RNA-seq transcriptome analysis of breast cancer cell lines under shikonin treatment

Abstract

Shikonin is a naphthoquinone isolated from the dried root of Lithospermum erythrorhizon, an herb used in Chinese medicine. Although several studies have indicated that shikonin exhibits antitumor activity in breast cancer, the mechanism of action remains unclear. In the present study, we performed transcriptome analysis using RNA-seq and explored the mechanism of action of shikonin in regulating the growth of different types of breast cancer cells. The IC₅₀ of shikonin on MCF-7, SKBR-3 and MDA-MB-231 cells were 10.3 μΜ, 15.0 μΜ, 15.0 μΜ respectively. Our results also demonstrated that shikonin arrests the progression of cell cycle and induces apoptosis in MDA-MB-231 cells. Using RNA-seq transcriptome analysis, we found 38 common genes that significantly express in different types of breast cancer cells under shikonin treatment. In particular, our results indicated that shikonin induces the expression of dual specificity phosphatase (DUSP)-1 and DUSP2 in both RNA and protein levels. In addition, shikonin also inhibits the phosphorylation of JNK and p38, the downstream signaling molecules of DUSP1 and DUSP2. Therefore, our results suggest that shikonin induces the expression of DUSP1 and DUSP2 which consequently switches off JNK and p38 MAPK pathways and causes cell cycle arrest and apoptosis in breast cancer cells.

Figure 1

Effect of shikonin on the cell viability in breast cancer cell lines. Different breast cancer cells. (A) MCF-7, (B) SK-BR-3, (C) MDA-MB231, and (D) human mammary epithelial cells, M10, were incubated with different concentrations of shikonin (0–50 μM) for 24 h. The cell viability was determined by the MTT assay. Data points represent the mean ± SD of three independent experiments. IC₅₀ values were calculated by GraphPad Prism 4.0 software using a sigmoidal curve fit based on nonlinear regression. Statistical significance was assessed by one-way ANOVA followed by Tukey post-hoc test and represented as follows: *P < 0.05 and **P < 0.01 vs. shikonin 0 μM (DMSO control).

Figure 2

Effect of shikonin on the cell cycle progression and apoptosis in breast cancer cell lines. (A) Different breast cancer cells, MCF-7, SK-BR-3 and MDA-MB231, were incubated with different concentrations of shikonin (0–10 μM) for 24 h. Representative cell cycle distribution of each cell type was analyzed by flow cytometry. (B) Percentage of sub-G1 in different breast cancer cells under shikonin treatment was assessed by Student’s t-test. The statistical significance of the difference between two experimental measurements was represented as follows: *P < 0.05 vs. shikonin 0 μM (DMSO control). (C) MDA-MB231 cells were treated with different concentrations of shikonin (0–10 μM) for 24 h. Cells were collected, stained with Annexin V and PI, and analyzed by flow cytometry. Data are representative of at least three independent experiments with similar results.

Figure 3

Intersectional analysis of SDE genes from breast cancer cells after shikonin treatment identified by RNA-seq and analysis of common genes using KEGG enrichment analysis. (A) Numbers of SDE genes from MCF-7, SK-BR-3, and MDA-MB-231 cells after shikonin treatment for 6 h were 408, 714, and 323, respectively. (B) 38 common genes were further used for KEGG enrichment analysis. The blue circles indicate the common genes from different types of breast cancer cells after shikonin treatment. The yellow circles show the biological functions and signaling pathway regulated by shikonin in breast cancer cells.

Figure 4

Correlation of gene expression ratios between RNA-seq and qRT-PCR. A total of 15 RNA-seq samples were validated by qRT-PCR (5 representative genes, DUSP1, DUSP2, CDKN1A, SESN2, and PGF, in three different types of breast human breast cancer cells. Data from both RNA-seq and qRT-PCR were normalized by setting the expression level of untreated control.

Figure 5

Effect of shikonin on the expression level of DUSP1 and DUSP2 and the activation of MAPKs pathway in breast cancer cells. (A) Different breast cancer cells, MCF-7, SK-BR-3 and MDA-MB231, and human mammary epithelial cells, M10, were incubated with or without shikonin 10 μM for 6 h. The expressions of DUSP1 and DUSP2 were determined by qRT-PCR. Data are presented as mean ± SD from three independent experiments. The statistical significance of the difference between two experimental measurements was assessed by Student’s t-test and represented as follows: ***P < 0.001 vs. shikonin 0 μM (DMSO control). (B) MB-231 cells were treated with different concentrations of shikonin for 6 hr. The expressions of DUSP1 and DUSP2 were analyzed by Western blot. The expression of β-actin was used as a loading control. (C) MB-231 cells were treated with different concentrations of shikonin for 24 hr. The expressions of phospho-JNK 1/2, JNK 1/2, phospho-p38, p38, phospho-ERK1/2, and ERK 1/2 were analyzed by Western blot. The Western blotting results are representative of results obtained in three separate experiments. (D) Gene expression levels of DUSP1 and DUSP2 from TCGA RNA-seq data in many types of cancer were analyzed by DriverDB^,. The list of abbreviations is shown as follows: BLCA: bladder urothelial carcinoma; BRCA: breast invasive carcinoma; BRCA-T: BRCA-associated triple-negative breast cancer; CESC: cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL: COAD: colon adenocarcinoma; GBM: glioblastoma multiforme; HNSC: head-neck squamous cell carcinoma; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; PAAD: pancreatic adenocarcinoma; PCPG: pheochromocytoma and paraganglioma; PRAD: prostate adenocarcinoma; READ: rectum adenocarcinoma; SARC: sarcoma; SKCM: skin cutaneous melanoma; THCA: thyroid cancer; THYM: thymoma; UCEC: uterine corpus endometrial carcinoma. (E) Shikonin inhibits cell growth and induces apoptosis in different types of breast cancer cells through enhances the expression of DUSP1 and DUSP2 and reduces the activity of their downstream signaling molecules, JNK and p38.