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

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.

neoplastic cells such as inducing cellular apoptosis through mitochondria-mediated pathway in human prostate cancer cells 9 , leukemia cells 10 and gastric cancer cells 11 , inhibiting migration and metastasis in human prostate cancer cells 12 , breast cancer cells 13 and lung cancer cells 14 , attenuating angiogenesis in murine melanoma 15 and lung carcinoma 16 .
Transcriptome analysis associated with bioinformatics data mining tools provides an opportunity to simultaneously analyze a large number of genes/targets and identify the mechanisms of action after treatments. RNA-seq has many advantages over microarray due to it being free from the probe-specific hybridization of microarrays and has expansive coverage, allowing the unbiased detection of both coding and noncoding novel transcripts as well as low-abundance transcripts 17 .
Several breast cancer cell lines used in biological studies have been classified based on the following measures: histological type, tumour grade, lymph node status and the presence of predictive markers such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) 18 . Yet, some studies have provided intriguing insights into anti-breast cancer activity of shikonin 13,[19][20][21][22] 22 . Although shikonin has been demonstrated as inhibiting the proliferation in different types of breast cancer cells, the mechanism of action has not been investigated. Herein, the aim of this study is to examine the mechanism of action of shikonin in regulating the growth of different types of breast cancer cells using the RNA-seq approach.

Results
Shikonin inhibits the growth of human breast cancer cells. To examine whether shikonin affects the growth of human breast cancer cells, different types of human breast cancer cell lines, MCF-7, SK-BR-3 and MDA-MB-231 were treated with shikonin for 24 hr. The cell viability was analyzed by MTT assay. As shown in Fig. 1, shikonin revealed significant cytotoxic effects in different types of human breast cancer cells in a dose-dependent manner. The IC 50 of shikonin at 24 hr on MCF-7, SKBR-3 and MDA-MB-231 cells were 10.3 μΜ, 15.0 μΜ, 15.0 μΜ respectively ( Fig. 1 A-C). In addition, we also examined whether shikonin affects the growth The cell viability was determined by the MTT assay. Data points represent the mean ± SD of three independent experiments. IC 50 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).

Shikonin arrests the progression of cell cycle and induces apoptosis in human breast cancer cells.
To investigate the mechanisms underlying shikonin-induced inhibition of cell growth, changes in cell cycle progression of human breast cancer cells were determined after shikonin treatment using flow cytometry. As shown in Fig. 2A,B, cells in sub-G1 phase were increased under shikonin treatment in a dose-dependent manner. These results suggest that shikonin inhibited cellular proliferation of human breast cancer cell lines, MCF-7, SKBR-3 and MDA-MB-231 via arrested G1 phase of the cell cycle. Moreover, we also performed Annexin V/PI apoptosis assay. Our results showed that shikonin induced apoptosis in MDA-MB-231 cells (Fig. 2C).

RNA-seq transcriptome analysis of different human breast cancer cell lines under shikonin treatment.
To study the gene expression profiling of human breast cancer cells under shikonin treatment, different human breast cancer cell lines, MCF-7, SK-BR-3 and MDA-MB-231 were treated with 10 μΜ shikonin for 6 hr. The gene expression profiling was performed using RNA-seq. As shown in Fig. 3A, numbers of significantly differentially expressed (SDE) genes (>2-fold change) in different human breast cancer cell lines under shikonin treatment were identified. The results of Venn diagrams analysis showed 38 SDE genes (termed as common genes), which were expressed in different types of breast cancer cells under shikonin treatment (Fig. 3A). Thirty-six common genes were consistently upregulated and one common gene was consistently downregulated in different types of breast cancer cells under shikonin treatment (Table 1). Only RN7SL1 was inconsistently expressed in different types of breast cancer cells under shikonin treatment ( Table 1).

Analysis of common genes using both functional enrichment analysis and KEGG enrichment analysis after shikonin treatment by MCF-7, SK-BR-3 and MDA-MB-231 cells.
We analyzed the common genes by functional enrichment analysis. The results showed that these genes participated mainly in adjustment of cell death, apoptosis, cell cycle and cell growth ( Table 2). We further analyzed the common genes by KEGG enrichment analysis. The results showed that the common SDE genes regulated by shikonin were significantly involved in MAPK signaling pathway, P53 signaling pathway, antigen processing and presentation, spliceosome, bladder cancer, endocytosis, HIF1 signaling pathway, cell cycle, pathways in cancer and the PI3K-AKT signaling pathway (Table 3). Several common genes were involved in regulating these pathways such as HSPA1B, HSPA1A, HSPA6, GADD45G, DUSP1, DUSP2, CDKN1A, SESN2, PGF, HMOX1 (Table 3 and Fig. 3B).

Validation of RNA-seq data by qRT-PCR.
To further validate the results of RNA-seq, qRT-PCR was performed on 5 genes (DUSP1, DUSP2, CDKN1A, SESN2, PGF) randomly selected from common genes using the same RNA samples that were used in RNA-seq. A total of 15 RNA-seq samples were validated by qRT-PCR (5 representative genes in three different types of breast human breast cancer cells. Correlation of the expression ratios from the RNA-seq and qRT-PCR data were highly correlated (R = 0.9; P = 5.7 × 10 −6 ) (Fig. 4).
Shikonin enhances the expression of DUSP1 and DUSP2 in both RNA and protein levels and decreases the phosphorylation of JNK and p38. The results of RNA-seq showed that shikonin induces the expression of DUSP1 and DUSP2 in breast cancer cells (Table 1). We confirmed the results of RNA-seq using qRT-PCR. As shown in Fig. 5A, the expression of DUSP1 and DUSP2 was increased in MCF-7, SK-BR-3 and MDA-MB-231 cells after shikonin treatment. However, there was no effect on the expression of DUSP1 and DUSP2 in M10 cells after shikonin treatment. In addition, we examined the expression of DUSP1 and DUSP2 in MDA-MB-231 after shikonin treatment. As shown in Fig. 5B, shikonin induced the expression of DUSP1 and DUSP2 in MDA-MB-231 cells. Furthermore, our results also showed that shikonin decreased the phosphorylation of JNK 1/2 and p38 in MDA-MB-231 cells, whereas the phosphorylation of ERK 1/2 exhibited no effect after shikonin treatment in MDA-MB-231 cells (Fig. 5C). On the other hand, we analyzed the expression of DUSP1 and DUSP2 using DriverDB 23,24 . As shown in Fig. 5D, DUSP1 and DUSP2 were down-regulated in several types of cancers.

