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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

The epithelial–mesenchymal transition (EMT) is a process in which polarized epithelial cells are converted into motile mesenchymal cells. During cancer development, EMT is conducive to tumor dissemination and metastatic spread. While overexpression of metadherin (MTDH) in breast cancer cell lines and tissues has been found to be associated with aggressive tumor behavior, its precise role in invasion and metastasis is largely unknown. Here we report that MTDH overexpression could significantly enhance the invasion and migration of breast cancer cells by inducing EMT. Metadherin overexpression led to upregulation of mesenchymal marker fibronectin, downregulation of epithelial marker E-cadherin, and the nuclear accumulation of beta-catenin. Also, transcription factors Snail and Slug were upregulated in breast cancer cells overexpressing MTDH. Overexpression of MTDH enhanced the invasiveness and migration ability of breast cancer cells in vitro. In addition, overexpression of MTDH led to increased acquisition of CD44+/CD24−/low markers that are characteristic of breast cancer stem cells. We also showed that NF-kappa was involved in the expression of EMT-related markers. Taken together, our results suggest that MTDH could promote EMT in breast cancer cells in driving the progression of their aggressive behavior. (Cancer Sci 2011; 102: 1151–1157)

Breast cancer has become the most common malignancy in women around the world. Each year there are over 1 million women diagnosed with breast cancer, with approximately 400 000 deaths.(1) Whereas a more sophisticated combination of surgery, chemotherapy, radiotherapy and endocrine therapy has led to improvements in disease-free survival and overall survival in general, invasion and metastasis remain the main obstacles in the effective treatment of this disease. Therefore, more attention is being paid to unraveling the molecular mechanisms leading to invasive and metastatic dissemination of carcinoma cells.

Epithelial to mesenchymal transition (EMT) is a morphogenetic process in which cells undergo a developmental switch from a polarized epithelial phenotype to a highly motile mesenchymal phenotype.(2) It is characterized by the loss of cell–cell adhesion molecules (such as E-cadherin), downregulation of epithelial differentiation markers (CK-18) and transcriptional induction of mesenchymal markers (such as vimentin and fibronectin).(3) Epithelial to mesenchymal transition has been considered to be essential in embryonic development, organ fibrosis, tumor cell migration and the evasion of apoptosis.(4–10) Several transcription factors including Snail, Slug and Twist have been reported to induce EMT by repressing E-cadherin transcription.(8,9) In addition, recent studies have shown that the population of breast cancer cells with the CD44+/CD24−/low phenotype can be expanded by EMT, Twist overexpression or exposure to transforming growth factor beta,(11) and that the CD44+/CD24−/low cells exhibited enhanced the invasive properties in breast cancer cells.(12,13)

