Method for synthesizing BMECs casein by regulating leucine through DDIT3 gene

Abstract

The invention relates to the technical field of animal nutrition science, and discloses a method for synthesizing casein of primary bovine mammary gland cells (BMECs) by regulating leucine through a DDIT3 gene, which comprises the following steps: S1, preparation and pretreatment of primary BMECs; s2, the pretreated primary BMECs are subjected to grouped culture, a control group, an essential amino acid (EAA) lack model group and a leucine treatment group are set, and a culture medium which does not contain all EAA and is supplemented with leucine is adopted in the leucine treatment group for culture; s3, regulating and controlling DDIT3 gene expression and combining a signal channel formed by upstream and downstream genes to realize regulation and control on BMECs casein synthesis. By supplementing leucine with a specific concentration in an EAA lacking environment and combining pathway regulation and control, beta-casein synthesis of BMECs is accurately activated, the daily ration nitrogen utilization efficiency is effectively improved, and the breeding cost and the environmental pressure are reduced.

Description

Technical Field

[0001] This invention relates to the field of animal nutrition science and technology, specifically to a method for regulating BMECs casein synthesis via the DDIT3 gene regulation of leucine. Background Technology

[0002] Milk protein synthesizers (BMECs) are the primary sites for the synthesis and secretion of milk proteins in lactating cows. β-casein is one of the most abundant and nutritionally valuable components of milk protein, and its synthesis efficiency directly determines milk quality and yield. Leucine, as an essential amino acid (EAA), is not only a substrate for protein synthesis but also an important signaling molecule. Currently, dairy farming relies heavily on high-protein diets to maintain milk production. However, research shows that dairy cows have low nitrogen utilization efficiency in their diets, with only about 25% of dietary nitrogen converted into milk protein. The majority is excreted as fecal and urinary nitrogen, resulting in significant waste of feed resources and severe environmental nitrogen pollution. To address this issue, developing low-protein feeds for dairy cows and improving nitrogen utilization efficiency after absorption has become an urgent need for the industry. The core strategy lies in reducing dietary crude protein levels while precisely supplementing with limiting EAAs to maintain milk protein synthesis capacity.

[0003] Leucine has a good stimulating effect on the milk protein synthesis pathway, but the key gene targets that regulate the efficient synthesis of β-casein under low EAA conditions need to be further explored. The relevant results provide important reference for the development of low-protein amino acid balanced diets for dairy cows. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for regulating casein synthesis in breast cancer cells via the DDIT3 gene using leucine. It identifies and intervenes in key molecular targets that regulate casein synthesis under EAA-deficient conditions, thereby promoting the efficient utilization of amino acids in breast cells.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for regulating BMECs casein synthesis via leucine regulation using the DDIT3 gene, comprising the following steps:

[0006] S1. Preparation and pretreatment of primary BMECs;

[0007] S2. The pretreated primary BMECs were cultured in groups, including a control group, an EAA-deficient model group, and a leucine treatment group. The leucine treatment group was cultured in a medium that did not contain any EAA and was supplemented with leucine.

[0008] S3. By regulating the expression of the DDIT3 gene and combining the signaling pathways formed by upstream and downstream genes, efficient regulation of casein synthesis in BMECs can be achieved.

[0009] S4. Detect casein synthesis-related indicators, cell proliferation indicators, apoptosis indicators, cell cycle indicators, and intracellular amino acid concentrations in BMECs.

[0010] Preferably, the preparation and pretreatment of primary BMECs in step S1 includes the following steps:

[0011] Primary BMECs were isolated from the mammary gland tissue of healthy dairy cows, and after digestion, filtration, centrifugation, and collection, they were inoculated into culture dishes and passaged to the 3rd generation.

[0012] Third-generation primary BMECs were seeded into culture dishes. When the cells reached a confluence of 70%-80%, they were replaced with serum-free medium and starved for 12 hours.

[0013] Preferably, the culture medium used in steps S1 and S2 is DMEM-F12 culture medium, which contains 1% L-glutamine, 10% fetal bovine serum, 5 μg / mL transferrin, 5 μg / mL insulin, 1 μg / mL hydrocortisone, 10 ng / mL epidermal growth factor and 1% penicillin-streptomycin mixture.

[0014] Preferably, during the group culture in step S2, the control group is cultured in standard DMEM-F12 medium containing all EAAs, the EAA-deficient model group is cultured in DMEM-F12 medium without all EAAs, and the leucine treatment group is cultured in DMEM-F12 medium without all EAAs and supplemented with leucine. The concentration of leucine added is 1, 4, 8, 12, 24, 36, 48, or 60 times the standard leucine concentration in DMEM-F12 medium.

[0015] Preferably, the leucine concentration in the leucine treatment group is 24 times the standard leucine concentration in DMEM-F12 medium. All three groups are cultured for 6 hours at 37°C, 5% carbon dioxide, and 95% humidity, with 6 biological replicates in each group.

