Hello,

Considering that MSigDB databases such as GO and Hallmark contain gene sets for different pathways, these gene sets may include genes that regulate the pathway, including both upregulators and downregulators. Unless this is specifically indicated by “up” and “down” labels, both types of genes may be present in the same gene set.

In that case, when we perform DEG analysis between two conditions, does it make sense to include only significantly upregulated genes in the enrichment analysis? Or is it more appropriate to include both upregulated and downregulated genes?

Even if we include only upregulated genes from the DEG analysis, I believe we can only say that “upregulated genes are enriched in the X pathway.” We cannot necessarily say that the pathway itself is truly upregulated in the first condition. We may not be able to infer the direction of the pathway. It would only indicate that the experiment has affected that specific pathway.