Annexin A4-nuclear factor-κB feedback circuit regulates cell malignant behavior and tumor growth in gallbladder cancer

Gallbladder cancer (GBC) is the most common malignant tumor of the biliary system. However, the mechanisms underlying its tumor initiation, progression, and metastasis are not yet fully understood. The annexin A4 (ANXA4) gene is highly expressed in GBC tissues and may play an important role in the initiation and progression of this disease. In this study, we examined the up-regulation of ANXA4 in human GBC tissues and cell lines. Elevated ANXA4 correlated well with invasion depth in GBC patients and predicted a poor prognosis. In vitro, GBC-SD and NOZ cells with ANXA4 knockdown demonstrated increased apoptosis and inhibited cell growth, migration, and invasion. Interactions between ANXA4 and nuclear factor-κB (NF-κB) p65 proteins were detected. In vivo, ANXA4 knockdown inhibited tumor growth of GBC cells in nude mice and down-regulated the expression of downstream factors in the NF-κB signaling pathway. Taken together, these data indicate that up-regulation of ANXA4 leads to activation of the NF-κB pathway and its target genes in a feedback regulatory mechanism via the p65 subunit, resulting in tumor growth in GBC.

ANXA4 overexpression correlated with invasion depth and lymph node metastasis and predicted poor prognosis in GBC patients. We further investigated the clinical significance of differential ANXA4 expression in GBC tissues by analyzing expression levels in a larger patient population. High expression of ANXA4 was detected in 30/60 (50.0%) GBC samples. As shown in Table 1, high expression of ANXA4 was positively associated with lymph node metastasis (P < 0.001), invasion depth (P = 0.028), and TNM stage (P = 0.010). Kaplan-Meier survival analysis showed that patients with high ANXA4 expression had poorer overall survival (OS) than patients with low ANXA4 expression (P < 0.001) (Fig. 1E). The median OS of GBC patients with low ANXA4 expression was 39 months (95% confidence interval (CI), 34.7-43.3) compared with 8 months (95% CI, 5.7-10.3) in patients with high ANXA4 expression. The actual 3-year and 5-year OS rats of patients with low ANXA4 expression were 63.3% and 33.3%, respectively, compared with only 13.3% and 10.0%, respectively, in patients with high ANXA4 expression. Multivariate Cox proportional hazards analysis revealed that ANXA4 expression was an independent prognostic marker for OS in GBC patients (P < 0.001), and that invasion depth (P < 0.001) and lymph node metastasis (P = 0.003) were also significantly associated with OS.

ANXA4 knockdown inhibited gallbladder cell growth and increased apoptosis. The high
ANXA4-expressing GBC cell lines, GBC-SD and NOZ, were transfected with lentivirus-based short hairpin RNA (shRNA) targeting human ANXA4 (shA4) or the corresponding scrambled construct, with non-transfected cells as a control. To rule out clonal effects, in vitro and in vivo experiments were performed in two ANXA4 knockdown clones. RT-PCR and western blot analyses revealed that ANXA4 mRNA ( Fig. 2A) and protein (Fig. 2B) levels were reduced, while the expression levels of other annexins, ANXA1, ANXA2 and ANXA3 (Supplementary Figure S1), remained unchanged in the ANXA4-knockdown clones. ANXA4-knockdown cells proliferated (Fig. 2C) and formed colonies (Fig. 2D) at significantly slower rates than non-transfected cells. Flow cytometric analysis revealed significantly higher levels of annexin V binding in ANXA4 shRNA-transfected cells compared with control cells, indicating increased apoptosis (Fig. 2E). We determined which caspase molecules were involved in ANXA4 shRNA-induced apoptosis by measuring caspase-3 and -9-activities in ANXA4 shRNA-transfected cells by caspase activity assays. Compared with untreated control cells, ANXA4 shRNA significantly induced caspase-3 and -9 activities in GBC-SD and NOZ cells (Fig. 3A,B). Western blot analysis showed that full-length procaspase-3 and procaspase-9 were significantly decreased in ANXA4-knockdown cells, while their cleaved forms were increased (Fig. 3C).

ANXA4 knockdown inhibited GBC cell migration and invasion.