Metadherin (MTDH, also known as astrocyte elevated gene-1 [AEG-1] and Lysine-rich CEACAM-1-associated protein [Lyric]) was originally identified as an oncogene whose expression can be induced in primary human fetal astrocytes by human immunodeficiency virus type 1 (HIV-1) or treated with HIV envelope glycoprotein (gp120) or tumor necrosis factor-α (TNF-α).(14–16) It has been found that the expression level of MTDH was elevated in many types of human malignancy, including breast cancer, prostate cancer, hepatocellular carcinoma, neuroblastoma, esophageal squamous cell carcinoma and non-small-cell lung cancer.(15,17–21) In our previous study, we found that overexpression of MTDH promoted metastatic seeding as well as the chemoresistance of breast tumors and was correlated with poor prognosis in breast cancer patients.(22) By comparing the expression of MTDH in normal, usual ductal hyperplasia, atypical ductal hyperplasia, ductal carcinoma in situ and invasive breast cancer tissues, we also found that MTDH was involved in the progression of breast precancerous lesions to cancer.(23) Although several signaling pathways, including Ha-ras, PI3K/Akt, nuclear factor – kappaB (NF-kappaB) and Wnt/beta-catenin,(24–27) were found to be involved in the function of MTDH, the precise mechanism of MTDH in invasion and metastasis is largely unknown. In the present study, we show that overexpression of MTDH could enhance the invasion ability by inducing EMT in human breast cancer cells and that the NF-kappaB signaling pathway was involved in MTDH-induced EMT.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Cell lines and reagents.  Breast cancer cell line MCF-7 and mouse fibroblast cell line NIH3T3 were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Gibco-BRL (Rockville, IN, USA). Fetal bovine serum (FBS) was supplied by Haoyang Biological Manufacturer Co., Ltd (Tianjin, China). Rabbit anti-E-cadherin, anti-NF-kappaB p65 and anti-beta-catenin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit anti-fibronectin was from Abcam (Cambrigeshire, England). Rabbit anti-MTDH antibody was from Invitrogen (Carlsbad, CA, USA). Mouse anti-human CD44 and CD24 was from BD Biosciences (Franklin Lakes, NJ, USA). Anti-mouse IgG horseradish peroxidase (HRP) antibody was from ZhongShan Goldenbridge (Beijing, China). Pro-lighting HRP agent for western blotting detection was from Tiangen Biotech Co. Ltd (Beijing, China). Cell lysis buffer for western blotting and the nuclear and cytoplasmic extraction reagents were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Other reagents were from Sigma–Aldrich (St. Louis, MO, USA) unless specifically described.

Cell culture.  MCF-7 and NIH3T3 cells were routinely cultured in DMEM supplemented with 10% FBS Gibco-BRL (HyClone, Rockville, MD, USA) and 100 units of penicillin–streptomycin at 37°C with 5% CO2 in a humidified incubator. For inhibitor treatment, 10 μM NF-kappaB inhibitor BAY 11-7028 (Beyotime Institute of Biotechnology) was added to the cultured cells for 2 days.

Plasmid construction and transfection.  The plasmid construction was performed as previously described.(22) Briefly, the cDNA representing the complete open reading frame of MTDH was cloned into the BamHI-XhoI vector fragment derived from the pcDNA3.1 vector (Invitrogen) to generate pcDNA3.1-MTDH (3.1-MTDH) cells. The expression plasmid was verified by sequencing of both strands and was used to transfect the MCF-7 cells using lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s protocol. Cells were then selected in 600 μg/mL G418 (Invitrogen) for 2 weeks and individual colonies were isolated, expanded and maintained in 300 μg/mL G418. The overexpression of MTDH in these clones was confirmed by western blot analysis and RT-PCR, and then mixed for further experiments. Empty pcDNA3.1 plasmid was used similarly to establish pcDNA3.1-vector (3.1-vector) cells.

RNA interference of NF-kappaB p65.  The sense shRNA target sequences are as follows: AAGGACATATGAGACCTTCAA.(28) The pSuper-Neo (OligoEngine) vector was linearized by digestion with BgIII and HindIII (TaKaRa, Dalian, China), and a 64-mers containing a hairpin loop was cloned into pSuper-Neo. For this procedure, a double-stranded oligodeoxyribonucleotide was synthesized as follows: 5′-GATCCCCAAGGACATATGAGACCTTCAATTCAAGAGATTGAAGGTCTCATATGTCCTTTTTTTA-3′ and 5′-AGCTAAAAAAAGGACATATGAGACCTTCAATCTCTTGAATTTGAAGGTCTCATATGTCCTTGGG-3′. The plasmid was verified by sequencing of both strands. Transient transfection of the pSN-shRNAp65 vector or the empty vector was performed using lipofectamine 2000 to establish pSN-shRNAp65 cell lines (pSN- shRNAp65) and the control cell lines (pSN-vector).

Quantitative reverse-transcription PCR analysis.  Total RNA were extracted with TRIZOL reagents according to the manufacturer’s protocol. cDNA was synthesized from 1 μg of total RNA by a PrimerScript RT Reagent kit (TaKaRa). Real-time quantitative RT-PCR (QRT-PCR) was performed using a SYBR green PCR mix in Applied Biosystems StepOne and StepOnePlus Real-Time PCR Systems. The gene expression ΔCt values of mRNA from each sample were calculated by normalizing with endogenous control GAPDH. The experiments were repeated in triplicate to confirm the findings.

