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Introduction

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females worldwide, with an estimated 1.7 million cases and 521,900 deaths in 2012 (1,2). One third of novel cancer diagnoses in females are breast cancer and one in eight women will be diagnosed with breast cancer, with a lifetime risk of mortality due to breast cancer of 3.4% (3). Systematic chemotherapy plays a critical role in treatment of breast cancer. Doxorubicin (Dox) treatment is one of the established clinically effective strategies for the treatment of breast cancer, and primarily functions by inhibiting topoisomerases and intercalating into the DNA double helix to interfere with DNA uncoiling, which induces cell death (4). However, the efficacy of Dox is limited by its drug resistance as well as side-effects. Increasing research indicates that multidrug resistance (MDR) contributes to the failure of chemotherapeutic drugs as treatment (5). There are at least two molecular pumps in tumor cell membranes, multi-drug resistance-associated protein (MRP) and P-glycoprotein (P-gp), to expel the antitumor drugs out of the cancer cells and attenuate the drug effect. Some P-gp inhibitors used to reverse MDR were able to enhance chemosensitivity in resistant cells but caused cytotoxicity and a number of complications (6). Therefore, finding key target molecules and novel therapeutic strategies to overcome resistance and diminish the side-effects of chemotherapeutic agents are the main goals of any ideal cancer treatment protocol.

The protein phosphatase 2A (PP2A) is a key tumor suppressor that regulates signaling pathways with a high relevance in human cancer (7). Consistent with its role as a tumor suppressor, PP2A plays a critical role in the regulation of survival, cell cycle progression, and differentiation by negatively regulating the PI3K/Akt pathway and dephosphorylating and inactivating ERK and MEK1 family kinases (8). Aberrant expression, mutations, and somatic alterations of the PP2A scaffold and regulatory subunits are frequently found in human breast, lung, colon and skin cancers (9). Therefore, reactivation of PP2A activity based on its tumor suppressor properties is considered to be an attractive therapeutic strategy for human cancer treatment (10,11).

Cancerous inhibitor of protein phosphatase 2A (CIP2A), a human oncoprotein that stabilizes c-Myc by inhibiting PP2A-mediated dephosphorylation of Myc at serine 62 (8). CIP2A promotes the proliferation and aggressiveness of several cancer types including head and neck squamous cell carcinoma, oral squamous cell carcinoma, esophageal squamous cell carcinoma, colon, gastric, breast, prostate, tongue, lung, cervical cancer and acute myeloid leukemia (8,12–15). In 2011, Choi et al (16) reported that CIP2A expression is associated with Dox resistance. CIP2A has been found to involve in regulating MDR of cervical adenocarcinoma upon chemotherapy by enhancing MDR gene encoded P-gp expression through E2F1 (17). Thus, effective and discerning CIP2A inhibitors would be beneficial for adjuvant therapy to reduce the development of cancer resistance to Dox and reactivation of PP2A activity in breast cancer.

Cucurbitacins are tetracyclic triterpene natural products that are mainly found in the members of family Cucurbitaceae. It has been used as a medicinal herb because it exhibits different biological activities such as anti-diabetic, anti-inflammatory, and anticancer activities against different cancer cell lines (18). Cucurbitacin B (CuB) is one of the most abundant forms of cucurbitacins which is shown to inhibit the growth of numerous human cancer cell lines such as breast, colon, leukemia, hepatic, pancreatic and glioblastoma, and xenografts (19–21). The effect of CuB on Dox-resistant breast cancer cells has not been previously evaluated. The aim of the present study was to investigate antitumor effects and possible mechanisms of CuB on Dox resistant human breast cancer cells.

Materials and methods

Reagents

Cucurbitacin B (CuB) with a purity of up to 98% was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). CuB was dissolved in dimethyl sulf-oxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) at a stock solution of 40 mM and stored at −20°C. Doxorubicin (Dox) was purchased from Sigma-Aldrich.

Cell culture

The human breast cancer cell line MCF-7 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Human Dox-resistant breast cancer cell line MCF-7/Adriamycin (MCF-7/Adr) was purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). MCF-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, Inc., Logan, UT, USA) and antibiotics. MCF-7/Adr cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum. Additionally, 2 µg/ml Adriamycin was added into MCF-7/Adr medium. All the cells were cultured in a humidified atmosphere with 5% CO2 at 37°C.