[0016] Preferably, the specific method for regulating DDIT3 gene expression in step S3 is siRNA transfection. The siRNA is a specific siRNA targeting the DDIT3 gene. The specific siRNA is siRNA-453. The nucleotide sequence of the sense strand of siRNA-453 is shown in SEQ ID NO:1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:2.

[0017] The transfection process involves mixing the specific siRNA with the CALNP™ RNAi transfection reagent, incubating at room temperature for 20 minutes to form a transfection complex, adding it to primary BMECs in the logarithmic growth phase, and verifying the silencing efficiency 24 hours after transfection.

[0018] Preferably, the regulation process in step S3 further includes the regulation of the ATF4 gene and the TRIB3 gene, wherein the specific siRNA for the ATF4 gene is siRNA-662, the sense strand nucleotide sequence of which is shown in SEQ ID NO:3 and the antisense strand nucleotide sequence of which is shown in SEQ ID NO:4; the specific siRNA for the TRIB3 gene is siRNA-1149, the sense strand nucleotide sequence of which is shown in SEQ ID NO:5 and the antisense strand nucleotide sequence of which is shown in SEQ ID NO:6;

[0019] The regulation of the ATF4 and TRIB3 genes was achieved through siRNA transfection. The transfection process involved mixing the corresponding specific siRNA with CALNP™ RNAi transfection reagent in a certain proportion, incubating at room temperature for 20 minutes to form a transfection complex, and then adding it to primary BMECs in the logarithmic growth phase. The silencing efficiency was verified 24 hours after transfection.

[0020] Preferably, the intracellular amino acid concentration detection in step S4 employs targeted metabolomics analysis, including the following steps:

[0021] Cells from each group were collected, washed twice with pre-cooled PBS, and then lysed in an ice bath by sonication with a 4:1 (v / v) mixture of methanol and water. After centrifugation at 12000×g for 10 minutes at 4°C, the supernatant was filtered through a 0.22 μm organic phase membrane. Separation was performed using an Agilent 1290 Infinity LCU HPLC system. Mobile phase A was an aqueous solution containing 25 mmol / L ammonium formate and 0.1% formic acid, and mobile phase B was an acetonitrile solution containing 0.1% formic acid. Quantitative detection was performed using mass spectrometry in electrospray ionization positive / negative ion mode and multiple reaction monitoring (MRM) scanning mode.

[0022] Preferably, in step S4, casein synthesis-related indicators are detected by Western blotting, and the proteins detected include β-casein, mTOR, phosphorylated mTOR, S6K1, and phosphorylated S6K1, with β-actin as an internal reference protein. Cell proliferation indicators are detected by CCK-8 assay, and apoptosis indicators are detected by Annexin V-FITC / PI double staining combined with the GuavaEasyCyteHT system. Before detection, cells are washed twice with pre-cooled PBS and then suspended in 0.1 mL binding buffer, with 10 μL of FITC-labeled Annexin V and 5 μL of LPI added, and incubated in the dark for 15 minutes. Cell cycle indicators are detected by PI staining combined with the GuavaEasyCyteHT system. Before detection, cells are fixed in 75% ethanol at 4°C for 12 hours, and then resuspended in PBS containing 5 μg / mL PI and cultured in the dark for 10 minutes.

[0023] Preferably, the method further includes a bioinformatics analysis step, specifically including:

[0024] Total RNA was extracted from cells in the control group, EAA-deficient model group, and leucine-treated group at concentrations of 4, 8, 12, and 24 times the standard concentration, and transcriptome sequencing was performed to screen for differentially expressed genes.

[0025] We constructed gene co-expression modules using weighted gene co-expression network analysis and screened modules that were significantly associated with β-casein expression.

[0026] Key genes were screened by combining screening criteria of gene significance greater than 0.60 and module membership greater than 0.80, protein-protein interaction networks, and machine learning algorithms.

[0027] The soft threshold for weighted gene co-expression network analysis was set to 6, and the scale-free network R... 2 The value is 0.85, and the height of the merged module is 0.25.

[0028] This invention provides a method for regulating casein synthesis in BMECs via the DDIT3 gene using leucine. It offers the following advantages:

[0029] 1. This invention provides a clear technical path for the development of low-protein diets for dairy cows by supplementing a specific concentration of leucine in EAA-deficient environments and combining it with the regulation of the ATF-4 / DDIT-3 / TRB-3 / mTORC1 pathway, thereby effectively promoting the synthesis of β-casein in BMECs, improving dietary nitrogen utilization efficiency, and reducing breeding costs and environmental pressure.

[0030] 2. This invention utilizes siRNA-targeted regulation technology of DDIT3 and its upstream and downstream genes to clarify the molecular mechanism by which leucine regulates milk protein synthesis, provides a reproducible method for verifying the function of key genes, and offers a technical reference for research on the regulation of milk protein synthesis by similar amino acids.

