*3.7. KEGG Enrichment Analysis of DEGs*

KEGG is a database for the systematic analysis of gene function and genome information, which can be used as a whole network to study gene and expression information [48]. As shown in Figure 5C and Supplementary Table S5, the significant DEGs were mainly enriched in 11 KEGG pathways (*P*value ≤ 0.05), and most of them were related to immune regulation and signal transduction. Among them, the hematopoietic cell lineage, hematopoietic cell lineage, the FoxO signaling pathway, and the cytokine-cytokine receptor interaction are closely related to the occurrence of cancer in humans. Cytokines are crucial intercellular regulators and mobilizers of cells engaged in innate and adaptive inflammatory host defenses, cell growth, differentiation, cell death, angiogenesis, and development and repair processes aimed at the restoration of homeostasis [49]. FOXO (Forkhead Box O) is a subgroup of Fox transcription factors that are considered to play a key role as tumor suppressors in a variety of cancers [50]. Complement and coagulation cascades could interact with systemic lupus erythematosus (SLE), and this interaction may lead to aggravation of the disease, which is more obvious in inflamed patients [51]. In the complement and coagulation cascades pathway, the expression of the important gene *SERPINB2* was increased in both L4 vs. Ctr and M21 vs. Ctr. A previous study showed that the protein SerpinB2 is substantially upregulated under multiple inflammatory conditions, and dysregulated expression and polymorphisms are associated with several human inflammatory diseases [52]. The above results further indicated that DMM exposure reduces the immune resistance of the body, even at a low concentration. DMM has certain immunotoxicity to the human body. Even low-dose exposure can cause immune reactions and cause potential harm to the body.

**Figure 5.** Gene ontology enrichment analysis for the significantly differentially expressed genes (**A**), simultaneously significantly different expressed genes for both comparison of L4 vs. Ctr and M21 vs. Ctr (**B**), and KEGG enrichment analysis for the significantly differentially expressed genes (**C**).

#### *3.8. Target Gene Screening and Quantitative Real-Time PCR Validation*

To validate the results obtained from the transcriptome analysis, four important genes related to immune regulation were selected. As shown in Figure 6, there was general accordance between the RNA sequence and the real-time qPCR data for all the tested genes (*CD40LG* and *EGR1* downregulated, *SERPINB2* and *RAG1* upregulated), although the fold changes differed between the analytical methods. Compared to the high concentration exposure (M21), the effect of the low concentration DMM exposure (L4) on gene expression was more significant. Therefore, the low-dose chronic toxicity of DMM needs to be further studied.

**Figure 6.** Quantitative real-time PCR results for the selected genes. Data are expressed as the mean ± SD from triplicate exposures. Different letters on the different bars indicate significant differences (*p* < 0.05).