We determined the influence of ANXA4 down-regulation on the migration and invasion abilities of GBC-SD and NOZ cells using the Transwell system. Compared with control cells, cell migration (Fig. 4A,B) and invasion (Fig. 4C,D) were significantly reduced in ANXA4-knockdown cells. p65 subunit is required for feedback regulation between ANXA4 and NF-κB. We explored the effects of ANXA4 knockdown on transcriptional activation of NF-κ B in GBC cells by luciferase assays in GBC-SD and NOZ cells transfected with ANXA4 shRNA or scrambled constructs, with non-transfected cells as a control. As shown in Fig. 5A, ANXA4-knockdown significantly reduced NF-κ B transcriptional activity both before and after tumor necrosis factor (TNF)-α stimulation, compared with controls. ANXA4-knockdown significantly reduced mRNA levels of the NF-κ B downstream target genes COX-2, iNOS, cyclin D1 and VEGF before and after TNF-α stimulation (Fig. 5B-E). Western blot analysis indicated that the corresponding protein levels were also notably reduced in ANXA4-knockdown clones (Fig. 5F).
We investigated the interaction between ANXA4 and NF-κ B subunits by co-immunoprecipitation of endogenous proteins in extracts from ANXA4-knockdown cells, scrambled control cells, and non-transfected control cells. Immunoprecipitation of endogenous ANXA4 revealed a strong association with endogenous p65 in GBC-SD and NOZ cells (Fig. 6A), and this interaction was reduced in ANXA4-knockdown cells. We also detected p50 and inhibitor of κ B (Iκ Bα ) in this immunoprecipitate, but not the other subunits RelB, cRel and p52 (Fig. 6A). The interaction of p65 with ANXA4 was confirmed by His pull-down assays (Fig. 6B). His pull-down experiments also showed no direct interaction of ANXA4 with p50 or Iκ Bα , suggesting that the associations of p50 and Iκ Bα with ANXA4 were indirect and occurred through an inhibitory p50-p65-Iκ Bα complex (Fig. 6B). We examined any effects of feedback regulation of p65 on ANXA4 expression using NF-κ B p65 small interfering RNA (siRNA) knockdown GBC-SD and NOZ cells. The presence of NF-κ B p65 siRNA reduced p65 and ANXA4 mRNA (Fig. 6C,D) and protein (Fig. 6E) expression levels. Moreover, the expression levels of the NF-κ B downstream targets COX-2, iNOS, cyclin D1, and VEGF proteins were down-regulated in p65-knockdown cells. Taken together, these results suggest that the p65 subunit is required for feedback regulation between ANXA4 and NF-κ B.
ANXA4 knockdown inhibited tumor growth of GBC cells in nude mice by inhibiting the NF-κB signaling pathway. Nude mice were injected with ANXA4-knockdown, scrambled control, and non-transfected control cells to induce tumors, and the tumors were harvested every week for 6 weeks (Fig. 7A). Tumor volume and weight were significantly decreased in mice injected with ANXA4-knockdown cells 3) months. The actual 3-year and 5-year OS rates for patients with low ANXA4 expression (63.3% and 33.3%, respectively) were higher than those for patients with high ANXA4 expression (13.3% and 10.0%, respectively, P < 0.001).

Discussion
The dismal prognosis of GBC has been attributed to typical delays in diagnosis, the difficulty of radical resections (currently the only potentially curative therapy), and insufficient therapies for advanced-stage patients. In addition, few predictable biomarkers are available for GBC prognosis in the clinical environment. A recent analysis comparing the complete exomes from 57 gall-bladder tumors with normal tissues aimed at identifying the somatic mutation spectrum reported a high rate of C > T/G > A somatic mutation in GBC tissue 23 . This study identified several GBC driver mutations and indicated ErbB signaling as a core pathway, thus providing potentially important information for the development of targeted therapies. We previously provided the first report identifying ANXA4 as a potential biomarker for GBC using proteomic analysis 11 . In the current study, we showed that overexpression of ANXA4 correlated well with invasion depth as well as lymph node metastasis, and predicted poor survival in GBC patients. ANXA4 knockdown in GBC cells inhibited cell proliferation and increased apoptosis compared with control GBC cells. These results are consistent with the reported role of ANXA4 in both of these processes. ANXA4 knockdown also inhibited cell migration and invasion, in accordance with the reported involvement of this gene in pathogenic proliferation and invasion of adenocarcinoma cells 12 .