[0031] 3. This invention integrates multi-dimensional detection and verification of cell proliferation, apoptosis, cell cycle and amino acid metabolism, while promoting milk protein synthesis and optimizing the growth status of BMECs, thereby improving the comprehensiveness and reliability of the regulatory effect and contributing to the healthy development of the dairy industry. Attached Figure Description

[0032] Figure 1 This is a flowchart of the method of the present invention;

[0033] Figure 2 This is a regression analysis diagram showing the relationship between β-casein expression and intracellular amino acid concentration in this invention.

[0034] Figure 3This is a schematic diagram illustrating the effect of adding different concentrations of leucine on the mTOR signaling pathway under EAA deficiency according to the present invention.

[0035] Figure 4 This is a transcriptomic analysis diagram of BMECs treated with different leucine concentrations under EAA deficiency according to the present invention;

[0036] Figure 5 This is a weighted gene co-expression network analysis diagram of BMECs cultured with different leucine concentrations under EAA-deficient conditions, according to the present invention.

[0037] Figure 6 This is a schematic diagram illustrating the identification and verification of the DDIT3 key gene in β-casein synthesis according to the present invention.

[0038] Figure 7 This is a schematic diagram illustrating the identification and verification of the ATF4 key gene in β-casein synthesis according to the present invention.

[0039] Figure 8 This is a schematic diagram illustrating the identification and verification of the TRIB3 key gene in β-casein synthesis according to the present invention.

[0040] Figure 9 This is a schematic diagram illustrating the effects of amino acid supply and the DDIT-3 / TRB-3 pathway on BMEC apoptosis and cell cycle in this invention.

[0041] Figure 10 This is a schematic model diagram illustrating the effect of leucine supplementation in EAA deficiency on BMECs proliferation and β-casein synthesis according to the present invention.

[0042] Figure 11 A schematic diagram illustrating the effect of different Leu treatments on intracellular amino acid concentrations according to the present invention;

[0043] in, Figure 4 (A) shows the PCA diagrams of all experimental groups; (B) shows the number of differentially expressed genes in different treatment groups compared to the EAA-deficient group; (C) shows the overlapping differentially expressed genes between different treatments; (D) shows the GO enrichment analysis of differentially expressed genes caused by different leucine concentration treatment groups; (E) shows the KEGG pathway analysis of differentially expressed genes caused by different leucine concentration treatment groups.

[0044] Figure 5 (A) shows the scale-free fit index and average connectivity at different soft thresholds; (B) is a dendrogram of gene clustering related to β-casein expression; (C) shows the correlation analysis between module feature genes and β-casein expression; the values ​​in each module indicate the correlation (P-value) between module feature values ​​and β-casein expression; (D) shows the GO enrichment analysis of gene enrichment in the black module; (E) is a chord diagram depicting... Figure 3Key genes involved in several GO entries in D;

[0045] Figure 6 (A) Selecting core genes related to β-casein expression based on gene significance and module membership in the black module; (B) Connectivity between different nodes within the black module; (C) Identifying Hub genes using the LASSO logistic regression algorithm; (D) Screening Hub genes using the random forest algorithm; (E) Overlap analysis of genes screened by different methods; (F) Validating the predicted value of the DDIT3 gene using receiver operating characteristic (ROC) curves; (G, H) The effect of leucine supplementation on DDIT-3 protein expression; (I) The interference efficiency of different siRNAs on the DDIT3 gene; (J, K) The effect of DDIT3 knockdown on mTOR pathway phosphorylation and β-casein expression; (L) KEGG pathway enrichment analysis of differentially expressed genes induced by DDIT3 knockdown.

[0046] Figure 7 (A) shows differentially expressed genes caused by EAA deficiency relative to standard culture medium; (B) shows potential upstream regulatory genes of DDIT3 screened according to the TRRUST database; (C) shows key overlapping genes between the three datasets in Venn diagrams; (D, E) shows the effect of leucine supplementation on ATF-4 protein expression; (F) shows the interference efficiency of different siRNAs on the ATF4 gene; (G, H) shows the effect of ATF4 gene knockdown on mTOR pathway phosphorylation and β-casein expression, respectively.

[0047] Figure 8 In the middle section, (A, B) show the differentially expressed genes induced by BMECs through interference from ATF4 and DDIT3 genes, respectively; (C) shows the potential downstream target genes of DDIT3 predicted based on the TRRUST database; (D) shows the key overlapping genes among the four datasets in the Venn diagram; (E, F) shows the effect of DDIT3 gene knockdown on TRB-3 protein expression; (G) shows the interference efficiency of different siRNAs on the TRIB3 gene; and (H, I) shows the effect of TRIB3 gene knockdown on phosphorylation and β-casein expression in the mTOR pathway, respectively.

[0048] Figure 9 In the middle (A, B), the effects of EAA deficiency and leucine supplementation on apoptosis are shown; (C, D) the effects of DDIT3 and TRIB3 gene knockdown on apoptosis are shown; (E, F) the effects of EAA deficiency and leucine supplementation on cell cycle distribution are shown; and (G, H) the effects of DDIT3 and TRIB3 gene knockdown on cell cycle distribution are shown. Detailed Implementation

[0049] The technical solution of the present invention will now be clearly and completely described 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.

