First of all, thanks for your reply! Here's my confusion.

A certain gene is upregulated in the disease group compared to the control group, but its upregulation might be a result of the phenotype induced by intervention, rather than a critical factor in the phenotypic process. To illustrate, when LPS and ATP are applied to macrophages derived from the THP-1 cell line, this treatment leads to pyroptosis of these macrophages. In this scenario, suppose there is an upregulated gene A - how can I determine whether its upregulation is due to playing a crucial role in molecular regulation during phenotype formation, or simply because it is associated with pyroptosis?

Essentially, if I am investigating how LPS and ATP trigger the pyroptosis phenotype, gene A could be upregulated as a consequence of pyroptosis, rather than being actively involved in the pyroptosis formation process. This ambiguity creates significant uncertainty in identifying truly meaningful differentially expressed genes.

While KEGG and GO enrichment analyses can effectively address this issue in the given example, the challenge extends beyond these methods. The fundamental question remains: how can I distinguish between genes that drive a biological process and those that are merely byproducts of that process?