ANXA4 has been implicated in chemoresistance in ovarian cancer 12 , lung cancer 24 and malignant mesothelioma 15 . The mechanism of ANXA4-induced platinum resistance may be mediated in part by increased efflux of cellular platinum via the copper transporter ATP7A 25,26 , while modulation of Ca 2+ -dependent NF-κ B transcriptional activity may also play a role. After etoposide treatment, ANXA4 co-translocated to the nucleus with the p50 subunit, subsequently enhancing the transcriptional activity of NF-κ B and ultimately inducing resistance to etoposide-associated apoptosis 27 . Morimoto et al. recently proposed a novel mechanism for ANXA4-induced chemoresistance whereby calcium-binding sites in the annexin repeats of ANXA4 were responsible for resistance to platinum-based drugs by elevating the intracellular chloride concentration 28 . The role of ANXA4 in cancer and chemoresistance suggests that it could represent a potential therapeutic target for cancer treatment.
ANXA4 has been shown to interact with members of the mammalian NF-κ B signaling pathway, including p105 and p50 27 . Jeon et al. revealed that ANXA4 interacted with the NF-κ B p50 subunit and modulated the transcriptional activity of NF-κ B in a calcium-dependent manner. In the presence of high Ca 2+ levels, ANXA4 co-translocated to the nucleus with the p50 subunit, consequently increasing the transcriptional activity of NF-κ B, and ultimately inducing resistance to etoposide-associated apoptosis. The NF-κ B family includes five proteins forming a protein complex that controls DNA transcription, and has been linked to a number of cancers 29 . Activation of NF-κ B p65 (also known as v-rel avian reticuloendotheliosis viral oncogene homolog A) is associated with several types of cancer and is believed to play a major role in tumorigenesis 30 . Lui and Brown showed that phosphorylation of p65 in follicular thyroid carcinomas correlated with translocation to the nucleus and gene expression of COX-2, interleukin-8 and glutathione S-transferase-π 31 . In addition, Rho-kinase mediates TNF-α -stimulated nuclear translocation of NF-κ B p65 and subsequent DNA binding activity, and this mediation may be an important step in macrophage proliferation 32 . We conclude that p65 can interact with some annexins, and these interactions play an important role in carcinogenesis.
Receptor interacting protein (RIP)-1 has been reported to be up-regulated in GBC, promoting NF-kB (p65) activation 33 . Ubiquitination of RIP1 plays an important role in TNF-α -induced IKK/NF-κ B activation 34,35 . The expression of many genes encoding transcription factors and cell signaling modulators are also altered during GBC pathogenesis, suggesting that the interactions with their corresponding partners are also affected. However, in terms of NF-κ B activation, the association between RIP1 upregulation and ANXA4 upregulation in GBC is unknown. The interaction mechanisms between ANXA4 and other genes, including RIP1 will be an interesting goal for future studies aimed at extending our understanding of GBC pathogenesis.
The results of this study showed that the p65 subunit was required for the feedback circuit between ANXA4 and NF-κ B, which has been implicated in tumor development and progress. To the best of our knowledge, this is the first report of a feedback circuit between ANXA4 and NF-κ B. The existence of this feedback circuit suggests that ANXA4 interacts with the p65 subunit in such a way that loss of ANXA4 decreases the transcriptional activity of NF-κ B, and inhibition of NF-κ B subsequently suppresses ANXA4 expression. This hypothesis is strengthened by the fact that deregulation and knockdown of ANXA4 in GBC cells corresponded to changes in expression levels of NF-κ B downstream target genes, such as COX-2, iNOS, cyclin D1, and VEGF. These NF-κ B downstream target genes were down-regulated in tumors from mice injected with ANXA4-knockdown clones, indicating that NF-κ B plays an important role in tumor growth regulated by ANXA4 in GBC. In addition, NF-κ B reported that ANXA6 interacted with p65, and that ANXA6 overexpression resulted in increased NF-κ B activity, increased catabolic events in articular chondrocytes, and accelerated cartilage destruction 36 . However, the mechanism whereby NF-kB regulates ANXA4 expression is unknown.
