Estrogen promotes stemness and invasiveness of ER-positive breast cancer cells through Gli1 activation
© Sun et al.; licensee BioMed Central Ltd. 2014
Received: 31 August 2013
Accepted: 28 May 2014
Published: 3 June 2014
Although long-term estrogen (E2) exposure is associated with increased breast cancer (BC) risk, and E2 appears to sustain growth of BC cells that express functional estrogen receptors (ERs), its role in promoting BC stem cells (CSCs) remains unclear. Considering that Gli1, part of the Sonic hedgehog (Shh) developmental pathway, has been shown to mediate CSCs, we investigated whether E2 and Gli1 could promote CSCs and epithelial-mesenchymal transition (EMT) in ER+ BC cell lines.
We knocked down Gli1 in several BC cells using a doxycycline-controlled vector, and compared Gli1-knockdown cells and Gli1 + cells in behavior and expression of ER, Gli1, ALDH1 (BC-CSC marker), Shh, Ptch1 (Shh receptor) and SOX2, Nanog and Bmi-1 (CSC-associated transcriptions factors), using PCR; tissue microarrays, western blot; chromatin immunoprecipitation q-PCR, confocal immunofluorescence microscopy; fluorescence-activated cell sorting; annexin–flow cytometry (for apoptosis); mammosphere culture; and colony formation, immunohistochemistry, Matrigel and wound-scratch assays.
Both mRNA and protein expressions of ER correlated with those of Gli1 and ALDH1. E2 induced Gli1 expression only in ER+ BC cells. E2 promoted CSC renewal, invasiveness and EMT in ER+/Gli1+ cells but not in Gli1-knockdown cells.
Our results indicate that estrogen acts via Gli1 to promote CSC development and EMT in ER+ BC cells. These findings also imply that Gli1 mediates cancer stem cells, and thus could be a target of a novel treatment for ER+ breast cancer.
KeywordsSonic hedgehog Shh Epithelial-mesenchymal transition Epithelial-mesenchymal transition Estrogen
Cancer stem cell
Aldehyde dehydrogenase activity
Protein patched homolog 1
Short hairpin RNA
Fetal bovine serum
Tris-buffered saline containing Tween 20
Bovine serum albumin
Fluorescence-activated cell sorting
Epithelial growth factor
Basic fibroblast growth factor
Mammary epithelial growth medium.
Serial studies have suggested that breast cancer stem cells (CSCs) play critical roles in tumor growth, invasion, metastasis and resistance to cytotoxic agents and radiation . CSCs, like other stem cells, have unique characteristics, such as the ability to self-renew, generate differentiated cells  and express key stemness-associated transcription factors, including SOX2, Nanog and Bmi-1 [3–5]. In human breast cancer, CSCs also appear to be enriched within cell subpopulations with a CD44+/CD24-/low surface marker profile or with high intracellular aldehyde dehydrogenase 1 (ALDH1) activity [6–8]. However, the molecular mechanisms regulating breast CSC frequency, localization and maintenance remain poorly understood.
Epithelial-mesenchymal transition (EMT) is a process that involves epithelial cells acquiring a mesenchymal phenotype and migratory capability and plays an important role in tumor metastasis [9, 10]. Importantly, the EMT process has been shown to be associated with the acquisition of stem cell properties in normal and cancer cells [11, 12]. This crucial event results from transcriptional repression of E-cadherin through overexpression of several different EMT-inducing factors, such as Snail, vimentin and Bmi-1 [5, 13]. The link between EMT and CSCs enables cancer cells to migrate from the primary tumor and colonize at distant sites.
Estrogen is vital for normal postpubertal mammary development and long-term exposure to estrogens is proven to be associated with an increased risk of breast cancer. Although abundant evidence has suggested that estrogen sustains the growth of breast cancer cells expressing functional estrogen receptors (ERs) , the role of estrogen in promoting breast CSCs remains controversial. Some researchers think that ER-positive cells contribute to the stem cell compartment directly stimulated by hormones [15, 16], while others consider that estrogen may stimulate the expansion of a specific stem cell compartment in a paracrine manner [17–19]. Similar to the dynamic spatio-temporal signaling that governs the specification and maintenance of normal mammary gland stem cells [18, 20], specific hormone-growth factor paracrine signaling pathways have also proven to regulate stem-like breast cancer cells [17, 18].