Cytotoxic assay and cell viability

Cells were seeded into a 96-well plate and pre-cultured for 24 h, then treated with CuB for 24 h. Cell cytotoxicity was determined by MTT assay. The absorbance was measured at 490 nm by automated micro-plated reader (BioTek Instruments, Inc., Winooski, VT, USA), and the inhibition rate was calculated as followed: Inhibition rate (%) = (average A490 of the control group − average A490 of the experimental group)/(average A490 of the control group − average A490 of the blank group) × 100%. Cell viability was estimated by trypan blue dye exclusion (22).

Soft-agar colony formation assay

Cells were suspended in 1 ml of RPMI-1640 containing 0.3% low-melting-point agarose (Amresco, Cleveland, OH, USA) and 10% FBS, and plated on a bottom layer containing 0.6% agarose and 10% FBS in 6-well plate in triplicate. After 2 weeks, plates were stained with 0.2% gentian violet and the colonies were counted under a light microscope (23).

Apoptosis determination by DAPI staining

Approximately 2×105 cells/well of cells in a 12-well plate was treated with CuB for 24 h. Then cells in each treatment and control were stained by DAPI and examined and photographed by fluorescence microscopy as described (24).

Western blot analysis

Cell pellets were lysed in RIPA buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, 1 mM DTT, 1 mM NaF, 1 mM sodium vanadate, 1 mM PMSF (Sigma-Aldrich) and 1% protease inhibitors cocktail (Merck). Protein extracts were quantitated and loaded on 8–12% sodium dodecyl sulfate poly-acrylamide gel, electrophoresed and transferred to a PVDF membrane (Millipore, Kenilworth, NJ, USA). The membrane was incubated with primary antibody, washed, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Pierce). Detection was performed by using a chemiluminescent western detection kit (Cell Signaling Technology, Danvers, MA, USA) (25). The antibodies used were anti-MRP1, anti-CIP2A, anti-Akt, anti-pAkt (Ser473) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-casp-9, anti-casp-3, anti-PARP, anti-PP2A, anti-ERK1/2, anti-pERK1/2 (Thr202/Tyr204), (Cell Signaling Technology), anti-P-gp (Abcam), and anti-GAPDH (AB10016; Sangon Biotech, Co., Ltd., Shanghai, China).

PP2A activity assay

PP2A immunoprecipitation phosphatase assay kit (Upstate-Millipore, Temecula, CA, USA) was used to measure phosphate release as an index of phosphatase activity according to the manufacturer's instructions. Briefly, 100 µg protein isolated from cells was incubated with 4 µg anti-PP2A monoclonal antibody overnight. A total of 40 µl of protein A agarose beads were added and the mixture was incubated at 4°C for 2 h. Subsequently, the beads were collected and washed three times with 700 µl of ice-cold TBS and one time with 500 µl Ser/Thr assay buffer. The beads were further incubated with 750 mM phosphopeptide in assay buffer for 10 min at 30°C with constant agitation. A total of 100 µl of Malachite Green Phosphate detection solution was added and the absorbance at 650 nm was measured on a microplate reader (26).

Transfection of siRNA

Two siRNAs targeting CIP2A were designed and synthesized by Shanghai GenePharma Co., Ltd., (Shanghai, China) referred to as siRNA1 and siRNA2. The siRNA sequences were as follows: 5′-CUGUGGUUGUGUU UGCACUTT-3′ (CIP2A siRNA1), 5′-ACCAUUGAUAUCCUUAGAATT-3′ (CIP2A siRNA2), 5′-UUCUCCGAACGUGUCACGUTT-3′ [negative control (NC) siRNA].

Using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol, MCF-7/Adr cells were transfected with 100 nM siRNA. In addition, 48 h after transfection, the cells were then harvested for western blot analysis and cell viability.

Statistical analysis

All experiments were repeated at least three times and the data are presented as the mean ± SD unless noted otherwise. Differences between data groups were evaluated for significance using Student's t-test of unpaired data or one way analysis of variance and Bonferroni post-test. P-values <0.05 indicate statistical significance.