Western blot analysis.  Cells were washed twice with cold phosphate-buffered saline (PBS) and lysed on ice in radio immunoprecipitation assay buffer (1× PBS, 1% NP40, 0.1% sodium dodecyl sulfate [SDS], 5 mM EDTA, 0.5% sodium deoxycholate and 1 mM sodium orthovanadate) with protease inhibitors and quantified using the bicinchonininc acid method. Nuclear and cytosolic extracts were prepared with a nuclear/cytosol fractionation system according to the manufacturer’s protocol (Beyotime Institute of Biotechnology, Jiangsu, China). Equal amounts of protein (80 μg) were separated by SDS polyacrylamide gel, electrotransferred to polyvinylidene fluoride membranes (ImmobilonP; Millipore, Bedford, MA, USA) and blocked in 5% non-fat dry milk in Tris-buffered saline, pH 7.5 (100 mM NaCl, 50 mM Tris and 0.1% Tween-20). Membranes were immunoblotted overnight at 4°C with anti-MTDH monoclonal antibody, anti-beta-catenin and anti-E-cadherin monoclonal antibody, followed by their respective horseradish peroxidase conjugated secondary antibodies. Signals were detected by enhanced chemiluminescence. Beta-actin was used as the loading control.

Immunofluorescence staining.  The pcDNA3.1-MTDH cells and pcDNA3.1-vector cells were grown on coverslips in the 24-well plates, washed in PBS and then fixed with 4% paraformaldehyde for 20 min at room temperature, and then permeabilized using 0.1% Triton-X100 in PBS (PBST) for 10 min. Cells were blocked with 10% normal goat serum in PBS, followed by rabbit anti-E-cadherin and anti-beta-catenin monoclonal antibody and rhodamine-conjugated anti-rabbit secondary antibody. After washing with PBST, cells were further stained with 4, 6-diamidino-2-pheny-lindole (DAPI) for 10 min. The coverslips were rinsed in PBST and then mounted on glass slides with antifade mounting medium. The fluorescence signal was examined with a DP71 fluorescence microscope (Olympus, Tokyo, Japan).

Invasion and migration assay.  Invasion assays were performed in 24-well transwell chambers (Corning, Acton, MA, USA) containing polycarbonate filters with 8-μm pores coated with matrigel (BD Biosciences, Bedford, MA, USA). First, the invasion chambers were rehydrated with DMEM (serum free) for 2 h at 37°C in 5% CO2 atmosphere. Five hundred microlitres of balanced mixture of the conditional medium from NIH3T3 fibroblasts and the complete medium was added to the lower compartment as the chemotactic factor. Then, 1 × 105 3.1-MTDH cells and 3.1-vector cells in serum-free DMEM were added to the upper compartment of the chamber. Each cell group was plated in three duplicate wells. After incubation for 18 h, the noninvasive cells were removed with a cotton swab. Cells that had migrated through the membrane and stuck to the lower surface of the membrane were fixed with methanol and stained with hematoxylin–eosin. Finally, the cells in the lower compartment of the chamber were counted under a light microscope in at least 10 random visual fields. The migration assay was similar to the invasion assay described above, except that the upper side of the membranes was not coated with the matrigel.