[0050] Please see the appendix Figure 1 - Appendix Figure 11 This invention provides a method for regulating BMECs casein synthesis via DDIT3 gene regulation of leucine, comprising the following steps:

[0051] S1. Preparation and pretreatment of primary BMECs;

[0052] S2. The pretreated primary BMECs were cultured in groups, including a control group, an EAA-deficient model group, and a leucine treatment group. The leucine treatment group was cultured in a medium that did not contain any EAA and was supplemented with leucine.

[0053] S3. By regulating the expression of the DDIT3 gene and combining the signaling pathways formed by upstream and downstream genes, the synthesis of casein in BMECs can be regulated.

[0054] S4. Detect casein synthesis-related indicators, cell proliferation indicators, apoptosis indicators, cell cycle indicators, and intracellular amino acid concentrations in BMECs.

[0055] Specifically, in S1, the primary BMECs were first prepared and pretreated by isolating cells from the mammary gland tissue of healthy dairy cows, and then collecting them through digestion, filtration, centrifugation, culture, and passage to ensure the stability and purity of cell function and to ensure the consistency of experimental procedures and the reliability of results.

[0056] In S2, the pretreated cells were divided into a control group, an EAA-deficient model group, and a leucine-treated group. By setting up a complete nutritional control, an EAA-deficient model, and a leucine gradient intervention, a differential experimental system was constructed to simulate the nutritional environment of BMECs under a low-protein diet, thus creating conditions for elucidating the regulatory role of leucine.

[0057] S3 targets and regulates the DDIT3 gene and its upstream and downstream ATF4 and TRIB3 genes through siRNA transfection, and combines with the ATF-4 / DDIT-3 / TRB-3 / mTORC1 signaling pathway to directly act on the casein synthesis process of BMECs, establishing a clear link between gene expression, signaling pathways and casein synthesis.

[0058] S4 utilizes multi-dimensional detection methods such as targeted metabolomics, Western blotting, and flow cytometry to obtain data on intracellular amino acid concentration, casein synthesis-related proteins, cell proliferation, apoptosis, and cell cycle, providing objective evidence for verifying regulatory effects and clarifying regulatory mechanisms. Simultaneously, a bioinformatics analysis step is added, using transcriptome sequencing, weighted gene co-expression network analysis, and machine learning algorithms to screen key genes, further supporting the elucidation of the molecular mechanism by which leucine regulates milk protein synthesis, and providing comprehensive technical support for research related to the development of low-protein dairy cow diets.

[0059] The preparation and pretreatment of primary BMECs in step S1 includes the following steps:

[0060] Primary BMECs were isolated from the mammary gland tissue of healthy dairy cows, and after digestion, filtration, centrifugation, and collection, they were inoculated into culture dishes and passaged to the 3rd generation.

[0061] Third-generation primary BMECs were inoculated and cultured until the confluence reached 70%-80%, then the culture was replaced with serum-free medium and starved for 12 hours.

[0062] Specifically, primary BMECs were isolated from healthy cow mammary gland tissue, digested with a mixture of 0.25% trypsin and collagenase I / II, filtered through a 70-mesh cell sieve, and collected by centrifugation at 1000 rpm. The collected cells were then seeded into DMEM-F12 culture dishes containing complete additives and cultured. The cells were passaged to the third generation to screen for functionally stable and uniformly pure cell samples. The cells were positive for keratin 18 immunofluorescence and had a purity ≥90%. The third-generation primary BMECs were then seeded into culture dishes and cultured until confluence reached 70%-80%. Then, the culture was replaced with serum-free DMEM-F12 medium and starved for 12 hours. This reduced the interference of amino acid components in the original medium on subsequent experiments, ensuring the cells were in a uniform metabolic initiation state.

[0063] The culture medium used in both steps S1 and S2 is DMEM-F12 medium, which contains 1% L-glutamine, 10% fetal bovine serum, 5 μg / mL transferrin, 5 μg / mL insulin, 1 μg / mL hydrocortisone, 10 ng / mL epidermal growth factor, and 1% penicillin-streptomycin mixture.

[0064] Specifically, DMEM-F12 medium serves as the basic system, providing a fundamental environment for the growth and functional maintenance of primary BMECs. The added L-glutamine, fetal bovine serum, transferrin, insulin, hydrocortisone, and epidermal growth factor work synergistically to support cell proliferation and maintain its physiological functions related to casein synthesis. The penicillin-streptomycin mixture inhibits bacterial contamination, ensuring the stability of the cell culture process. It provides standardized nutritional and environmental support for cell preparation and pretreatment in step S1 and group culture in step S2, ensuring that subsequent experiments on regulating the DDIT3 gene and related signaling pathways and detecting various indicators can be carried out based on functionally stable cell samples.