Results

Chemical structure of CuB and characterization of MCF-7 and MCF-7/Adr cells

The chemical structure of CuB is shown in Fig. 1A. First, the P-gp and CIP2A expression level was compared between MCF-7/Adr and MCF-7 cells by western blot analysis, which confirmed P-gp and CIP2A overexpression in MCF-7/Adr cells (Fig. 1B). MCF-7 and MCF-7/Adr cells were exposed to various concentrations of Dox (2.5–80 µM) for 24 h. The half-maximal inhibitory concentration (IC50) of Dox against MCF-7 cells is 6.2 µM, while IC50 of Dox against MCF-7/Adr cells is 37.78 µM. As shown in Fig. 1C, the Dox cytotoxicity was higher in MCF-7 cells than in MCF-7/Adr cells.

Figure 1 Chemical structure of CuB and characterization of MCF-7 and MCF-7/Adr cells. (A) Chemical structure of CuB. (B) Western blot analysis showing protein expression in MCF-7 and MCF-7/Adr cells using the indicated antibodies. (C) Cell viability was determined by MTT assay after Dox treatment in MCF-7 and MCF-7/Adr cells for 24 h.

Effects of CuB on MCF-7 and MCF-7/Adr breast cancer cells

MCF-7 and MCF-7/Adr cells were seeded in 96-well plates for 24 and 48 h and then treated with different concentrations of CuB and Dox (Fig. 2A–C). After 24 or 48 h, the cell viability was evaluated by the MTT assay according to the manual. Absorbance at 490 nm was measured on an automated micro-plate reader. We found that CuB had moderate cytotoxicity to MCF-7 and MCF-7/Adr cells with an IC50 of 0.45 and 0.66 µM (Table I). By trypan blue exclusion assay, we found that CuB rapidly reduced viable MCF-7/Adr (Fig. 2D) in a dose- and time-dependent manner. We investigated CuB's effect on cell colony formation activity, and the results showed that CuB significantly inhibited the clonogenic ability of MCF-7/Adr (Fig. 2E). These results suggested that CuB inhibited the anchorage-dependent (cell proliferation) and anchorage-independent (colony formation) growth of MCF-7/Adr cells.

Figure 2 Effect of CuB on MCF-7 and MCF-7/Adr breast cancer cells. (A–C) The inhibitory effects of CuB on MCF-7 and MCF-7/Adr cells analyzed by MTT assay. CuB, cucurbitacin B; Dox, doxorubicin. (D) Inhibitory effects of CuB and Dox on cell viability of MCF-7/Adr cells assayed by trypan blue exclusion assay. (E) The colony formation assays of MCF-7/Adr cells treated with CuB and Dox at indicated concentration.

Table I IC50s of CuB on breast cancer cell lines.

Table I

IC50s of CuB on breast cancer cell lines.

Cell lines	MCF-7	MCF-7/Adr
IC50 (µM)	0.45±0.03	0.66±0.05

[i] The cells were treated with CuB at various concentrations for 24 h, the cell cytotoxicity was analyzed by MTT assay, and the IC₅₀ was calculated using CalcuSyn (version 2.0; Biosoft, Cambridge, UK). Values shown are means plus or minus SD of quadruplicate determination.

CuB reverses the resistance of MCF-7/Adr cells to Dox

MTT assay revealed a significant difference between the growth-inhibiting effect of Dox on normal MCF-7 cells and on Dox resistant MCF-7/Adr cells (Fig. 1C). The IC50 of MCF-7 cells was 6.2 µM, vs. an IC50 of 37.78 µM for the MCF-7/Adr cells. However, the Dox IC50 of MCF-7/Adr cell was 18.09 µM (0.05 µM CuB treatment) and 12.94 µM (0.1 µM CuB treatment) (Table II). The resistance index (RI) of MCF-7/Adr parent group was 6.09, the MCF-7/Adr CuB 0.05 µM group 2.91 and the MCF-7/Adr CuB 0.1 µM group 2.09. The RI calculation formula was used to find the IC50 of resistant cells/IC50 of sensitive cells. Following the treatment with CuB (0.05 and 0.1 µM), the IC50 of Dox to the MCF-7/Adr cells was reduced from 37.78 to 18.09 and 12.94 µM by reversion fold (RF) 2.09- and 2.92-fold. The RF calculation formula was used to find the IC50 of Dox on MCF-7/Adr cells/IC50 of Dox (with 0.05 or 0.10 µM CuB) on MCF-7/Adr cells. Thus, low doses of CuB (0.05 and 0.1 µM) can reverse Dox resistance.

