Computational Molecular Biology 2025, Vol.15, No.5, 235-244 http://bioscipublisher.com/index.php/cmb 239 and the regulation of milk secretion. For instance, COL6A1 and NFKB2 have been found to be closely related to milk fat synthesis, and they exert regulatory functions on mammary epithelial cells through these pathways (Han et al., 2025). However, the role of the JAK-STAT pathway cannot be ignored. It is usually activated under the action of prolactin or certain cytokines and plays a key role in the immune regulation and lactation function of the breast. As for pathways such as MAPK, mTOR, Rap1 and TGF-β, they are more involved in milk protein synthesis, cell cycle control, and even reflected in breast structure adjustment (Zheng et al., 2025). Not every pathway plays the same role at all stages. Such regulation is mostly phased and cross-cutting. 5.2 Identification of core regulatory factors in gene networks In the complex gene expression network, those that truly play a "key role" may only be in the minority. Genes such as GAPDH, KDR, CSF1, PYGM, RET, PPP2CA, GUSB and PRKCA are frequently named in the lactation-related pathways (Roudbari et al., 2023). Some transcription factors - such as HHEX, HOXA7, NOTCH3/4 and members of the SMAD family - usually appear at the core position in the miRNA and mRNA co-expression network. They affect not only milk production but also indicators such as lactose content and somatic cell count. These regulatory factors do not act alone and are also strongly associated with the expression of mammary mammary specific genes (such as DGAT1, GLYCAM1) and certain specific lncrnas (Cai et al., 2025). Network analysis merely offers a starting point; more verification still relies on functional experiments. 5.3 Interaction patterns between miRNAs, mRNAs, and metabolites Sometimes, it is difficult to explain the mechanism of lactation regulation by looking at a single gene or pathway, especially when it involves the interaction of multiple molecular layers. Mirnas such as miR-148a, miR-186, miR-200a and miR-152 often affect fat synthesis, milk production and breast development through signaling pathways such as PTEN, PI3K-Akt or PPAR (Xia et al., 2021; Li et al., 2024). WGCNA et al. Co-expression analysis methods have identified miRNA modules closely related to milk volume, protein, milk fat and even lactose, as well as their target mrnas. However, it is not only miRNA but also lncRNA and circRNA that are frequently involved in regulation. ceRNA networks such as lincRNA TCONS_00082721 combined with FABP4 or circ11103/miR-128/PPARGC1A are structures that cannot be avoided when studying milk fat metabolism. These interaction patterns are not necessarily all direct; sometimes they are chain-like or feedback-like, and as a whole, they demonstrate the high complexity of the lactation regulation mechanism. 6 Case Study: Multi-Omics Integration Reveals Key Genes for Milk Production 6.1 Multi-omics sample collection and analysis during lactation in Holstein cattle Omics analysis does not start from a single perspective but requires repeated sampling from multiple tissues and at multiple time points. Tissues such as the mammary gland, liver and gastrointestinal tract are often collected both during lactation and non-lactation periods, and the collection involves more than just the transcriptome and genome. Epigenetic information such as DNA methylation, histone modification, and non-coding RNA is also crucial (Gao et al., 2024; Zheng et al., 2025). Especially in the large-scale sample study of Holstein dairy cows, researchers have dealt with millions of variant sites (Figure 2). Although these data are complex in hierarchy, the ultimate goal is the same - to identify which genes and regulatory elements play a leading role in the lactation stage, especially those candidate factors specifically expressed in the breast. 6.2 Omics comparison between high-and low-yielding dairy cattle groups Sometimes, the difference between high and low milk production is not only reflected in the quantity, but also at the molecular level behind it. It can be seen from the comparison of transcriptome and metabolome that the pathways related to energy metabolism and amino acid synthesis are often upregulated in the mammary glands and livers of high-yield dairy cows. Conversely, those individuals with lower milk production were more active in immune pathways or certain splicing forms (Nguyen, 2025). Furthermore, some key metabolites, such as citrulline and N-acetylornithine, were significantly elevated in the high-yield group, which might imply stronger gluconeogenesis and energy utilization capabilities. However, not all differences can be directly attributed to the expression of a certain gene. It is necessary to consider the regulatory network together.
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