*2.7. Genistein Inhibited Cell Migration and Invasion of HeLa Cells in Transwell® Assays*

The inhibition of cell migration by genistein was verified using the Transwell assay. A remarkable decrease in cell migration activity was observed with increasing concentrations of genistein; 12.5 μM–100 μM of genistein decreased cell migration activity by 81%, 85%, 50%, and 36% compared with the solvent control, respectively (Figure 9A–C). As shown in Figure 9B–D, genistein (12.5–100 μM) also significantly inhibited cell-invasive activity; 12.5 μM, 25 μM, 50 μM, and 100 μM of genistein decreased cell invasion activity compared with the solvent control by 36%, 39%, 30%, and 27%, respectively. Compared with the inhibition of cell migration activity, genistein exerted a stronger inhibitory effect on cell invasion activity. Moreover, compared with the cell viability results, the inhibition of cell migration and invasion was not entirely due to the inhibition of cell viability.

**Figure 9.** Effect of genistein on the migration and invasion activities of HeLa cells. Cells were seeded on membranes and co-cultured with different doses of genistein for 24 h. (**A**) Cells that migrated to the lower surface of the filter were fixed and stained with crystal violet, and then photographed by an inverted microscope at ×100. (**B**) Cell invasion was assessed by following cell movement through the Matrigel to the lower surface of the filter. Invading cells were fixed, stained with crystal violet, and photographed under an inverted microscope at ×100. (**C**) Cell migration was quantified from at least three images randomly using Image J software. (**D**) Cell invasion was quantified from at least three images randomly using the Image J software. The experiment was repeated three times. \*\* *p* < 0.01 vs. DMSO control. \* *p* < 0.05 vs. DMSO control; scale bar: 200 μm.

#### *2.8. Identification of Differentially Expressed Genes (DEGs) Associated with Genistein Treatment*

To further decipher the mechanism of genistein's action on cervical cancer cells, we analyzed global gene expression profiles in genistein- and DMSO-treated cells by using RNA sequencing. Approximately 59–67 million (M) clean reads from six samples (three for genistein treatment; three for control) were obtained after deletion of the low-quality and adaptor sequences; Q30 bases ranged from 92.72% to 93.32% (data not shown). These results demonstrate that the samples were of good quality, and that the coverage of the cervical cancer cell genome was high.

We previously observed significant changes in the proliferation and metastasis of cervical cancer cells after genistein treatment. The transcriptome screening results provide strong evidence that these changes were accompanied by significant differences in gene expression. A total list of expressed genes was determined using RNA-Seq data (Figure 10).

Among these, genes that were upregulated (orange) and downregulated (blue) following genistein treatment (compared with control) were identified (*p* < 0.05; |log2FoldChange| > 1).

**Figure 10.** (**A**) Volcano plot of DEGs identified following genistein treatment (compared with control). Grey dots, genes with no significant difference in expression; blue dots, downregulated genes; and orange dots, upregulated genes. Fold-change was calculated using gene-normalized expression of the genistein group/gene-normalized expression of the control group. Differences in expression with a *p* value < 0.05 and a Log2 (fold change) > 1 were considered statistically significant. (**B**,**C**) Gene ontology enrichment of up- and downregulated DEGs after genistein treatment. (**D**) KEGG enrichment of upregulated DEGs after genistein treatment.

Gene ontology of the DEGs was analyzed by GESA (Gene Set Enrichment Analysis), and the upregulated and downregulated DEGs were enriched for biological process analysis. The upregulated DEGs are involved in morphological processes, including cilium organization, strand displacement, and cilium morphogenesis. In contrast, the downregulated DEGs are associated with ribosomal subunits, the ribosome, the multiorganism metabolic process, and mitochondrial translation. GESA analysis revealed that the gene clusters involved in the regulation of mitochondrion organization, substrate adhesion-dependent cell spreading, and focal adhesion were significantly downregulated (Figures 10B,C and 11A,B,E,F). Moreover, the top KEGG pathways are listed in Figure 10D. The downregulated DEGs are involved in the spliceosome, ribosome, GAP junction, proteasome, cell cycle, purine metabolism, glycolysis metabolism, and oxidative phosphorylation. Previously, we predicted that the focal adhesion pathway was a promising target for genistein treatment in silico (Figures 3 and 4). The RNA sequencing results validated these

predictions, and numerous genes in the focal adhesion pathway were downregulated after genistein treatment, including FAK, PAK, Src, Shc, Actinin, Talin, and ILK. Moreover, mitochondrion function was also significantly inhibited by genistein, while adhesion-related pathways involving adherent junctions were also downregulated (Figure 11C,D,G,H). Interestingly, our data provide evidence that genistein mainly exerted a downregulation effect on cervical cancer cells.

**Figure 11.** (**A**−**D**) Representative enrichment of gene signatures in genistein and control group by gene set enrichment analysis (GESA). Representative enriched gene sets are shown (FDR q value < 0.05). (**E**−**H**). Heatmap of the representative DEGs between genistein and control group in parallel with GESA analysis.

#### *2.9. Genistein Inhibits Activation of the FAK–Paxillin Pathway*

To investigate whether genistein inhibited cell migration and invasion through inhibition of the FAK−paxillin pathway, we performed Western blot analyses to detect the expression of the relevant proteins. As shown in Figure 12, genistein treatment strongly decreased the phosphorylation of paxillin and FAK. In addition, the expression of βcatenin and vimentin was inhibited by genistein. The results suggest that the molecular mechanism of genistein on cell proliferation and metastasis involves inactivation of the FAK−paxillin pathway.

**Figure 12.** Effect of genistein on the focal adhesion protein expression in HeLa cells. (**A**) Cells were grown with or without different doses of genistein for 30 min. The expression levels of specific proteins were detected by Western blot analysis. GAPDH was used as a control. All first antibodies were used at a dilution of 1:1000. Secondary antibodies were used at a concentration of 1:3000. (**B**–**E**) Integrated optical intensity of the bands was determined by Image J software (https://imagej.net, accessed on 3 January 2023). The experiment was repeated three times. \*\* *p* < 0.01 vs. DMSO control.