Table II Reversing effect of CuB on MCF-7/Adr cells.

Table II

Reversing effect of CuB on MCF-7/Adr cells.

Groups, CuB (µM)+Dox (µM)	Inhibition rate (%)	IC50 (µM)	Resistance index	Reversion fold
0+5	9.51±0.53	37.78	6.09	1
0+10	14.84±2.94
0+20	23.75±3.61
0+30	52.03±5.36
0+50	71.64±8.34
0+80	83.35±3.89
0.05+5	15.35±2.74	18.09	2.91	2.09
0.05+10	25.62±3.88
0.05+20	36.48±4.92
0.05+30	63.65±5.12
0.05+50	86.46±3.71
0.05+80	89.61±7.15
0.1+5	20.17±3.58	12.94	2.09	2.92
0.1+10	35.72±5.46
0.1+20	48.69±5.77
0.1+30	75.47±4.83
0.1+50	89.43±6.72
0.1+80	98.51±6.02

[i] The Dox IC₅₀ of MCF-7 cell was 6.20 µM. Data are presented as the mean ± SD; n=4. The RI calculation formula was used to find the IC₅₀ of resistant cell/IC₅₀ of sensitive cells. The RF calculation formula was used to find the IC₅₀ of Dox on MCF-7/Adr cells/IC₅₀ of Dox (with 0.05 or 0.10 µM CuB) on MCF-7/Adr cells.

CuB induces apoptosis and influences the expression of P-gp, MRP1 in MCF-7/Adr cells

We next tested whether or not CuB induces apoptosis of MCF-7/Adr cells. By an optical light microscope, we found some dead MCF-7/Adr cells floating in the medium treated with CuB. The cell death is reminiscent of the phenomena induced by apoptosis. We investigated the nucleus morphological changes by DAPI staining. As shown in Fig. 3A, we observed the nuclear condensation and fragmentation with CuB treatment which are typical changes in cell apoptosis. Furthermore, a western blot analysis was used to detect the activation of the caspase-9 (casp-9) initiator caspase, caspase-3 (casp-3) effector caspase and its substrate, poly(ADP-ribose) polymerase (PARP) (Fig. 3B). CuB was demonstrated to induce a significant dose-dependent decrease in pro-casp-9, pro-casp-3 and the cleavage of PARP, in MCF-7/Adr cells, indicating that CuB induced caspase-dependent apoptosis. We detected expression levels of MDR related factors P-gp and MRP1 by western blot analysis (Fig. 3C). The results indicated that P-gp, and MRP1 expression of MCF-7/Adr cells were downregulated by treatment with increasing concentration of CuB. In addition, the decreased expression of P-gp and MRP1 in MCF-7/Adr cells may in part contribute to the reversal of MDR.

Figure 3 CuB induces apoptosis and influences the expression of P-gp, MRP1 in MCF-7/Adr cells. (A) MCF-7/Adr cells were incubated with various doses of CuB for 24 h. Cells were examined by DAPI staining. (B and C) MCF-7/Adr cells were treated with increasing concentrations of CuB for 24 h. Western blot analysis was conducted using the indicated antibodies.

CuB targets CIP2A to reactivate protein phosphatase 2A

Phosphorylated Akt (pAkt) is a cancer MDR locus (27). We next investigated the role of Akt in CuB-induced apoptosis in MCF-7/Adr cells. As shown in Fig. 4A, CuB decreased Akt phosphorylation in a dose-dependent manner. PP2A, one of the main serine-threonine phosphatases, plays a critical role in the regulation of cell cycle progression, survival, and differentiation by negatively regulating the PI3K/Akt pathway and dephosphorylating and inactivating MEK1 and ERK family kinases (26). We tested effects of CuB on PP2A activity, and found that CuB upregulated PP2A activity (Fig. 4B). CIP2A is an oncogenic PP2A inhibitor protein that is highly expressed in malignant cancers (28). Furthermore, we tested effects of CuB on CIP2A expression and found that the protein level of CIP2A decreased (Fig. 4C), indicating that CuB targeted CIP2A, at least in part, to reactivate PP2A. Taken together, these data indicate that the CIP2A/PP2A/Akt pathway may mediate the sensitizing effect of CuB.