Flow cytometry.  Cells were washed twice with phosphate-buffered saline (PBS) and then harvested and resuspended in the wash buffer (1 × 106 cells/100 μL). Combinations of fluorochrome-conjugated mouse anti-human CD44 and CD24 or their respective isotype controls were added to the cell suspension at concentrations recommended by the manufacturer and incubated at 4°C in the dark for 30 min. The labeled cells were washed in the wash buffer, then fixed in PBS containing 1% paraformaldehyde, and then analyzed on a FACS-Scan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Statistical analysis.  Statistical analysis was carried out using SPSS 13.0 for Windows. Student’s t-test was chosen to analyze the statistical difference. Results were presented as mean ± standard deviation (SD). P < 0.05 was considered statistically significant and the experiments were repeated at least three times.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Overexpression of MTDH in breast cancer cell lines.  To determine whether MTDH plays an important role in breast cancer invasion by inducing EMT, we transfected the 3.1-MTDH expression plasmids to MCF-7 cell lines to generate the MTDH overexpression cells. The overexpression of MTDH in transfected MCF-7 cells was confirmed by RT-PCR and western blot analysis. As shown in Figure 1, cells transfected with 3.1-MTDH showed significantly increased MTDH in both mRNA and protein levels compared with the control 3.1-vector cell lines.

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Figure 1.  Overexpression of metadherin (MTDH) in breast cancer cell lines. (A) Relative MTDH mRNA level in breast cancer cell lines transfected with 3.1 and 3.1-MTDH vector, respectively. Error bars represent means ± SD of triplicate measurements. (B) MTDH protein levels measured by western blot analysis.

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Overexpression of MTDH induced phenotypic changes in breast cancer cells.  The typical phenotypic change associated with EMT is the conversion from the status of polarized epithelial cells to that of highly motile mesenchymal cells. As shown in Figure 2, while 3.1-vector cells exhibited a cobblestone-like appearance and tight cell–cell junction, which are typical of the epithelial phenotype, the 3.1-MTDH cells showed spindle-like morphology, loss of cell–cell contact and increased cell scattering that are typical of fibroblasts.

image

Figure 2.  Metadherin (MTDH) overexpression induced phenotypic changes. Cells were examined with phase contrast microscopy. (A) 3.1-vector. (B) 3.1-MTDH.

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Overexpression of MTDH led to acquisition of mesenchymal markers and reduction of epithelial markers.  We then examined the expression of EMT markers in cells that overexpress MTDH. RT-PCR showed that epithelial marker E-cadherin and CK-18 were downregulated while the mesenchymal vimentin and fibronectin were upregulated in 3.1-MTDH cells (Fig. 3A). Western blot analysis further showed that the expression of E-cadherin was decreased in 3.1-MTDH cells, which was accompanied by increased nuclear accumulation of beta-catenin (Fig. 3B). It is known that a number of transcriptional repressors such as Snail, Twist and Slug inhibit E-cadherin transcription, and increases in these repressors are closely associated with EMT during cancer cell growth and invasiveness.(29,30) Hence, we examined whether the expression of transcriptional repressors was changed in 3.1-MTDH cells. Overexpression of MTDH upregulated two EMT transcription factors, Snail and Slug (P = 0.004 and P = 0.0001, respectively), while the level of Twist was not significantly changed (P = 0.058) (Fig. 3A). Immunofluorescence staining showed that expression of E-cadherin was lower in 3.1-MTDH cells than in 3.1-vector cells (Fig. 3C). Thus, these data suggest that MTDH was actively involved in inducing EMT in breast cancer cells.

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Figure 3.  Changes in epithelial to mesenchymal transition (EMT) markers induced by metadherin (MTDH) overexpression. (A) Total RNA was prepared and the expression of EMT markers (E-cadherin, CK-18, vimentin, fibronectin) and transcription factors (Snail, Twist, Slug) were examined by reverse transcription real-time PCR. Data are shown as mean ± SD. (B) Extracts from the cells were analyzed for the expression of epithelial marker protein E-cadherin and nuclear beta-catenin by western blot analysis. The diagram shows a quantification of three assays. (C) Reduced expression of E-cadherin in MTDH-overexpressing cells revealed by immunofluorescence staining. Cells were grown on coverslips and stained with the E-cadherin antibodies using a rhodamine-coupled secondary antibody; nuclear staining with Nuclei were counterstained with DAPI. Left, 3.1-vector; Right, 3.1-MTDH.