To confirm the feedback regulatory mechanism inhibiting tumor growth in vivo, we injected GBC cells into nude mice to induce tumors. ANXA4-knockdown GBC cells produced smaller tumors than GBC cell lines expressing high levels of ANXA4, associated with lower accumulation of ANXA4 and NF-κ B, including COX-2, iNOS, cyclin D1, and VEGF. Taken together, these observations suggest that ANXA4 interacts with the NF-κ B p65 subunit and modulates the transcriptional activity of the NF-κ B pathway in an ANXA4-NF-κ B feedback circuit. ANXA4 has been implicated in several cancers, and the current results suggest that therapy targeting ANXA4 may suppress the NF-κ B signaling pathway, further reversing ANXA4 overexpression in a feedback regulatory mechanism and resulting in inhibition of tumor growth. We postulate that overexpression of ANXA4 during GBC development and progress activates the NF-κ B signaling pathway, which in turn drives ANXA4 expression, resulting in carcinogenesis and invasiveness. ANXA4 knockdown thus provides promising prospects for developing GBC therapies based on the ANXA4-NF-κ B feedback circuit in gallbladder cells.

Materials and Methods
Patients and tissues. Tissue specimens, including 60 GBC tumor specimens and 20 normal tumor-adjacent tissues, were obtained from 60 primary GBC patients who underwent surgery at Shanghai Chang Zheng Hospital affiliated with the Second Military Medical University, None of the patients had received any preoperative antineoplastic therapy. The study was conducted in accordance with the human subject guidelines approved by the Scientific and Ethical Committee of the Second Military Medical University, Shanghai, China. All samples were obtained by experienced surgeons and examined by experienced pathologists from 2006 to 2008. Informed consent was obtained from all patients or their relatives for the use of tissues in experimental procedures. Formalinfixed, paraffin-embedded samples were sectioned at 4-μ m thicknesses, and immunohistochemical staining was performed as described below. In addition, 20 self-paired GBC and normal tumor-adjacent tissue specimens were snap frozen in liquid nitrogen and stored at − 80 °C following surgery for quantitative real-time RT-PCR analysis.
Clinical staging was performed according to the American Joint Committee on Cancer, 6th Edition guidelines 37 . No distant metastasis was detected in any of the patients, and all patients achieved clear resection margins

Figure 3. Effect of ANXA4 knockdown on caspase activation in GBC-SD and NOZ cells.
Caspase-3 and -9 activities were determined by caspase activity assay in GBC-SD (A) and NOZ (B) cells. Caspase-3 and -9 activities were significantly increased in ANXA4-knockdown cells. Data are presented as mean ± SD from three independent experiments. * * P < 0.01 compared with control. (C) Pprotein expression levels of total caspase-3 and -9 were decreased in ANXA4-knockdown clones, while their cleaved forms increased, as shown by western blot analysis.
(R0), defined as complete removal of the neoplasm without macroscopic residual tumor and with negative histologic margins. Stage I GBC patients underwent cholecystectomy; stage II patients underwent radical surgeries including cholecystectomy, regional lymphadenectomy, and resection of the gallbladder fossa; stage III patients underwent radical tumor resection combined with regional lymphadenectomy and wedge resection of the liver. Information on relevant patient clinicopathological variables including age, sex, tumor size, histology, differentiation, depth of tumor and lymph node metastasis was collected, and follow-up information was also recorded. OS was calculated from the day of surgery to death or January 2013. All patients were followed-up until death or January 2013. After surgery, patients with high-risk stage III GBC were treated with chemotherapy using gemcitabine.
Immunohistochemical staining and assessment. Immunohistochemical staining was performed using the standard immunoperoxidase procedure 38 . Briefly, serial 4 μ m sections were cut from formalin-fixed and paraffin-embedded tissue, dewaxed in xylene, rehydrated in alcohols, and then incubated with fresh 3% hydrogen peroxide for 20 min at room temperature. After washing with phosphate-buffered saline (PBS), the tissue sections were antigen-retrieved by heating in a microwave for 13 min in a citric acid buffer solution (pH 6.0). Sections were blocked with appropriate normal serum in PBS. The specific antibodies were incubated with the sections overnight in humidified boxes at 4 °C. The sections were then washed with PBS for 5 min followed by incubation with an UltraSensitive S-P Kit (Maixin-Bio, Fuzhou, China), according to the manufacturer's instructions. After exposure to stable 3,3-diaminobenzidine for 5-10 min, slides were counterstained with hematoxylin, dehydrated, and mounted. Control sections were incubated with PBS instead of the primary antibody. Microscopic images were captured with a BX41 light microscope (Olympus Optical Co., Tokyo, Japan) equipped with an Olympus DP70 digital camera and image analysis software (analySIS 3.2, Soft Imaging System, Hanover, Germany). Three sections were randomly selected for each group, and five high-power (× 400) fields were observed randomly for each section.