Figure 4 CuB targets CIP2A to reactivate protein phosphatase 2A. (A) MCF-7/Adr cells were treated with increasing concentrations of CuB for 24 h. Western blot analysis was conducted using the indicated antibodies. (B) MCF-7/Adr cells were treated with increasing concentrations of CuB for 24 h. PP2A activity was measured by PP2A immunoprecipitation phosphatase assay. *P<0.05. (C) MCF-7/Adr cells were treated with increasing concentrations of CuB for 24 h. Western blot analysis was conducted using the indicated antibodies.

Silencing CIP2A enhances CuB-induced growth inhibition and apoptosis in MCF-7/Adr

We next examined whether CIP2A knockdown would alter cellular sensitivity to CuB. Two siRNAs targeting CIP2A were synthesized and used. As shown in Fig. 5A, knockdown by both siRNA1 and siRNA2 markedly decreased CIP2A protein in MCF-7/Adr cells. These data showed that CIP2A siRNA1 and 2 were specific and efficient in reducing CIP2A expression. To evaluate the role of CIP2A in CuB-induced proliferation inhibition, MCF-7/Adr cells were transfected with siRNA1 or siRNA2 targeting CIP2A, followed by CuB treatment. Cell viability and western blot analysis were used to detect cell growth and protein expression. CIP2A silencing enhanced CuB-induced growth inhibition (Fig. 5B) and promoted CuB-induced apoptotic effect (Fig. 5C). Notably, CIP2A depletion also enhanced CuB-induced MDR inhibition. These data demonstrate that CIP2A plays a critical role in CuB-reversed MCF-7/Adr multidrug resistance.

Figure 5 Silencing CIP2A enhances CuB-induced growth inhibition and apoptosis in MCF-7/Adr. (A) MCF-7/Adr cells were transfected with 100 nM CIP2A-specific siRNA or NC siRNA for 48 h. Cells were harvested for western blot analyses. (B) MCF-7/Adr cells were transfected with CIP2A-specific siRNA or NC siRNA, followed by treatment with CuB (0.4 µM) for indicated times, and analyzed by trypan blue exclusion assay. *P<0.05. (C) MCF-7/Adr cells were transfected with CIP2A-specific siRNA or NC siRNA, followed by treatment with CuB (0, 0.2 and 0.4 µM) for 24 h. Western blot analysis was conducted using the indicated antibodies.

Discussion

Tumorigenesis and chemoresistance is tightly related to malfunction of the cellular signaling pathways that control cell proliferation, survival, or death. The malfunction contains increased expression of ATP-dependent drug efflux pumps and decreased influx, increased drug metabolism enzymes, impairment of cell death pathways, enhancement of cell survival pathways, alternation of drug metabolism, mutations in cell cycle pathways and superior DNA repair (29). The aims of the present study were to identify an effective MDR reversing agent with fewer side-effects and to gain insight regarding its molecular mechanism. Compounds from natural source constitute an indispensable candidate drug library for pharmacotherapy. CuB is a representative therapeutic agent for anticancer activities. Recently, CuB was reported to enhance the anticancer effects of cisplatin, gemcitabine, methotrexate, docetaxel, and gemcitabine in laryngeal squamous, pancreatic, and breast cancers and osteosarcoma (30). However, the relationship between CuB and Dox-resistance in MCF-7/Adr cells has yet to be firmly established. Inhibition of cell proliferation is an efficient strategy in cancer therapy. In the present study, we first showed that CuB inhibited MCF-7/Adr cell proliferation (Fig. 2B and C), cell viability (Fig. 2D) and soft-agar colony formation (Fig. 2E).