Discussion
The use of Chinese herbal medicine for health promotion and adjuvant therapy is becoming increasingly popular worldwide. Zicao, the dried root of Lithospermum erythrorhizon, is a Chinese herbal medicine widely used for its anti-inflammatory properties in China, Japan, Korea, etc. 25 . Shikonin is a major component of zicao and has been reported to suppress the growth of several types of cancer through a wide spectrum of anticancer mechanisms 4 . However, the mechanism of action of shikonin in regulating the growth of breast cancer cells is limited.
Various subtypes of breast cancer have distinct prevalence and outcomes. In the present study, we used three different types of breast cancer cell lines, MCF-7 (Luminal A; ER + , PR +/− , HER2 − , good outcome), SK-BR-3 (HER2; ER − , PR − , HER2 + , poor outcome), MDA-MB-231 (Claudin-low; ER − , PR − , HER2 − , poor outcome) 26 and demonstrated that shikonin inhibits the growth of these cancer cells including arresting the progression of the cell cycle and inducing apoptosis. To further explore the mechanism of action of shikonin in regulating the growth of different types of breast cancer cells, we analyzed the gene expression profiling of different types of breast cancer cells using RNA-seq. We found 38 common genes regulated by shikonin in different types of breast cancer cells and further analyzed these common genes using KEGG enrichment analysis. The analytic results indicated that these common genes were significantly involved in the MAPK signaling pathway, P53 signaling pathway, antigen processing and presentation, spliceosome, bladder cancer, endocytosis, HIF1 signaling pathway, cell cycle, pathways in cancer and the PI3K-AKT signaling pathway.
In particular, the results of RNA-seq pointed out that shikonin induced the expression of both DUSP1 and DUSP2, the upstream regulators of MAPK signaling pathway. Also, the results of qRT-PCR confirmed that shikonin induced the expression of both DUSP1 and DUSP2 in different types of breast cancer cells. The expression ratios from RNA-seq and qRT-PCR data were highly correlated. Moreover, our experimental results also demonstrated that shikonin induced the protein expression of both DUSP1 and DUSP2 in different types of breast cancer cells. In addition, we also found that DUSP1 and DUSP2 were down-regulated in several types of cancers. Therefore, induction of DUSP1 and DUSP2 might be a therapeutic strategy for treating cancer.
DUSP1 and DUSP2 are the members of the threonine-tyrosine dual-specificity phosphatase family which play an important role in regulating the dephosphorylation of threonine and tyrosine residues on MAPKs 27 . MAPKs are signaling components that link extracellular signals to regulate a wide range of cellular processes in cancer cells including growth, differentiation, migration and apoptosis 28 . Our experimental results indicated that shikonin reduced the phosphorylation of JNK 1/2 and P38 in MDA-MB-231 cells. Previous studies pointed out that JNK and P38 MAPK pathways regulated the progression of cell cycle, modulated the cell survival and differentiation, and controled the balance of apoptosis and autophagy in response to chemotherapeutic agents in cancer cells 29,30 . Therefore, we 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.
In summary, our results showed that shikonin inhibits cell growth and induces apoptosis in different types of breast cancer cells. We further examined the transcriptome regulation of shikonin in different types of breast cancer cells using the RNA-seq. We firstly reported that shikonin affects the expression of common genes among different types of breast cancer cells and is involved in regulating several anticancer mechanisms of action. Particularly, our results indicated that shikonin induces the expression DUSP1 and DUSP2 and reduces the activity of their downstream signaling molecules, JNK and p38. These results suggest that shikonin induces apoptosis through enhancing the expression of DUSP1 and DUSP2 (Fig. 5E).

Materials and Methods
Chemicals and reagents. Cell HSPA1A, HSPA1B, SESN2, HSPH1, TSPYL2, SLC25A25,  MAP1LC3B, HMOX1, BAG3, ETAA1, SIK1, MAFF, ARC RNA preparation and RNA-seq. Total Table 3. KEGG enrichment analysis of common genes after shikonin treatment by MCF-7, SK-BR-3 and MDA-MB-231 cells.  Functional enrichment analysis. For the differentially expressed genes associated with treatment responses, we performed functional enrichment analysis, as described in our previous studies 23,24 to interpret their biological functions. In brief, we used the topGO and GeneAnswers packages of Bioconductor to calculate the topology of the GO graph, as well as to visualize the many-to-many relationships between GO terms and genes. In the "Pathway" analysis, we used collections from KEGG 31 , PID 32 , Biocarta (http://www.biocarta.com/), REACTOME 33 , and MSigDB 34 to annotate driver genes.
Statistical analysis. Data were obtained from at least three independent experiments and expressed as the mean ± standard deviation for each group. Statistical analyses, including Student's t-test, one-way analysis of variance and regression analysis were performed using GraphPad Prism 4.0 software (GraphPad, Inc., La Jolla, CA, USA). P < 0.05 was considered to indicate a statistically significant difference.