[0065] During the group culture in step S2, the control group was cultured in standard DMEM-F12 medium containing all EAAs, the EAA-deficient model group was cultured in DMEM-F12 medium without all EAAs, and the leucine treatment group was cultured in DMEM-F12 medium without all EAAs but supplemented with leucine. The leucine concentration was 1, 4, 8, 12, 24, 36, 48, or 60 times the standard leucine concentration in DMEM-F12 medium.

[0066] Specifically, the control group was cultured in standard DMEM-F12 medium containing all EAAs to provide a complete nutritional environment for primary BMECs, serving as a reference for the experiment; the EAA-deficient model group was cultured in DMEM-F12 medium without all EAAs to construct an EAA-deficient experimental environment, simulating the nutritional status of BMECs under a low-protein diet; the leucine treatment group was supplemented with different concentrations of leucine in DMEM-F12 medium without all EAAs, and a correlation system between leucine concentration and cell response was established through gradient intervention, with the 4-fold, 8-fold, 12-fold, and 24-fold groups subsequently used for transcriptomics analysis.

[0067] The leucine concentration in the leucine treatment group was 24 times that of the standard leucine concentration in DMEM-F12 medium. All three groups were cultured for 6 hours at 37°C, 5% carbon dioxide, and 95% humidity, with 6 biological replicates in each group.

[0068] Specifically, the leucine-treated group was treated with DMEM-F12 medium at 24 times the standard leucine concentration. At this concentration, β-casein expression tended to stabilize, and mTOR phosphorylation level was significantly increased compared to the EAA-deficient group, providing a clear and effective intervention dose for elucidating the regulatory role of leucine on the DDIT3 gene and related signaling pathways. All three groups were cultured at 37℃, 5% carbon dioxide, and 95% humidity for 6 hours to eliminate the interference of environmental variables on the experimental results and ensure that the differences between groups were only caused by the EAA and leucine settings in the culture medium. Six biological replicates were set for each group, which increased the sample size of the experimental data and reduced random errors.

[0069] The specific way to regulate DDIT3 gene expression in step S3 is by siRNA transfection. The siRNA is a specific siRNA targeting the DDIT3 gene. This specific siRNA is siRNA-453. The nucleotide sequence of the sense strand of siRNA-453 is shown in SEQ ID NO:1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:2.

[0070] The transfection process involves mixing specific siRNA with CALNP™ RNAi transfection reagent, incubating at room temperature for 20 minutes to form a transfection complex, and then adding it to primary BMECs in the logarithmic growth phase. The silencing efficiency is verified 24 hours after transfection.

[0071] Specifically, transfection was performed using a specific siRNA (siRNA-453) targeting the DDIT3 gene. This siRNA was experimentally verified to be the sequence with the highest silencing efficiency (≥70%), enabling targeted regulation of DDIT3 gene expression and avoiding non-specific interference. The specific siRNA was mixed with CALNP™ RNAi transfection reagent and incubated at room temperature for 20 minutes to form a transfection complex, providing a vector support for siRNA entry into cells and ensuring the feasibility of the transfection operation. The transfection complex was added to primary BMECs in the logarithmic growth phase to utilize the cell viability at this stage to improve the transfection success rate. The silencing efficiency was verified by Western blotting 24 hours after transfection to ensure that DDIT3 gene expression was effectively regulated. After silencing, the phosphorylation levels of mTOR and S6K1 proteins and the expression of β-casein were significantly increased.

[0072] The S3 step also includes the regulation of the ATF4 and TRIB3 genes. The specific siRNA for the ATF4 gene is siRNA-662, whose sense strand nucleotide sequence is shown in SEQ ID NO:3 and its antisense strand nucleotide sequence is shown in SEQ ID NO:4. The specific siRNA for the TRIB3 gene is siRNA-1149, whose sense strand nucleotide sequence is shown in SEQ ID NO:5 and its antisense strand nucleotide sequence is shown in SEQ ID NO:6.

[0073] The regulation of the ATF4 and TRIB3 genes was achieved through siRNA transfection. The transfection process involved mixing the corresponding specific siRNA with CALNP™ RNAi transfection reagent in a certain proportion, incubating at room temperature for 20 minutes to form a transfection complex, and then adding it to primary BMECs in the logarithmic growth phase. The silencing efficiency was verified 24 hours after transfection.

