ATP7A in porcine intramuscular preadipocytes on day 8 after induction of differentiation; G: Relative expression level of ATP7A in the longissimus dorsi and leg muscles of Queshan Black pigs compared with Large White pigs;

[0025] Figure 4 The attached figure shows the results of verifying the inhibition of adipogenic differentiation of porcine intramuscular preadipocytes by interfering with ATP7A; A: Detection of the efficiency of ATP7A interference during the differentiation of porcine intramuscular preadipocytes; B: Relative expression levels of adipogenic differentiation marker genes of porcine intramuscular preadipocytes after interfering with ATP7A; C: Quantitative analysis of the state of porcine intramuscular preadipocytes and lipid droplet area 8 days after induction of differentiation. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] In an embodiment of the present invention, porcine intramuscular preadipocytes were isolated from the longissimus dorsi muscle of Queshan black pigs, transfected with miR-149-5p, and induced to differentiate into adipocytes. Transcriptomic analysis was conducted to identify genes with lower expression levels in the miR-149-5p overexpression group compared to the control group, and the molecular characteristics of porcine intramuscular preadipocytes were analyzed, as detailed in the following embodiments.

[0028] Example 1: Isolation, culture, and induced differentiation of porcine intramuscular preadipocytes

[0029] The longissimus dorsi muscle of 3-day-old piglets was collected under aseptic conditions. The piglet skin was washed with warm water and disinfected with 75% alcohol. The tissue was immersed in 75% alcohol twice and washed three times in PBS (2% penicillin-streptomycin) at 37°C. Blood vessels, fascia, and other non-muscle tissues were removed in PBS using ophthalmic scissors, and the tissue was cut into pieces approximately 1 mm thick. 3 Tissue blocks were collected. Using a cut pipette tip, the tissue blocks were transferred to 50 mL sterile centrifuge tubes. Then, twice the volume of the tissue blocks were incubated in a 37°C water bath for 90 min with 0.2% type I collagenase, 0.1% bovine serum albumin, and 2% penicillin-streptomycin (prepared in DMEM), shaking the centrifuge tube several times during this period. Digestion was terminated with an equal volume of DMEM containing 10% fetal bovine serum, and the cells were filtered sequentially through 75, 100, 200, and 400 mesh cell sieves into 100 mL beakers. The filtrate was centrifuged at 1500 rpm for 10 min and washed three times in culture medium. After resuspending, the cells were seeded into T25 cell culture flasks and cultured at 37°C and 5% CO2 for 1.5 h. The supernatant was discarded, and the cells were washed twice with PBS at 37°C (at this point, the flasks mainly contained porcine intramuscular adipocytes). The culture medium was changed for the first time after 48 h, and then replaced with complete culture medium (20% FBS) for the next 48 h. When cells reached approximately 80% confluence, they were transfected with miR-149-5p mimics or NC. After confluence, differentiation was induced after 2 days of contact inhibition. Subsequent experiments were performed using cells transfected with induced differentiation. RNA-seq sequencing was performed on cells transfected with miR-149-5p mimics and NC to screen for genes expressed at lower levels in the miR-149-5p mimics group compared to the control group (|log2-fold change|>log2 1.2, P<0.05). RNA-seq results are shown below. Figure 1It can be seen that the expression level of ATP7A gene in the miR-149-5p mimics group is significantly lower than that in the NC group, so it is speculated that the target gene of miR-149-5p may be ATP7A.

[0030] Induction of differentiation of porcine intramuscular preadipocytes: After the cells reached confluence, differentiation was induced by contact inhibition for 2 days, and then the medium was changed to adipogenic induction medium. After 2 days, the medium was changed to adipogenic maintenance medium. The medium was changed every 2 days until differentiation was induced for 8 days (Table 1).

[0031] Table 1. Preparation of culture medium

[0032] 10% FBS 10% FBS 10% FBS 89% DMEM / High Sugar 89% DMEM / High Sugar 89% DMEM / High Sugar 1% penicillin-streptomycin <![CDATA[5ug·mL -1 Insulin <![CDATA[5ug·mL -1 Insulin <![CDATA[1umol·L -1 DEX]]> 1% penicillin-streptomycin <![CDATA[0.5mmol·L -1 IBMX]]> 1% penicillin-streptomycin

[0033] Example 2: MiR-149-5p significantly inhibited adipogenic differentiation of porcine intramuscular preadipocytes.