Immunohistochemical reactions were analyzed using a previously described semiquantitative scoring system, in which the final immunoreaction score was expressed as the product of the intensity and quantity scores 39 . The percentage of positive cells was graded as follows: 0, negative; 1, < 10% positive cells; 2, 11-50% positive cells; 3, 51-80% positive cells; 4, > 80% positive cells. The staining intensity was graded as follows: 0, negative; 1, weakly positive; 2, moderately positive; and 3, strongly positive. The product of the above two scores was defined as the protein expression level, and was given a numerical value from 0-12 for further analysis. High ANXA4 expression in the tumor was defined by a score ≥ 6, and low expression as a score < 6. All staining results were independently evaluated by three different pathologists who were blinded to patient outcomes.  NF-κ B transcriptional activity was assayed in ANXA4-knockdown clones of (i) GBC-SD and (ii) NOZ after TNF-α stimulation using a reporter assay system. NF-κ B transcriptional activity in ANXA4 shRNA cells was significantly decreased compared with the control. The expression levels of NF-κ B downstream targets COX-2, iNOS, cyclin D1, and VEGF were determined by RT-PCR (B-E) and western blot analyses (F) in ANXA4-knockdown (i) GBC-SD and (ii) NOZ cells after TNF-α stimulation. ANXA4 shRNA significantly reduced the expression of these factors at the mRNA and protein levels. β -actin expression was used as a control. All data are presented as mean ± SD from three independent experiments. * P < 0.05, * * P < 0.01, compared with control. Reagents and plasmids. C-terminal FLAG-tagged ANXA4 expression plasmids (cFLAG-ANXA4) were generated by PCR and subcloned into pcDNA3.1/Zeo (Thermo Fisher Scientific, MA, USA). His-tagged p65 expression plasmids (His-p65) were generated by PCR and subcloned into pcDNA4/HisMax (Thermo Fisher Scientific). Knockdown of NF-κ B p65 was performed using SignalSilence ® NF-κ B p65 siRNA I (Cell Signaling Technology, MA, USA).
The following antibodies were used in this study: goat anti-ANXA4, mouse anti-NF-κ B p65, p50, c-Rel, RelB, cRel and p52, goat anti-COX-2, mouse anti-iNOS, rabbit anti-VEGF, and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology, Inc., CA, USA), as well as rabbit anti-cyclin D1, rabbit anti-6X His tag, mouse anti-β -actin (Abcam, Cambridge, England), and mouse anti-FLAG (Sigma, MO, USA). ) and non-transfected cells (Control) and subjected to western blot analysis with antibodies specific for the indicated proteins, or β -actin as a control. Right panels: untreated cell lysates; left panels: immunoprecipitates (IP) produced with antibodies specific for ANXA4 or IgG as a control. (B) Nuclear extracts from GBC-SD or NOZ cells transfected with vectors containing cFLAG-tagged ANXA4 and/or Histagged p65 were subjected to western blot analysis with His-, FLAG-, p50-, Iκ Bα -or control β -actin antibodies. Right panels: untreated extract; left panels: pellets from His-tag pull-down analysis. Cells transfected with vectors containing NF-κ B p65 siRNA, the corresponding scrambled siRNA, or non-transfected controls were subjected to RT-PCR (C) or western blot analysis (D) using primers for p65 and ANXA4 mRNA, or antibodies specific for p65 or ANXA4 proteins, respectively. NF-κ B p65 siRNA down-regulated p65 and ANXA4 mRNA expression. Data are presented as mean ± SD from three independent experiments. * P < 0.05, * * P < 0.01 compared with control. (E) Cells transfected with vectors containing NF-κ B p65 siRNA, the corresponding scrambled siRNA, or non-transfected controls were subjected to western blot analysis using antibodies specific for COX-2, iNOS, cyclin D1, and VEGF or control β -actin antibodies. NF-κ B p65 siRNA reduced the protein expression levels of the NF-κ B downstream targets.