Sonic hedgehog (Shh)/Gli signaling controls a variety of developmental processes, such as pattern formation, differentiation, proliferation and organogenesis. The net effect of pathway stimulation by Shh protein binding to the protein patched homolog 1 receptor (Ptch1) is the activation of members of the Gli family of zinc finger transcription factors, which translate the extracellular Shh stimulus into defined transcriptional programs in a context-dependent and cell type-specific manner . Gli1, a member of the Gli family, was originally identified as an amplified gene in malignant glioma  and was proven to be able to regulate stem cells and cancer stem cells . Moreover, Gli1 can be expressed without active Shh signaling . However, whether Gli1 is involved in estrogen-induced stemness and invasiveness in breast cancer remains uninvestigated.
In this study, we investigated whether estrogen could promote CSC maintenance and EMT through Gli1 in human ER-positive breast cancer cell lines MCF-7 and HCC1428, and ER-negative cell lines BT549 and MDA-MB-231. Here, we provide direct evidence that estrogen promoted the acquisition of the CSC phenotype and EMT through transcriptional activation of Gli1 only in ER-positive breast cancer cells.
ER expression is positively correlated with Gli1 and ALDH1 in human breast cancer cell lines
Estrogen-induced Gli1 expression only in ER-positive breast cancer cells
We also treated breast cancer cells with the Shh ligand to examine the effect of Shh on Gli1 and Ptch1 mRNA expression. Addition of various concentrations of Shh to these cells for 24 h increased both Gli1 and Ptch1 mRNA expression levels relative to untreated cells (Additional file 2: Figure S2). These results indicated that Gli1 activation was not mediated by canonical Shh signaling. Given that E2 modulated Gli1 transcription, quantitative chromatin immunoprecipitation (qChIP) assays were performed in ETOH and E2-treated MCF7 cells to determine the mechanism of this E2 effect. We found increased ER protein binding to the Gli1 promoter (area #1), as well as to the Gli1 gene body (area #3) in E2-treated MCF7 cells compared with ETOH-treated control cells (Figure 3H). The occupancy of IgG at the Gli1 gene promoter was not changed by E2 treatment (Figure 3I). These results indicated that E2 induced transcriptional activation of Gli1, probably through enriching ER occupancy at the Gli1 gene promoter and gene body.
Estrogen-induced CSC survival and self-renewal in ER-positive breast cancer cells is mediated by Gli1
Cells were then transfected with shVEC, shGli1-1 or shGli1-2 (the latter two representing Gli1-knockdown cells) and treated with E2 for 4 days, following which the CD44+/CD24–/low cell population was observed using flow cytometry. We found that treatment of shVEC cells with E2 induced a statistically significant expansion of CD44+/CD24–/low stem-like cells. In Gli1-knockdown cells, E2 failed to significantly increase the proportion of CD44+/CD24–/low cells (Figure 4B, C and Additional file 1: Figure S1D & E). The parallel expression patterns of Gli1 and CSC markers indicated that Gli1 may regulate the expansion of CSCs in estrogen-treated ER-positive breast cancer cells (Figure 4A–C & Additional file 1: Figure S1C–E).
Next, shVEC and Gli1-knockdown cells were cultured at very low densities (1 cell/μL) in 96-well plates containing serum-free medium with or without E2 for seven days. In the presence of E2, shVEC cells produced more and larger spheres compared with control cells (p < 0.01; Figure 4D–F). In Gli1-knockdown cells, E2 failed to significantly increase the number and size of these spheres (p < 0.01; Figure 4D–F). The significant inhibition in Gli1-knockdown cells of the estrogen-mediated increase in sphere formation suggested that Gli1 was indeed involved in the regulation of estrogen-induced self-renewal of breast CSCs.