[0074] Specifically, specific siRNAs targeting the ATF4 gene (siRNA-662) and the TRIB3 gene (siRNA-1149) were used for targeted regulation. Both siRNAs were experimentally verified to have a silencing efficiency ≥40%, effectively targeting the ATF4 and TRIB3 genes respectively, avoiding non-specific interference with other genes. The corresponding specific siRNAs were mixed with CALNP™ RNAi transfection reagent in a specific ratio and incubated at room temperature for 20 minutes to form a transfection complex, providing a vector for siRNA entry into cells and ensuring the feasibility of the transfection operation. The transfection complex was then added... Primary BMECs in the logarithmic growth phase were used to improve transfection success rate by utilizing the cell's activity state during this period. Silencing efficiency was verified by Western blotting 24 hours after transfection to ensure effective regulation of ATF4 and TRIB3 gene expression. ATF4, as an upstream regulator of DDIT3, downregulates DDIT3 expression and activates the mTOR pathway upon silencing. TRIB3, as a downstream target gene of DDIT3, directly activates the mTOR pathway and promotes β-casein synthesis upon silencing. This provides experimental support for elucidating the complete regulatory relationship of the ATF-4 / DDIT-3 / TRB-3 / mTORC1 pathway.

[0075] The intracellular amino acid concentration detection in step S4 employs targeted metabolomics analysis, including the following steps:

[0076] Cells from each group were collected, washed twice with pre-cooled PBS, and then lysed in an ice bath by sonication with a 4:1 (v / v) mixture of methanol and water. After centrifugation at 12000×g for 10 minutes at 4°C, the supernatant was filtered through a 0.22 μm organic phase membrane. Separation was performed using an Agilent 1290 Infinity LCU HPLC system. Mobile phase A was an aqueous solution containing 25 mmol / L ammonium formate and 0.1% formic acid, and mobile phase B was an acetonitrile solution containing 0.1% formic acid. Quantitative detection was performed using mass spectrometry in electrospray ionization positive / negative ion mode and multiple reaction monitoring (MRM) scanning mode.

[0077] Specifically, after collecting cells from each group and washing them twice with pre-cooled PBS, a mixture of methanol and water (4:1 volume ratio) was added and sonicated on ice for lysis. The sonication was performed at 200W, with a 3-second sonication cycle followed by a 3-second pause. After centrifugation at 4°C and 12000×g for 10 minutes, the supernatant was filtered through a 0.22μm organic phase filter to remove extracellular impurities and release intracellular amino acids, obtaining a pure sample. Separation was performed using an Agilent 1290 Infinity LCU HPLC system. Mobile phase A was an aqueous solution containing 25 mmol / L ammonium formate and 0.1% formic acid, and mobile phase B was an acetonitrile solution containing 0.1% formic acid. The separation was performed according to the set gradient. The high-elution elution method enables the effective separation of different amino acids. Quantitative detection is performed using a 6500 / 5500QTRAP mass spectrometer in electrospray ionization positive / negative ion mode and multiple reaction monitoring (MRM) scanning mode, accurately acquiring the concentration data of various amino acids in cells. The results show that the concentrations of leucine and isoleucine increase linearly with leucine supplementation, while the concentrations of 5 EAAs and 11 non-EAAs show a secondary decreasing trend. Furthermore, β-casein expression is positively correlated with leucine and isoleucine concentrations, providing data support for analyzing changes in intracellular amino acid metabolism after leucine supplementation and the regulatory role of the DDIT3 gene and related signaling pathways in casein synthesis.

[0078] In step S4, casein synthesis-related indicators were detected by Western blotting. The proteins detected included β-casein, mTOR, phosphorylated mTOR, S6K1, and phosphorylated S6K1, with β-actin as an internal control protein. Cell proliferation indicators were detected by CCK-8 assay. Cell apoptosis indicators were detected by Annexin V-FITC / PI double staining combined with the GuavaEasyCyteHT system. Before detection, cells were washed twice with pre-cooled PBS and then suspended in 0.1 mL binding buffer. 10 μL of FITC-labeled Annexin V and 5 μL of LPI were added, and the cells were incubated in the dark for 15 minutes. Cell cycle indicators were detected by PI staining combined with the GuavaEasyCyteHT system. Before detection, cells were fixed in 75% ethanol at 4°C for 12 hours and then resuspended in PBS containing 5 μg / mL PI and incubated in the dark for 10 minutes.

[0079] Specifically, Western blotting was used to detect β-casein, mTOR, phosphorylated mTOR (Ser2448 site), S6K1, and phosphorylated S6K1 (Thr389 site), with β-actin as the internal control protein. The protein loading amount was 10 μg, and the primary antibody dilution ratio was 1:1000 for all proteins except β-actin, which was 1:5000. The secondary antibody was HRP-conjugated goat anti-rabbit or anti-mouse IgG. This method can accurately quantify the expression levels of casein synthesis products and key proteins in the ATF-4 / DDIT-3 / TRB-3 / mTORC1 pathway. Cell proliferation indicators were detected using the CCK-8 assay, which can reflect cell growth status and proliferative capacity. Cell apoptosis markers were detected using Annexin V-FITC / PI double staining combined with the GuavaEasyCyteHT system. Before detection, cells were washed twice with pre-cooled PBS and then suspended in 0.1 mL binding buffer. 10 μL of FITC-labeled Annexin V and 5 μL of LPI were added, and the cells were incubated in the dark for 15 minutes. This method accurately distinguished between early and late apoptotic cells and quantified the apoptosis ratio. Results showed that leucine supplementation reduced the apoptosis rate. Cell cycle markers were detected using PI staining combined with the GuavaEasyCyteHT system. Before detection, cells were fixed in 75% ethanol at 4°C for 12 hours and then resuspended in PBS containing 5 μg / mL PI and 100 μg / mL RNase A and incubated in the dark for 10 minutes. This method clearly identified the distribution of cells in the G0 / G1, S, and G2 / M phases. These multidimensional detection methods obtained experimental data on casein synthesis, pathway activity, cell proliferation, apoptosis, and cell cycle.

