Computational Molecular Biology 2025, Vol.15, No.5, 227-234 http://bioscipublisher.com/index.php/cmb 231 accumulation of ROS in its roots and to some extent becomes the "villain" in regulating resistance. Many mirnas target key transcription factors such as MYB, SPL, and ARF, which serve as the links connecting growth, development, and stress responses. The entire network is actually interwoven with multiple pathways. Modules such as phenylpropanin metabolism, glutathione cycle, and carbohydrate metabolism are also frequently driven by miRNA, indicating that it is not a single pathway working alone, but rather a whole system working in coordination (Li, 2025). 5.3 Network topology analysis and identification of core regulatory modules The network has been built. The next step is not to look at how many "points" there are, but to see which nodes are more "important". Like miR164, miR156, and miR159, they almost always take the center stage in all the diagrams because they regulate too many downstream areas. If you don't look at them, you'll miss a large part. Through the analysis of network topology structure, these "hub" mirnas can be identified, which are often the intersection points of multiple pathways (Aravind et al., 2017; Tang et al., 2022). Meanwhile, the WGCNA method can also identify several modules that are particularly closely related to drought. For instance, the miR164-MYB/NAC module is linked to the ABA pathway, and miR408a is associated with ROS balance. While miR156-SPL is more related to development and growth regulation (Yang et al., 2025). These modules, to put it bluntly, are the "high-incidence areas" of regulation. If genetic improvement is to be carried out in the future, they will be very promising targets. 6 Case Study: Experimental Validation and Functional Analysis of Specific miRNAs 6.1 Drought response mechanism of miR164 targeting NAC transcription factors Not all transcription factors are related to drought, but proteins like NAC, which play the role of "dispatchers" in plants, are often targets of miR164. In some drought-tolerant corn varieties, the regulation between miR164 and NAC seems to be centered around the ABA hormone - as soon as ABA is activated, miR164 will suppress the expression of NAC. In this way, both the structure of the root system and the reaction speed can be "adjusted more precisely". However, the situation is not always stable. For instance, miR164 overly strongly inhibits NAC, which instead makes plants more sensitive to drought. However, once the effect of miR164 is weakened or the expression of NAC is simply increased, sometimes plants become more drought-tolerant (Figure 2). Experimental evidence of this "waxing and waning" relationship has already been obtained for corn and other cereal crops (Liu et al., 2019; Tang et al., 2022). 6.2 Role of miR398 in regulating ROS scavenging and cellular protection When it comes to drought resistance, ROS (reactive oxygen species) cannot be overlooked. Once they accumulate in excessive amounts, plant cells won't be able to handle them. And miR398 plays a somewhat "subtle" role in this process. It will target those genes encoding SOD (copper/zinc superoxide dismutase) and control their expression on a regular basis. However, once drought stress occurs, miR398 will decline, the amount of SOD will increase, and the antioxidant capacity of cells will also be enhanced. In this way, the plants can last a little longer. This mechanism is not uncommon and has similar results in many plants. Some people conducted regulatory experiments and found that when miR398 was lowered, its resistance increased, indicating that regulating its level can affect the ROS clearance system (Li et al., 2022; Zheng et al., 2023). 6.3 Expression profiling of key miRNAs via qRT-PCR and degradome validation Sequencing results alone cannot explain all the issues; experimental evidence is needed to confirm them. So qRT-PCR and degradation group analysis came in handy. In the study, qRT-PCR was used to verify whether miR164 and miR398 would "rise" or "fall" under drought conditions, and the results were indeed consistent with the sequencing data. The degradation group further confirmed exactly who they had cut - the target genes, the cutting sites, all of which were paired up one by one. These methods combined are like piecing together a graph of the miRNA regulatory network from different angles, especially in the area of drought response, providing very solid experimental support (Seeve et al., 2019; Jiao et al., 2022).
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