Estrogen-induced EMT in breast cancer cells is mediated by Gli1
Correlation of ER, Gli1 and ALDH1 expression in human breast cancer tissues
The incidence of breast cancer is increasing worldwide, and breast cancer is quickly becoming one of the most serious diseases for women today. Currently, treatments for breast cancer mainly include chemotherapy, surgery, endocrine and radiation therapies. However, metastases and recurrences have become the major bottlenecks in all of these treatment options. The CSC hypothesis has shed new light on the development of resistance to therapy, proposing that a pool of malignant cells with stem cell-like properties exists and that this pool of malignant cells increases the capacity for resistance to common chemotherapeutic treatments as compared with their more differentiated nontumorigenic counterparts; these CSCs are therefore thought to be responsible for tumor recurrence after treatment . Thus, therapeutic strategies that specifically target breast CSCs can be effective in eradicating tumors and reducing the risk of relapse and metastasis.
Estrogens are essential mitogens for normal mammary epithelial cells, and prolonged exposure to estrogen is considered an important risk factor for breast cancer development. Although 60–70% of human breast cancers are ER-positive  and respond to anti-estrogen therapy, many of them will inevitably progress to the estrogen-independent stage and develop resistance to anti-estrogen therapy . The existence of CSCs offers a simple explanation as to the presence of endocrine resistance in breast cancer [28–30]. There have been conflicting reports about the effects of estrogen on breast CSCs; some studies have reported that estrogen can increase or decrease CSC numbers in breast cancer cell lines [18, 31]. Here, we identified estrogen as an important positive modulator of CSC properties in ER-positive breast cancer cell lines.
Emerging studies have demonstrated that CSC-enriched populations exhibit low/no expression of ER in ER-positive cell lines [17, 18, 32], suggesting that the effects of estrogen on breast CSCs are not direct. Our data described here demonstrated that breast CSC activity and cell surface marker expression were increased by estrogen exposure through its effects on the estrogen/Gli1-signaling axis. We found that estrogen induced transcriptional activation of Gli1 through enriching ER occupancy at the Gli1 gene promoter and gene body. It may represent a novel mechanism underlying this phenomenon, which we will try to address in future studies. Moreover, because cyclopamine inhibited Smo, which is a critical component of the canonical pathway, but did not inhibit the expression of Gli1, our current data strongly suggested the involvement of a noncanonical pathway.
EMT, a process through which epithelial cells acquire characteristics of mesenchymal cells, is thought to play an important role in invasion and metastasis  and is closely associated with the “stemness” of cancer cells [12, 34]. Our experiments demonstrated that estrogen induced cellular morphological changes in breast cancer cells, consistent with the acquisition of the EMT phenotype, as characterized by the upregulation of vimentin and downregulation of E-cadherin expression. EMT was significantly decreased when Gli1 was knocked down, and this was associated with reduced CSC numbers, as indicated in the mammosphere assay, and accompanied by decreased expression of Gli1. These data indicated that Gli1 mediated the estrogen-induced EMT in ER-positive breast cancer cells.
ER-negative tumors, especially triple-negative breast cancer (TNBC), are more aggressive and tend to harbor more CSCs . While much of our data were collected using the ER-positive MCF7 and HCC1428 cell lines, we were able to observe upregulation of Gli1 in many different primary human tumor tissue samples (Figure 1B), suggesting the relevance of this pathway in primary tumors. Our results showed that the expression levels of Shh and Gli1 were both high in MDA-MB-231 and BT549 cells; however, the expression of these targets was not affected by estrogen. Goel et al. showed that a novel autocrine pathway involving vascular endothelial growth factor (VEGF)/VEGF receptor neuropilin-2 (NRP2), α6β1 and Gli1 contributed to the initiation of TNBC and demonstrated that Gli1 was activated in noncanonical Shh signaling . Additionally, DiMeo et al. provided evidence of a molecular link between Wnt signaling, self-renewal, EMT and metastasis in basal-like breast cancers .
Taken together, the data presented in our study provided evidence that Shh signaling pathway components were widely expressed in breast cancer. Estrogen had the potential to promote the viability of breast CSCs via binding to the Gli1 promoter in ER-positive breast cancer in vitro. It will be important to also test whether these results can be translated in animal studies. A critical property of CSCs is their ability to repopulate heterogeneous tumor populations and to functionally demonstrate tumor-initiating capacity in vivo. There is significant interest in understanding the biology behind solid tumor stem cells and identifying drug targets and therapeutic approaches for eliminating these tumor subpopulations.
