Transcriptome Analysis of Triple-Negative HCC1937 and MDA-MB-231 Breast Cancer Cells Treated with Revealed the Regulation of Migration and Invasion via the Downregulation of the Genes JAK2, ROCK1 and ROCK2

Abstract

Triple-negative breast cancer (TNBC) is a type of breast cancer with high mortality due to aggressive tumor behavior and limited treatment options. Natural products have been studied for their potential as alternatives to combine with cancer drugs to improve treatment efficacy. Plant phytoconstituents can regulate cellular processes such as proliferation, differentiation, invasion, angiogenesis, and migration. has been used in traditional medicine; it contains flavonoids, phenols, alkaloids, glycosides, bufadienolides, and tannins, among others. In the present investigation, we evaluated the effects of aqueous extract and the underlying molecular mechanisms in two breast cancer cell lines, HCC1937 and MDA-MB-231, with a focus on cellular migration. The cytotoxic activity results revealed that HCC1937 cells are more sensitive to aqueous extracts than are MDA-MB-231 cells. Compared with that of 48 h-treated MDA-MB-231 cells, cell migration was affected in HCC1937 cells after 24 h of treatment. The extract had greater anti-invasion effects on HCC1937 cells (37.7%, p < 0.01) than on MDA-MB-231 cells (47%, p < 0.05). An RNA-seq assay revealed 1850 differentially expressed genes (DEGs) in HCC1937 cells versus 1534 in MDA-MB-231 cells. WebGestalt analysis revealed the downregulation of genes involved in cell-cell adhesion, ruffle organization, and cell-substrate junctions, with more downregulated genes in HCC1937 cells. The downregulated genes analyzed with KOBAS-i were associated with MAPK signaling, focal adhesion, PI3K-Akt signaling, regulation of the actin cytoskeleton, and the ECM-receptor interaction pathway. In addition, IPA network analysis revealed that the RHO GTPase cycle, RHOA signaling, JAK/STAT signaling, actin cytoskeleton signaling and Integrin signaling pathways were inhibited. The data from the docking study indicated the binding potential of quercetin-3-O-arabinoside, kaempferol, quercetin, and apigenin with the JAK2, ROCK1, ROCK2, and PIK3CA proteins. Thus, our results demonstrate that inhibits the proliferation and invasion of TNBC cells by targeting genes involved in these important processes; thus, has the potential to be used in combination with chemotherapeutic drugs for cancer treatment.

1

Effect of the aqueous extract of K. pinnata on cell migration after 72 h of treatment. Microscopy images of wound healing assays in control and treated HCC1937 (A) and MDA-MB-231 (B) cells. Images were taken at 0, 24, 48, and 72 h after scratch formation (scale bar = 100 μm). Percentage of wound closure in HCC1937 (C) and MDA-MB-231 (D) cells. The statistical significance is represented by asterisks (*p < 0.05; **p < 0.01) compared with the control without extract.

2

Transwell assay for cell invasion after treatment with the aqueous extract of K. pinnata. Microscopy images of HCC1937 and MDA-MB-231 cells. Images were taken 20 h after treatment (scale bar = 200 μm). The statistical significance is represented by asterisks (*p < 0.05; **p < 0.01) compared with the control without extract.

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Shared genes between HCC1937 and MDA-MB-231 cells after treatment with aqueous extracts of K. pinnata. HCC1937 and MDA-MB-231 cells were treated with aqueous extracts. There were 737 shared genes when the cells were treated with the aqueous extract.

4

GO Slim summary of overrepresentation analysis (ORA) of HCC1937 cells after 72 h of aqueous extract of K. pinnata treatment (A) Biological process, cellular component, and molecular function categories are represented by red, blue, and green bars, respectively. The height represents the enriched number of genes in each category (B) Network structure for functional categories via a directed acyclic graph (DAG).

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GO analysis of MDA-MB-231 cells after 72 h of aqueous extract of K. pinnata treatment. GO analysis (A) Biological process, cellular component, and molecular function categories are represented by red, blue, and green bars, respectively. The height represents the enriched number of genes in each category. (B) Network structure for functional categories via a directed acyclic graph (DAG).

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JAK/STAT signaling pathway. JAK/STAT signaling in HCC1937 cells treated with aqueous extracts of K. pinnata and MDA-MB-231 cells. Genes downregulated (green) Molecules predicted to be downregulated (blue), molecules predicted to be inhibited (blue line), and indirect relationships (dashed lines). The intensity of the color indicates the magnitude of gene expression regulation. The gloss glow indicates activity when the measurement is the opposite, on the basis of the IPA knowledgebase.

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RHOA signaling pathway RHOA signaling in HCC1937 cells treated with aqueous extracts of K. pinnata and MDA-MB-231 cells. Genes that are overexpressed (red), downregulated (green), molecules that are predicted to be activated (orange line), molecules that are predicted to be downregulated (blue), molecules that are predicted to be inhibited (blue line), and indirect relationships (dashed lines). The intensity of the color indicates the magnitude of gene expression regulation. The gloss glow indicates activity when the measurement is the opposite, on the basis of the IPA knowledgebase.

8

2D interaction of (a) apigenin in JAK2, (b) quercetin in JAK2, (c) quercetin-3-O-arabinoside in JAK2, (d) kaempferol in JAK2, (e) apigenin in PIK3CA, (f) quercetin in PIK3CA, and (g) quercetin-3-O-arabinoside in PIK3CA.

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2D interaction of (a) apigenin in ROCK1, (b) quercetin in ROCK1, (c) quercetin-3-O-arabinoside in ROCK1, (d) kaempferol in ROCK1, (e) apigenin in ROCK2, (f) quercetin in ROCK2, (g) quercetin-3-O-arabinoside in ROCK2 and (h) kaempferol in ROCK2.

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Relevant gene–gene interactions in the HCC1937 cell line were constructed on the basis of KEGG enrichment analysis by KOBAS (hsa05205, hsa04014, hsa04010, hsa04151, and hsa04510). Genes that were underexpressed (blue) or overexpressed (red) were involved in processes related to cell motility, cell proliferation and apoptosis.

11

Related gene–gene interactions in the MDA-MB-231 cell line were constructed on the basis of KEGG enrichment analysis by KOBAS (hsa04510, hsa04512, hsa05205, hsa04010, and hsa04151). Genes underexpressed (blue) were involved in processes related to cell motility and cell proliferation.