Scientific RepoRts | 6:31056 | DOI: 10.1038/srep31056 Lentiviral shRNA knockdown of ANXA4. ANXA4 knockdown in GBC-SD and NOZ cells was accomplished by stably expressing lentivirus-based shRNA targeting human ANXA4, in a manner similar to the method described in previous studies 40 . Briefly, the pLKO.1 vector (Open Biosystems, AL, USA) was employed, and ANXA4 shRNA sequence (sense: 5′ -CCG GGC ACA CTT CAA GAG ACT CTT CGG ATC CGA AGA GTC TCT TGA AGT GTG CTT TTT G-3′ ; antisense: 5′ -AAT TCA AAA AGC ACA CTT CAA GAG ACT CTT CGG ATC CGA AGA GTC TCT TGA AGT GTG C-3′ ) was subcloned into the pLKO.1 vector. A non-targeting control shRNA (scrambled control, Scr.) was obtained from Sigma-Aldrich (MO, USA). Recombinant lentivirus was produced by cotransfection with three helper vectors, pRSV-REV, pMDLg/pRRE and pMD2G, and a target vector pLKO.1-puro-shRNA into HEK-293T cells. GBC-SD and NOZ cells were then infected with recombinant lentivirus for 24-h, and stably expressing cells were selected with puromycin. The efficiency of knockdown was determined by quantitative real-time PCR and western blot analysis. Tumor xenograft animal model. Balb/c athymic nude mice (24 males, 6 weeks old, 18-21 g) were provided by the Shanghai Laboratory Animal Center (Chinese Academy of Science, China) and housed in specific pathogen-free conditions. All animal experimental procedures were approved by the Experimental Animal Center of the Second Military Medical University, and the methods were carried out in accordance with the approved guidelines. Tumor xenograft assay of GBC-SD cells in vivo was performed as described previously 41,42 . Mice were randomly assigned to four groups (six mice per group): shA4 clone-1, shA4 clone-4, scrambled control and GBC-SD (control). Lentivirus-transfected cells were administered by subcutaneous injection (0.2 ml PBS containing 5 × 10 6 GBC-SD cells/ml). After development of a palpable tumor, the tumor volume (V) was determined by measuring the two perpendicular dimensions with calipers every week and calculated using the formula: V (mm 3 ) = 1/6π ab 2 , where a was the larger and b the smaller dimension of the tumor. Six weeks after inoculation, the mice were sacrificed and tumors were resected and weighed. A portion of each tumor was selected for RT-PCR and western blot analysis. All measures were performed independently by two different technicians who were blinded to the group allocation during the experiment. Tumor volume was significantly decreased in mice injected with ANXA4-knockdown cells compared with the control group (* * P < 0.01). (C) Tumor weight was significantly reduced in mice injected with ANXA4knockdown cells (* * P < 0.01 compared to the control group). The mRNA and protein expression levels of ANXA4 and NF-κ B signaling pathway factors COX-2, iNOS, cyclin D1, and VEGF in tumors were determined by (D) RT-PCR and (E) western blot, respectively. mRNA and protein expression levels were significantly decreased in tumor tissues from mice injected with ANXA4 shRNA-transfected cells. Data are presented as mean ± SD; * P < 0.05, * * P < 0.01 compared with blank control levels.
Scientific RepoRts | 6:31056 | DOI: 10.1038/srep31056 Other methods. Quantitative real-time RT-PCR, western blot, cell proliferation assay, colony formation assay, cell migration and invasion assays, flow cytometric analysis of apoptosis, luciferase reporter assay, co-immunoprecipitation, and His pull-down assay were performed using standard protocols. See Supplementary Methods for more details.
Statistical analysis. Results are expressed as mean ± standard deviation (SD) based on a minimum of three replicates. Differences between groups were evaluated using SPSS version 19.0 statistical software (IBM Corporation, NY, USA). Results were compared between two groups using Student's t-tests (two-tailed), and among more than two groups by one-way analysis of variance. The relationships between ANXA4 expression levels and various clinicopathologic characteristics were analyzed by non-parametric Mann-Whitney tests. Survival rates were determined using the Kaplan-Meier method. P < 0.05 was considered statistically significant.