[0034] Previous studies have shown that overexpression and interference with miR-149-5p induce differentiation in porcine intramuscular preadipocytes. Cells were collected 8 days after differentiation, stained with Oil Red O, and the staining results were quantified. The results showed that, compared with the control group, the miR-149-5p overexpression group had a significantly reduced lipid droplet formation (P<0.01). Figure 2 A, C) indicate that overexpression of miR-149-5p inhibits adipogenic differentiation of porcine intramuscular preadipocytes; the group interfering with miR-149-5p produced more lipid droplets (P<0.01, Figure 2 B, D) indicates that interference with miR-149-5p promotes adipogenic differentiation of porcine intramuscular preadipocytes.

[0035] Example 3: MiR-149-5p regulates ATP7A to inhibit adipogenic differentiation of porcine intramuscular preadipocytes

[0036] To elucidate the mechanism by which miR-149-5p affects adipogenic differentiation of porcine intramuscular preadipocytes, transcriptomic analysis was performed on porcine intramuscular preadipocytes overexpressing miR-149-5p. The transcriptomic analysis showed that ATP7A expression was significantly decreased after miR-149-5p overexpression (P<0.05). The presence of a binding site between miR-149-5p and ATP7A was predicted using the RNA22v2 online website. Figure 3 A) Therefore, it is speculated that miR-149-5p and ATP7A have target binding sites. Dual-luciferase reporter assay results showed that when miR-149-5p mimics were co-transfected with the ATP7A wild-type vector, firefly luciferase activity was significantly reduced (P<0.01). Figure 3 B), however, transfection with NC or co-transfection with the ATP7A mutant vector and miR-149-5pmimics did not affect firefly luciferase activity (P>0.05). Figure 3 B). The results indicate that miR-149-5p and ATP7A have a targeted binding relationship. ATP7A is a Cu 2+ Transporter proteins may affect Cu 2+ It influences the formation of intramuscular fat through transport.

[0037] To further verify the targeted inhibitory effect of miR-149-5p on ATP7A, miR-149-5p mimics and miR-149-5p inhibitors were transfected into porcine intramuscular preadipocytes. After 8 days of adipogenic differentiation, cells were collected, and the expression levels of miR-149-5p and ATP7A were detected using qRT-PCR. The results showed that during adipogenic differentiation, the miR-149-5p mimics group significantly increased miR-149-5p expression (P<0.01). Figure 3 C), and overexpression of miR-149-5p significantly reduced the expression of ATP7A in porcine intramuscular preadipocytes (P<0.01, Figure 3 D). To verify the targeting relationship between miR-149-5p and ATP7A, miR-149-5p was interfered with. The miR-149-5p inhibitor group significantly reduced miR-149-5p expression (P<0.01). Figure 3 E), miR-149-5p inhibitors significantly increased ATP7A expression in porcine intramuscular preadipocytes (P<0.05, Figure 3 F) indicates that ATP7A is a target gene of miR-149-5p. Furthermore, compared to introduced pigs, the expression levels of ATP7A in the longissimus dorsi and leg muscles of Queshan black pigs were significantly increased (P<0.01). Figure 3 G).

[0038] To investigate the effect of ATP7A on porcine intramuscular preadipocytes, siATP7A was transfected into porcine intramuscular preadipocytes. Cells were collected 8 days after adipogenic differentiation, and the expression levels of ATP7A and adipogenic differentiation marker genes (CEBPA, PPARG, FABP4, PLIN1, and LIPE) were detected using qRT-PCR. The results showed that the siATP7A group significantly inhibited ATP7A expression (P<0.01). Figure 4 A), and the expression levels of adipogenic differentiation marker genes were significantly reduced after ATP7A interference (P<0.05). Figure 4 B); This experiment examined the cell status 8 days after differentiation and quantified the lipid droplet area. The results showed that the number of lipid droplets in the siATP7A group was significantly reduced (P<0.05). Figure 4 C) The results showed that interfering with ATP7A inhibited the adipogenic differentiation of porcine intramuscular preadipocytes.

[0039] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0040] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of miR-149-5p in inhibiting adipogenic differentiation of porcine intramuscular preadipocytes, characterized in that, miR-149-5p inhibits the expression of the ATP7A gene, the sequence of which is shown in SEQ ID NO.

1.

2. Application of miR-149-5p targeting and binding to the ATP7A gene in inhibiting adipogenic differentiation of porcine intramuscular preadipocytes, wherein the ATP7A gene sequence is shown in SEQ ID NO.

1.

3. The application according to claim 2, characterized in that, Overexpression of miR-149-5p decreases the expression of the ATP7A gene, thereby inhibiting the adipogenic differentiation of porcine intramuscular preadipocytes.

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