*2.2. PPI Network Construction and Identification of Hub Genes*

The 371 core targets were introduced into the STRING database to build the PPI (Protein-Protein Interaction) network of genistein's action against human cervical cancer. A confidence score of protein and protein interaction of >0.9 was set, and data were collected and visualized using Cytoscape. As shown in Figure 2, the network contained 317 nodes and 1754 edges; the cluster coefficient was 0.33, and the network centralization value was 0.170. The top 10 hub genes in this network were subsequently identified by MCC (Maximal Clique Centrality) algometrical analysis: FN1, TIMP1, GAS6, IL6, C3, IGFBP3, IGFBP4, IGFBP1, CST3 and SPP1. The increasing significance of genes in the network is represented by the color change (yellow to red) (Figure 3).

**Figure 2.** PPI network of the core genes in genistein's action against human cervical cancer (dark purple color indicates high degree; grey color indicates low degree).

**Figure 3.** Hub gene analysis of the PPI network. The color change from yellow to red indicates the increasing importance of related genes in the network. The red color indicates the highest significance.

#### *2.3. GO Enrichment Analyses of Core Targets*

The predicted core targets were further analyzed by GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment in the DAVID database. The top 20 biological processes of predicted core targets were identified (Figure 4A); the core targets are mainly involved in the response to organic substances, regulation of programmed cell death, regulation of cell death, regulation of apoptosis, response to endogenous stimulus, regulation of cell proliferation, response to hormone stimulus, and response to extracellular stimulus. The enriched KEGG pathways (Figure 4B) included the following: pathways in cancer; the p53 signaling pathway; the cell cycle; apoptosis; the MAPK (Mitogen Activated Protein Kinase) signaling pathway; the TLR (Toll Like Recepter) signaling pathway; and focal adhesion. From the hub gene analysis, FN1 was selected as the most significant hub gene in genistein's action against the cervical cancer network. Therefore, we predicted that the focal adhesion pathway is a novel pathway targeted by genistein in the treatment of cervical cancer. To gain a deeper insight, all the predicted genes in this pathway potentially regulated by genistein are highlighted in red (Figure 5).

**Figure 4.** GO ontology and KEGG pathway enrichment of the core targets. (**A**) Biological processes and (**B**) KEGG pathways involved in genistein's action against human cervical cancer.

**Figure 5.** Predicted targets of genistein's action in the focal adhesion pathway. Proteins highlighted in red indicate potential targets of genistein's action against human cervical cancer.

#### *2.4. Genistein Attenuates Cell Viability and Growth in HeLa Cells*

According to in silico studies, genistein showed multiple effects on cervical cancer. Next, we experimentally validated these effects using human cervical cancer cells in vitro. The high−risk HPV 16 and 18 are highly associated with the pathogenesis of cervical cancer. Moreover, according to the literature, HeLa cells are more sensitive to genistein treatment. Therefore, in this study, we used HeLa cells for the following experiments [27]. As shown in Figure 6A, genistein significantly inhibited cell growth in a time- and dose-dependent manner after 24 h and 48 h of treatment by CCK−8 (Cell Counting Kit−8) assay. Moreover, 5-fluorouracil (5−FU), clinically used as an anti-tumor drug, was used as a positive control. The concentration of 5−FU was chosen based on the literature [28]. The result showed that 80 μM of 5−FU inhibited the viability of HeLa cells compared with the DMSO (Dimethyl sulfoxide) control. The results indicate that genistein exerted the same inhibitory effect on the human cervical cancer cells as the anti-cancer drug 5−FU. Moreover, we further tested the effects of genistein on cell growth by using cell number counting. The results indicate a prominent effect of genistein on cell growth; the cell number was significantly decreased after genistein (12.5–100 μM) treatment for 24–48 h (Figure 6B). Furthermore, genistein strongly inhibited the colony formation ability of cervical cells (Figure 6C). While cervical cells formed large colonies in the control group, this colony-forming ability was significantly decreased after genistein treatment, indicating that genistein (12.5–100 μM) strongly inhibited HeLa cells' proliferation. Overall, these results demonstrate that 12.5–100 μM genistein strongly inhibited HeLa cells' viability and proliferation.

**Figure 6.** (**A**). Effects of genistein treatment on the proliferation of HeLa cells for 24–48 h were detected by CCK−8 assay. (**B**). Effects of genistein treatment on cell growth were counted by hemocytometer after 24–48 h. (**C**). Representative images of HeLa cell colony formation after genistein treatment (0–100 μM). The data shown are the average of three replicates; the experiments were performed three times independently. \*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.05 compared with solvent control. Scale bar: 200 μm.

#### *2.5. Genistein Inhibits HeLa Cell Adhesion*

Since adhesion is the first critical step in cancer metastasis, we initially investigated the influence of genistein on HeLa cells' adhesion. The data demonstrate that genistein dosedependently inhibited cell adhesion in cervical cancer cells (Figure 7A,B). Compared to the control, 92%, 82%, 81%, and 71% inhibition of adhesion was observed with 12.5 μM, 25 μM, 50 μM, and 100 μM of genistein, respectively. Thus, HeLa cell adhesion was significantly inhibited after genistein (25–100 μM) treatment (*p* < 0.01; *p* < 0.05) (Figure 7A).

**Figure 7.** Effects of genistein on HeLa cell adhesion. Cells were grown in the presence of different doses of genistein for 24 h, and reseeded for 3 h. Adherent cells were then fixed by PFA (Paraformaldehyde) and stained with crystal violet solution. Absorbance readings were detected at OD570 nm by microplate reader. (**A**). Percentage of adhesion was then calculated based on the OD value of the adhered cells in the genistein-treated group (compared to control values (100%)). The experiment was performed three times independently, and data shown are the average of all three replicates. (**B**). Representative images from the three independent experiments. \*\* *p* < 0.01 vs. DMSO control; \* *p* < 0.05 vs. DMSO control. Scale bar: 200 μm.