[0080] This method also includes a bioinformatics analysis step, specifically including:

[0081] Total RNA was extracted from cells in the control group, EAA-deficient model group, and leucine-treated group at concentrations of 4, 8, 12, and 24 times the standard concentration, and transcriptome sequencing was performed to screen for differentially expressed genes.

[0082] We constructed gene co-expression modules using weighted gene co-expression network analysis and screened modules that were significantly associated with β-casein expression.

[0083] Key genes were screened by combining screening criteria of gene significance greater than 0.60 and module membership greater than 0.80, protein-protein interaction networks, and machine learning algorithms.

[0084] The soft threshold for weighted gene co-expression network analysis was set to 6, and the scale-free network R... 2 The value is 0.85, and the height of the merged module is 0.25.

[0085] Specifically, total RNA was extracted from cells in the control group, EAA-deficient model group, and leucine-treated group at concentrations of 4, 8, 12, and 24 times the standard concentration. Five biological replicates were performed for each group, resulting in a total of 25 samples. After purity testing with Nanodrop 2000 and integrity verification by agarose gel electrophoresis, transcriptome sequencing was performed to screen for differentially expressed genes. The screening criterion was Padj < 0.05, which allowed for the acquisition of information on changes in cellular gene expression after leucine intervention. The L24 group showed 745 differentially expressed genes compared to the EAA-deficient group. A weighted gene co-expression network analysis was used to construct a gene co-expression module. During analysis, the top 7500 highly variable genes were selected based on the median absolute deviation, with a soft threshold set to 6. A scale-free network R0 was used. 2 The coefficient of performance (COP) was 0.85, and the module merging height was 0.25. A total of 28 gene co-expression modules were constructed. Among them, the black module showed the most significant positive correlation with β-casein expression. The genes in this module were mainly enriched in functions such as positive regulation of apoptosis and transcription factor complexes. Using screening criteria of gene significance greater than 0.60 and module membership greater than 0.80, protein-protein interaction networks, LASSO regression, random forest and other machine learning algorithms, key genes were screened. Multi-dimensional validation was used to improve the accuracy of key gene screening. Finally, DDIT3 was identified as the core gene involved in leucine regulation of β-casein synthesis. This gene has good predictive value and provides core gene target support for clarifying the molecular mechanism of leucine regulation of milk protein synthesis, which helps to establish a reproducible key gene function validation method.

[0086] The following is the siRNA sequence:

[0087]

[0088] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for regulating BMECs casein synthesis via DDIT3 gene regulation of leucine, characterized in that, Includes the following steps: S1. Preparation and pretreatment of primary BMECs; S2. The pretreated primary BMECs were cultured in groups, including a control group, an essential amino acid (EAA) deficiency model group, and a leucine treatment group. The leucine treatment group was cultured in a medium that did not contain all EAAs and was supplemented with leucine. S3. By regulating the expression of the DDIT3 gene and combining the signaling pathways formed by upstream and downstream genes, the synthesis of casein in BMECs can be regulated. S4. Detect casein synthesis-related indicators, cell proliferation indicators, apoptosis indicators, cell cycle indicators, and intracellular amino acid concentrations in BMECs.

2. The method for regulating BMECs casein synthesis via DDIT3 gene regulation of leucine according to claim 1, characterized in that, The preparation and pretreatment of primary BMECs in step S1 includes the following steps: Primary BMECs were isolated from the mammary gland tissue of healthy dairy cows, and after digestion, filtration, centrifugation, and collection, they were inoculated into culture dishes and passaged to the 3rd generation. Third-generation primary BMECs were inoculated and cultured until the confluence reached 70%-80%, then the culture was replaced with serum-free medium and starved for 12 hours.

3. The method for regulating BMECs casein synthesis via DDIT3 gene regulation of leucine according to claim 1, characterized in that, The culture medium used in steps S1 and S2 is DMEM-F12 medium, which contains 1% L-glutamine, 10% fetal bovine serum, 5 μg / mL transferrin, 5 μg / mL insulin, 1 μg / mL hydrocortisone, 10 ng / mL epidermal growth factor, and 1% penicillin-streptomycin mixture.

4. The method for regulating BMECs casein synthesis via DDIT3 gene regulation of leucine according to claim 1, characterized in that, During the group culture in step S2, the control group was cultured in standard DMEM-F12 medium containing all EAAs, the EAA-deficient model group was cultured in DMEM-F12 medium without all EAAs, and the leucine treatment group was cultured in DMEM-F12 medium without all EAAs but supplemented with leucine. The leucine concentration was 1, 4, 8, 12, 24, 36, 48, or 60 times the standard leucine concentration in DMEM-F12 medium.