We investigated the effect of the combination of CuB and Dox on the MCF-7/Adr cells and identified that CuB in combination with Dox had an improved effect compared with Dox or CuB alone. Following the treatment with CuB (0.05 and 0.1 µM), the IC50 of Dox to the MCF-7/Adr cells was significantly reduced from 37.78 to 18.09 and 12.94 µM by 2.09- and 2.92-fold (Table II). The RI of of MCF-7/Adr parent group was 6.09, the MCF-7/Adr CuB 0.05 µM group 2.91 and the MCF-7/Adr CuB 0.1 µM group 2.09, respectively. Furthermore, the present study explored the mechanisms of CuB in reversing Dox resistance.

Evading apoptosis is one of the hallmarks of drug resistance, and targeting apoptosis has become a cancer therapeutic strategy (31). Apoptosis is accompanied by various morphological changes, including nuclear condensation, apoptotic bodies, DNA fragmentation and cell surface changes. Nuclear morphology in MCF-7/Adr cells was analyzed using DAPI staining; we found changes in nucleus condensation, which are typical characteristics of apoptosis (Fig. 3A). Therefore, CuB might have the ability of induction of cell apoptosis. The mechanisms of apoptosis involve two signaling pathways: the mitochondrial pathway (intrinsic apoptotic pathway) and the cell death receptor pathway (extrinsic apoptotic pathway) (32). The extrinsic and intrinsic apoptotic pathways that ultimately lead to activation of effector caspases (casp-3, -2 and -7) have been characterized (33,34). The decrease of pro-casp-9, pro-casp-3, as well as the proteolysis of PARP (Fig. 3B), indicate that casp-3 is activated. Thus, CuB may trigger apoptosis by activating the intrinsic apoptosis pathway which results in activation of effector casp-9. Drug resistance is largely mediated through overexpression of MDR, MRP, drug resistance protein, and proteasome subunits, increases in antioxidant defenses, and TOP2 activity; these results have been widely verified (35–37). The present study identified that the treatment of CuB was able to reverse the MDR of the MCF-7/Adr cells via the downregulation of P-gp and MRP1 (Fig. 3C).

PP2A is a serine/threonine phosphatase that has a critical role in regulating various cellular processes, including signaling transduction, protein synthesis, cell cycle determination, metabolism, apoptosis and stress response (38). Because loss of PP2A function has been identified in various malignant diseases such as cancer of the colon, liver, lung and breast, it has been suggested that PP2A functions as a tumor suppressor and enhancing PP2A activity could be an effective approach for anticancer treatment (39). In this study, we showed that by inhibiting CIP2A, activity of PP2A was significantly enhanced and expression of pAkt was downregulated in MCF-7/Adr cells (Fig. 4). Several cellular inhibitors of PP2A have been identified, including SET (26) and CIP2A. CIP2A, originally named KIAA1524 or P90, has been cloned from patients with HCC (8). CIP2A is associated with clinical aggressiveness in human breast cancer and promotes the malignant growth and metastasis of breast cancer cells. Induction of CIP2A is often associated with chemoresistance in cancer cells, and the inhibition of CIP2A in combination with chemotherapy may enhance the efficacy of cancer treatment (40). We knocked down CIP2A expression in MCF-7/Adr cells and found that CIP2A depletion significantly promoted CuB induced apoptosis and reversed MDR (Fig. 5). Our data validated the mechanism by which CuB-reversed MDR and induced cancer cell apoptosis in MCF-7/Adr cells, that is, reversed MDR and induction of cancer cell apoptosis by inhibiting CIP2A to reactivate PP2A and enhance PP2A-dependent pAkt downregulation.

In conclusion, our results suggest that further studies investigating the detailed molecular modification of the PP2A/CIP2A signaling pathway by CuB and exploring its possible application in other malignant diseases are warranted.

Acknowledgments

The present study was supported by grants from the National Natural Sciences Foundation of China (grant no. 81400157); the Natural Science Foundation of Hubei Provincial Department of Education (grant no. Q20152106); the Faculty Development Grants from Hubei University of Medicine (grant nos. 2014QDJZR08, 2015QDJZR16); the Foundation for Innovative Research Team of Hubei University of Medicine (grant no. 2014CXX05); the Key Discipline Project of Hubei University of Medicine and the National Training Program of Innovation and Entrepreneurship for Undergraduates (grant no. 201610929001).

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