CSC self-renewal and EMT enable tumor metastasis, a key driving force for tumor growth and recurrence. Estrogen is a steroid hormone that has been closely linked to enhanced growth and invasion of breast cancer. Using breast cancer cells lines (MCF-7 and HCC1428), we demonstrated that estrogen may act via Gli1 to promote CSC development and EMT in ER-positive breast cancer cells, which may contribute to breast tumor malignancy. Thus, Gli1, which promotes the expansion of cancer stem cells, may be a potential therapeutic target for novel treatments for ER-positive breast cancer.
Chemicals and antibodies
Lipofectamine 2000 transfection reagent and TRIzol LS Reagent were purchased from Invitrogen (Grand Island, NY, USA). The DAB substrate kit for peroxidase was purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). Antibodies against Gli1, ERα, E-cadherin, Vimentin, Nanog, Bmi-1, SOX2, Smo and β-actin were from Cell Signaling Technology (Danvers, MA, USA). Anti-ALDH antibodies were from BD (Franklin Lakes, NJ, USA). Anti-Shh antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Amino-terminal Shh (ShhN) peptide was from R&D Systems (Minneapolis, MN, USA). Unless otherwise noted, all other chemicals were from Sigma (St. Louis, MO, USA).
All the human breast cancer cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). HCC1428, MDA-MB-231, BT549, HCC1937, HCC1569, HCC70, HCC1500, HS578T, SK-BR-3, AU565 and ZR-75-1 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. MCF-7, T47D, BT474, BT483, MDA-MB-468, MDA-MB-453, MDA-MB-435 and MDA-MB-361 cells were grown in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. MCF-10A cells were cultured in DMEM/F12 supplemented with 20 ng/mL epidermal growth factor, 0.01 mg/mL insulin, 500 ng/mL hydrocortisone, 5% heat-inactivated HS, 100 IU/mL penicillin and 100 μg streptomycin. All the cell lines were grown at 37°C in an atmosphere containing 5% CO2 and 95% air.
Gli1-specific shRNA inhibition
To knock down Gli1 expression, shRNA targeting Gli1 expressed in the pSingle vector was prepared as described previously . Cells were grown in culture dishes until they reached 75% confluence, at which point they were transfected for 24 h with pSingle-shRNA specific to Gli1 using the Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions. The tight on/off regulation of the pSingle vector system and coordinate inactivation of the target gene was mediated using doxycycline (Dox). Expression of the shRNA in the absence of induction was extremely low and prevented unwanted suppression of the target gene. When Dox was added to the culture medium, transcriptional suppression was relieved, permitting the shRNA to be transcribed. After transfection, cells were trypsinized, collected and subjected to various experiments.
Total RNA was extracted from different cell lines using the TRIzol reagent. Quantitative determination of RNA levels was performed in triplicate in three independent experiments. Real-time PCR and data collection were performed on the ABI PRISM 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA). The housekeeping gene GAPDH was used as an internal control to normalize the expression levels of different genes. Quantification of the relative expression of target genes was performed using the ΔΔCt method. The following gene-specific primers were used: ERα, forward 5′-AGA TGG TCA GTG CCT TGT TGG-3′ and reverse 5′-CCA AGA GCA AGT TAG GAG CAA ACA G-3′; Shh, forward 5′-GTG TAC TAC GAG TCC AAG GCA C-3′ and reverse 5′-AGG AAG TCG CTG TAG AGC AGC-3′; Gli1, forward 5′-GCG ATC TGT GAT GGA TGA GAT TCC C-3′ and reverse 5′-TGC CTT GTA CCC TCC TCC CGA A-3′; SOX2, forward 5′-GCT GTA TGG CTG CTG CAC TTC A-3′ and reverse 5′-GCA CAC GCA CCC AGC ACT GT-3′; Nanog, forward 5′-AAT ACC TCA GCC TCC AGC AGA TG-3′ and reverse 5′-TGC GTC ACA CCA TTG CTA TTC TTC-3′; Bmi-1, forward 5′-GAC CAC TAC TGA ATA TAA GG-3′ and reverse 5′-CAT TTG TCA GTC CAT CTC TC-3′; ALDH1, forward 5′-GTT AGC TGA TGC CGA CTT GG-3′ and reverse 5′-CCC ACT CTC AAT GAG GTC AAG-3′; Ptch1, forward 5′-TCG CTC TGG AGC AGA TTT CC-3′ and reverse 5′-TCT CGA GGT TCG CTG CTT TT -3′; and GAPDH, forward 5′-CAA GGT CAT CCA TGA CAA CTT TG-3′ and reverse 5′-GTC CAC CAC CCT GTT GCT GTA G-3′.