5. The method for regulating BMECs casein synthesis via DDIT3 gene regulation of leucine according to claim 4, characterized in that, The leucine concentration in the leucine treatment group was 24 times that of the standard leucine concentration in DMEM-F12 medium. All three groups were cultured for 6 hours at 37°C, 5% carbon dioxide, and 95% humidity, with 6 biological replicates in each group.

6. The method for regulating BMECs casein synthesis via DDIT3 gene regulation of leucine according to claim 1, characterized in that, The specific method for regulating DDIT3 gene expression in step S3 is siRNA transfection. The siRNA is a specific siRNA targeting the DDIT3 gene. This specific siRNA is siRNA-453. The nucleotide sequence of the sense strand of siRNA-453 is shown in SEQ ID NO:1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:

2. The transfection process involves mixing the specific siRNA with the CALNP™ RNAi transfection reagent, incubating at room temperature for 20 minutes to form a transfection complex, adding it to primary BMECs in the logarithmic growth phase, and verifying the silencing efficiency 24 hours after transfection.

7. The method for regulating BMECs casein synthesis via DDIT3 gene regulation of leucine according to claim 1, characterized in that, The regulatory process in step S3 also includes the regulation of the ATF4 gene and the TRIB3 gene. The specific siRNA for the ATF4 gene is siRNA-662, the nucleotide sequence of the sense strand of siRNA-662 is shown in SEQ ID NO:3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:

4. The specific siRNA for the TRIB3 gene is siRNA-1149, the nucleotide sequence of the sense strand of siRNA-1149 is shown in SEQ ID NO:5, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:

6. The regulation of the ATF4 and TRIB3 genes was achieved through siRNA transfection. The transfection process involved mixing the corresponding specific siRNA with CALNP™ RNAi transfection reagent in a certain proportion, incubating at room temperature for 20 minutes to form a transfection complex, and then adding it to primary BMECs in the logarithmic growth phase. The silencing efficiency was verified 24 hours after transfection.

8. The method for regulating BMECs casein synthesis via DDIT3 gene regulation of leucine according to claim 1, characterized in that, The intracellular amino acid concentration detection in step S4 employs targeted metabolomics analysis, including the following steps: Cells from each group were collected, washed twice with pre-cooled PBS, and then lysed in an ice bath by sonication with a 4:1 (v / v) mixture of methanol and water. After centrifugation at 12000×g for 10 minutes at 4°C, the supernatant was filtered through a 0.22 μm organic phase membrane. Separation was performed using an Agilent 1290 Infinity LCU HPLC system. Mobile phase A was an aqueous solution containing 25 mmol / L ammonium formate and 0.1% formic acid, and mobile phase B was an acetonitrile solution containing 0.1% formic acid. Quantitative detection was performed using mass spectrometry in electrospray ionization positive / negative ion mode and multiple reaction monitoring (MRM) scanning mode.

9. The method for regulating BMECs casein synthesis via DDIT3 gene regulation of leucine according to claim 1, characterized in that, In step S4, casein synthesis-related indicators were detected using Western blotting. The proteins detected included β-casein, mTOR, phosphorylated mTOR, S6K1, and phosphorylated S6K1, with β-actin as an internal control. Cell proliferation indicators were detected using the CCK-8 assay. Apoptosis indicators were detected using Annexin V-FITC / PI double staining combined with the GuavaEasyCyteHT system. Before detection, cells were washed twice with pre-cooled PBS and then suspended in 0.1 mL binding buffer. 10 μL of FITC-labeled Annexin V and 5 μL of propidium iodide (PI) were added, and the cells were incubated in the dark for 15 minutes. Cell cycle indicators were detected using PI staining combined with the GuavaEasyCyteHT system. Before detection, cells were fixed in 75% ethanol at 4°C for 12 hours and then resuspended in PBS containing 5 μg / mL PI and cultured in the dark for 10 minutes.

10. The method for regulating BMECs casein synthesis via DDIT3 gene regulation of leucine according to claim 1, characterized in that, This method also includes a bioinformatics analysis step, specifically including: Total RNA was extracted from cells in the control group, EAA-deficient model group, and leucine-treated group at concentrations of 4, 8, 12, and 24 times the standard concentration, and transcriptome sequencing was performed to screen for differentially expressed genes. We constructed gene co-expression modules using weighted gene co-expression network analysis and screened modules that were significantly associated with β-casein expression. Key genes were screened by combining screening criteria of gene significance greater than 0.60 and module membership greater than 0.80, protein-protein interaction networks, and machine learning algorithms. The soft threshold for weighted gene co-expression network analysis was set to 6, and the scale-free network R... 2 The value is 0.85, and the height of the merged module is 0.25.

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