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Overexpression of MTDH increased invasion and migration of breast cancer cells.  Increased migration and invasion is another key feature of cells that have undergo EMT. To assess whether stable MTDH overexpression affects migration and invasion of MCF-7 cells, we evaluated both cell migration and invasion by two-chamber assay. As indicated in Figure 4, migration of 3.1-MTDH cells (68.80 ± 3.77) was greatly enhanced compared with the control cells (27.60 ± 6.58; P < 0.0001). Similarly, 3.1-MTDH (10.80 ± 3.11) cells showed significantly increased invasive ability in comparison with the 3.1-vector cells (0.80 ± 0.45; < 0.0001).

image

Figure 4.  Metadherin (MTDH) overexpression promoted cell mobility. Migration assay of 3.1-vector (A) and 3.1-MTDH (B). Invasion assay of 3.1-vector (C) and 3.1-MTDH (D). Cells were stained with hematoxylin–eosin and counted in 10 representative fields. (E,F) Summary graphs for migration and invasion, respectively. Data are presented as mean ± SD.

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Overexpression of MTDH led to increased acquisition of surface markers that characterize breast cancer stem cells.  Breast cancers are thought to contain a rare population of tumor initiating/stem cell-like cells with high CD44 but low or undetectable levels of CD24 (CD44+/CD24−/low).(31) Many studies have reported that induction of EMT in immortalized human mammary epithelial cells is associated with the acquisition of stem-like characteristics.(11,32) To test whether MTDH overexpression will lead to an acquisition of stem-like characteristics, we examined the CD44+/CD24−/low expression pattern by flow cytometry. As shown in Figure 5, the proportion of CD44+/CD24−/low was increased significantly in 3.1-MTDH cells (14.64 ± 1.04 vs 59.08 ± 3.14; P < 0.0001). This finding further suggests that MTDH is critical for induction of EMT and substantiated the notion that cancer cells undergoing EMT shared the properties of breast tumor-initiating/stem cells.

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Figure 5.  Metadherin (MTDH) overexpression led to an increase of the CD44+/CD24−/low subpopulation. Cells were analyzed by flow cytometry. Upper left quadrant contains CD44+/CD24−/low cells. (A,C) Isotype of 3.1-vector and 3.1-MTDH, respectively. (B,D) CD44/CD24 expression pattern of 3.1-vector and 3.1-MTDH, respectively. The results shown are representative of three independent experiments.

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NF-kappaB pathway was involved in MTDH-induced EMT.  In previous reports,(24,26) the NF-kappaB signaling pathways were shown to be involved in the MTDH signal pathway. Metadherin could interact with the p65 subunit of NF-kappaB and CBP, thus activating NF-kappaB signaling. NF-kappaB has been suggested to be a regulator of Snail gene transcription and its activation is also involved in EMT.(33,34) To test if NF-kappaB was involved in the pathway of EMT induced by MTDH, cells was treated with 10 μM NF-kappaB inhibitor BAY 11-7028 for 2 days. Treatment of 3.1-MTDH cells with BAY 11-7028 could significantly abrogate the expression changes of E-cadherin and Snail (Fig. 6A,B). We further tested the role of NF-kappaB in MTDH-induced EMT by knocking down the expression of NF-kappaB p65 by RNAi. The RNAi efficiency of p65 was determined by western blotting 48 h after transfection (Fig. 6C). The protein level of E-cadherin and the mRNA level of Snail were measured (Fig. 6D,E). Downregulation of E-cadherin induced by MTDH was obviously attenuated by p65 RNAi (Fig. 6D). Similarly, upregulation of Snail induced by MTDH was also attenuated by p65 RNAi (Fig. 6E). The results suggest NF-kappaB was involved in EMT induced by MTDH in breast cancer cells.