Western blot analysis
Cells were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris–HCl [pH 7.4], 1 mM EDTA, 1 mM EGTA, 2 mM NaF, 1 mM sodium orthovanadate, 10 μg/mL leupeptin, 10 μg/mL pepstatin, 10 μg/mL aprotinin, 10 μg/mL E 64 and 1 mM Pefabloc; EMD). Protein concentrations were determined using an Enhanced BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Jiangsu, China) and then boiled for 5 min. Protein samples (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, USA). Membranes were rinsed in Tris-buffered saline containing Tween 20 (TBST), blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature, and incubated with the primary antibody at 4°C overnight. The membranes were then rinsed and incubated in peroxidase-conjugated secondary antibodies for 1 h at room temperature. After washing, proteins were detected using enhanced chemiluminescence (ECL) (Millipore Corporation). Membranes were stripped and reprobed with anti-β-actin mouse monoclonal antibodies to confirm equal loading of samples.
Confocal immunofluorescence microscopy
Cell lines were plated on culture slides (Costar, Manassas, VA, USA). After 4 days, cells were rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS, and permeabilized using 0.5% Triton X-100. Cells were then blocked for 30 min in 10% BSA (Sigma Aldrich, St. Louis, MO, USA) in PBS and then incubated with primary monoclonal antibodies in 10% BSA overnight at 4°C. After three washes in PBS, slides were incubated for 1 h in the dark with FITC-conjugated secondary goat anti-mouse or goat anti-rabbit antibodies (Invitrogen). After three additional washes, slides were stained with 4-,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich) for 5 min to visualize the nuclei and examined using a Carl Zeiss confocal imaging system (LSM 780; Carl Zeiss, Jena, Germany).
Fluorescence-activated cell sorting (FACS) analysis
Anti-CD44-APC and anti-CD24-PE antibodies used for FACS analysis were obtained from Biolegend (San Diego, CA, USA). Briefly, for each cell line, 1 × 106 cells were aliquoted into two tubes; tube 1 was stained with IgG isotype controls for APC and PE, and tube 2 was stained with anti-CD44-APC and anti-CD24-PE antibodies. Cells were incubated with the appropriate antibodies for 30 min on ice and then washed with PBS. Cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences); each sample required 10,000 cells for analysis.
Cell cycle assays
Aliquots of 1 × 105 cells were collected using trypsinization and treated with 50 μg/mL DNase-free RNase and 20 μg/mL propidium iodide (PI) following the manufacturer’s instructions. Cells were analyzed using an FC500 instrument (Beckman Coulter, Brea, CA) with MultiCycle for Windows software (Beckman Coulter) for detailed cell cycle status.
Apoptosis was determined using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences Pharmingen, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, cells were detached and resuspended in 100 μL binding buffer containing FITC-Annexin V and PI. After incubation for 15 min at room temperature in the dark, cells were analyzed using an FC500 instrument (Beckman Coulter). Annexin V-positive cells were classified as apoptotic.
