Baitouweng decoction suppresses growth of esophageal carcinoma cells through miR-495-3p/BUB1/STAT3 axis

Figure 1 Analysis of differentially expressed genes. A: Volcanic map of differentially expressed genes (DEGs). Red, upregulated; green, downregulated; gray, no significant difference; B: Correlation between genes detected by gene chip and qRT-PCR; C: Protein-protein interaction network for DEGs.

Figure 2 Enrichment analysis of differentially expressed genes. A: Distribution of changed genes in Gene Ontology analysis (P < 0.05); B: The canonical pathway of significant change was analyzed with IPA software. Yellow, upward; blue, down; C: Using Cytoscape to describe the hub gene network. The darker the node, the higher the score; D: Target genes of BUB1 predicted using iRegulon in Cytoscape. The nodes in red and green indicate upregulated and downregulated proteins; E: Binding motif of BUB1.

Figure 3 Ingenuity pathway analysis. A–C: Top-ranked enriched networks based on differentially abundant genes. Red, increased; green, decreased.

Figure 4 Baitouweng decoction inhibits proliferation of esophageal cancer cells in a dose- and time-dependent manner. A: Dose-effect relationship of Baitouweng decoction on esophageal cancer cells; B: Time-effect relationship of Baitouweng decoction on esophageal cancer cells.

Figure 5 BUB1 is a target gene of miR-495-3p. A: Diagram of the putative binding sites of miR-495-3p in the 3'-untranslated regions of BUB1; B and C: Dual luciferase activity in EC9706 cells and KYSE150 cells. ^aP < 0.01 compared with normal control. UTR: Untranslated region; WT: Wild type; MUT: Mutant.

Figure 6 Baitouweng decoction inhibits BUB1 expression. A: qRT-PCR analysis of the effect of Baitouweng decoction on the expression of BUB1 in EC9706 and KYSE150 cells; B: Western blot analysis of Baitouweng decoction on the expression of BUB1 in EC9706 and KYSE150 cells; C: Baitouweng decoction or miR495-3p mimics significantly upregulate the expression of miR-495-3p in EC9706 and KYSE150 cells. ^aP < 0.05, ^bP < 0.01 compared with normal control.

Figure 7 Effect of Baitouweng decoction on the cell cycle and apoptosis of esophageal cancer cells. A and B: Baitouweng decoction induces G2/M phase arrest in EC9706 and KYSE150 cells; C and D: Baitouweng decoction increases the apoptosis of EC9706 and KYSE150 cells. ^aP < 0.05, ^bP < 0.01 compared with normal control.

Figure 8 Effect of Baitouweng decoction on migration and invasion of esophageal cancer cells. A and B: Baitouweng decoction reduces the migration activity of both EC9706 and KYSE150 cells; C and D: Baitouweng decoction reduce the invasion activity of both EC9706 and KYSE150 cells. 1: Normal control; 2: NC mimics; 3: miR-495-3p mimics; 4: Baitouweng decoction; 5: NC mimics + Baitouweng decoction; 6: miR-495-3p mimics + Baitouweng decoction.

Figure 9 Protein expression of STAT3, p-STAT3, CCNB1, CDK1, Bax, Caspase3, and Caspase9 in different groups detected using Western blot. A and B: EC9706 cells; C and D: KYSE150 cells. ^bP < 0.01 compared with normal control.