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Figure 6.  Role of NF-kappaB in metadherin (MTDH)-induced expression changes of epithelial to mesenchymal transition (EMT) markers. (A) E-cadherin expression at the protein level. Protein extract was prepared from cells with or without BAY 11-7028 treatment for 2 days. (B) The mRNA levels of Snail measured by RT-PCR. Total RNA was extracted from cells with or without BAY 11-7028 treatment for 2 days. (C) P65 protein levels measured by western blot analysis after transfection. (D) E-cadherin expression level in cells transfected with the pSN-shRNA p65 plasmids and control vector, respectively. (E) The mRNA levels of Snail measured by RT-PCR in cells transfected with pSN-shRNA p65 plasmids and the control vector, respectively. The figures shown are representative of three independent experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Understanding the molecular mechanisms leading to invasiveness and metastatic dissemination of carcinoma cells is crucial to the development of new therapeutic strategies against breast cancer. Epithelial to mesenchymal transition is believed to be one of the critical steps in the progression of malignancy. Epithelial to mesenchymal transition enables carcinoma cells to detach themselves from the tumor mass, migrate to distant tissue sites and eventually form metastatic tumor masses. Previous studies have shown that MTDH overexpression is associated with increased aggressiveness and poor prognosis in breast cancer. In the present study, we presented several lines of evidence showing that MTDH is involved in EMT. We showed that transfection of MTDH in MCF-7 cells could induce the conversion of a cobblestone-like epithelial morphology to that of spindle-shaped mesenchymal cells. Consistent with the morphological change, E-Cadherin and CK18 were downregulated while vimentin and fibronectin were upregulated in breast cancer cells on MTDH transfection. We further showed that MTDH could upregulate the transcription of Snail and Slug, two transcription factors that were considered to be the key regulators of EMT.(35,36) In addition, we showed that MTDH could significantly enhance the invasion and migration ability of breast cancer cells in in vitro cell invasion and migration assays. Finally, by using NF-kappaB inhibitors and small interfering RNA against NF-kappaB p65, we demonstrated that NF-kappaB was involved in the EMT induced by MTDH in breast cancer cells. Thus, our findings shed some new light on the role of MTDH in the progression of malignant behavior of breast cancer cells.

Interestingly, we also found that overexpression of MTDH could lead to increased acquisition of CD44+/CD24−/low markers, which phenotypically define breast cancer stem cell-like cells. This is consistent with previous reports that cancer cells undergoing EMT share the properties of breast tumor-initiating stem cell-like cells and thus have more malignant properties.(11,37,38) The signaling pathways involved in EMT include those triggered by different members of the TGF-beta superfamily, Wnts, Notch, EGF, HGF, FGF, HIF and many others.(39–42) As the NF-kappaB signaling pathway was reported to be involved in the MTDH signal pathway in previous studies,(24,26) we determined if this signaling pathway participates in MTDH-induced EMT. When the 3.1-MTDH cells were treated with BAY 11-7028, an NF-kappaB inhibitor, the expression changes in Snail and E-cadherin induced by MTDH were greatly attenuated. This finding was further confirmed by RNAi of NF-kappaB p65. These findings suggest that the NF-kappaB pathway was involved in MTDH-induced EMT. However, further studies are required to identify the detailed mechanism involved in MTDH-inducing EMT.

In summary, MTDH could promote EMT in breast cancer cells in a NF-kappaB-dependent manner. To the best of our knowledge, we are the first to provide evidence that MTDH could induce EMT in breast cancer. Thus, MTDH may represent a promising target for developing a novel treatment strategy for breast cancer.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

This project is supported by Program for New Century Excellent Talents in University, Key Project of Chinese Ministry of Education (No. 108080), National Natural Science Foundation of China (No. 30772133; No. 81072150) and Independent Innovation Foundation of Shandong University (IIFSDU, No. 2009JQ007) to Q.Y., and by the National Basic Research Program of China (2011CB966200). The authors thank Zhaojian Liu, Qiao Liu, Ying Zhao, Jie Li, Xiangnan Kong and Jiang Zhu (Shandong University School of Medicine, Jinan, Shandong, China) for their technical support and critical discussions.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References