Mammosphere culture was performed as described by Dontu et al. with slight modifications . Single-cell suspensions were plated in ultralow attachment 96-well plates (Costar) at different densities of viable cells. Cells were grown in serum-free mammary epithelial growth medium (MEGM), supplemented with 1:50 B27 (Invitrogen), 20 ng/mL epithelial growth factor (EGF), 20 ng/mL basic fibroblast growth factor (bFGF; BD) and 10 μg/mL heparin (Sigma). The number of spheroids was counted after 7–10 days. For in vitro propagation, primary spheres were collected, dissociated into single-cell suspensions and plated in ultralow attachment 96-well plates. The secondary number of spheroids was counted 14 days after plating.
Colony formation assay
Cells were seeded in triplicate at 500 cells/6-cm dish in complete medium. After 3 weeks of growth, cells were fixed and stained with crystal violet (0.1% w/v in 20 nM 4-morpholinepropanesulfonic acid; Sigma), and visible colonies were counted according to the number of cells in each colony. All experiments were repeated at least three times. Plating efficiency was determined as the number of colonies formed divided by the total number of cells plated.
Wound scratch migration assay
Cells were seeded in 6-cm culture dishes, and cell monolayers were wounded by scratching with sterile plastic 200-μL micropipette tips and photographed using phase-contrast microscopy immediately following and 48 h after wounding. Migration assays were independently performed in triplicate. The migration distance of each cell was measured after the photographs were converted to Photoshop files.
Matrigel invasion assay
Invasion of cells was measured in Matrigel (BD)-coated transwell inserts (6.5 mm, Costar) containing polycarbonate filters with 8-μm pores, as detailed previously . Inserts were coated with 50 μL of 1 mg/mL Matrigel matrix according to the manufacturer’s recommendations. A total of 2 × 105 cells in 200 μL serum-free medium were plated in the upper chamber, and 600 μL of medium containing 10% FBS was added to the lower chamber. After 24 h incubation, top cells (noninvasive) were removed, and bottom cells (invasive) were counted. Cells that invaded to the lower surface of the membrane were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. For each membrane, five random fields were counted at 10× magnification. Data were presented as the mean ± SD from three independent experiments performed in triplicate.
Chromatin immunoprecipitation (ChIP)-qPCR
A chromatin immunoprecipitation kit (Cat. 17–371) was purchased from Millipore and ChIP experiments were carried out essentially as described (27). Immunoprecipitated DNA was analyzed on an ABI PRISM 7900HT sequence detection system. Primers used for detection of promoters following ChIP are located at -653 to -472 bp (1), -297 to -116 bp (2) and +167 to +359 bp (3) of the Gli1 promoter.
Tissue microarrays and immunohistochemical analyses
Tissue microarrays of breast samples (BC081116a) were from Alenabio (Xian, China). The manufacturer provided clinical and pathological information. Immunostaining was performed using the avidin-biotin-peroxidase complex method (UltrasensitiveTM, MaiXin, Fuzhou, China). Sections were deparaffinized in xylene, rehydrated in a graded series of alcohols and then boiled in 0.01 M citrate buffer (pH 6.0) for 2 min in an autoclave. Hydrogen peroxide (0.3%) was applied to block endogenous peroxide activity, and sections were incubated with normal goat serum to reduce nonspecific binding. Tissue sections were incubated with rabbit polyclonal anti-Gli1 antibodies (1:100 dilution), mouse monoclonal anti-ER antibodies (1:50 dilution) or mouse monoclonal anti-ALDH1 antibodies (1:100 dilution). Staining for these antibodies was performed at room temperature for 2 h. Biotinylated goat anti-mouse serum IgG was used as a secondary antibody. After washing, sections were incubated with streptavidin-biotin conjugated with horseradish peroxidase and the peroxidase reaction was developed with 3,30-diaminobenzidine tetrahydrochloride. Two independent, blinded investigators examined the slides randomly. Five views were examined per slide and 100 cells were observed per view at 400× magnification.
Data were described as the mean ± SD. Associations between protein expression levels in the breast tissue microarray were assessed using Spearman’s rank correlation test. Comparisons between different groups were carried out using the Student’s two-tailed t-test. The limit of statistical significance was set at a P-value <0.05. Statistical analysis was performed using SPSS/Win11.0 software (SPSS, Inc., Chicago, IL, USA).
This work was supported by grants from the National Natural Science Foundation of China (Nos. 30672434).
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