The expression of NOL6 in breast cancer tissues was analyzed using the TCGA database (https://portal.gdc.cancer.gov/) and prognostic analysis was conducted using the Gene Expression Profiling Interactive Analysis (GEPIA) tool (http://gepia.cancer-pku.cn/).

This study was conducted in accordance with the principles outlined in the Declaration of Helsinki. The serum samples were collected from 40 BC patients and 20 healthy controls at Lianyungang Affiliated Hospital of Nanjing University of Traditional Chinese Medicine. This study was approved by our hospital’s Ethics Committee. The informed consent has been obtained by the patients or their families/legal guardians. Peripheral blood was processed using Ficoll lymphocyte isolation solution, and the clinical and pathological characteristics of the BC patients and healthy controls were assessed (Table 1).

BC cell lines, including MCF-7 (SCSP-531), MDA-MB-231 (TCHu227), as well as the normal breast cell line HMEC (GNHu46), were purchased from Cell Bank, Chinese Academy of Sciences (Shanghai, China). SW527 (5922-60-1) was bought from ATCC (Manassas, VA, USA). They were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Carlsbad, CA, USA, Cat# 11965092, Lot# 2123456) supplemented with 10% fetal bovine serum (FBS; Gibco, Cat# 10099141, Lot# 3456789) and incubated at 37 °C in a 5% CO₂ atmosphere. All cell lines were authenticated by short tandem repeats (STR) profiling and tested negative for mycoplasma. NOL6 shRNA plasmids were constructed in our lab. For gene knockdown or overexpression, plasmid containing DNA (pcDNA) plasmids or shRNA constructs targeting NOL6 (5^′-CTGACCCTGGGACTCCTTC-3^′), and negative control (sh-NC) (5^′-GCAGGAATTGGAACATTAT-3^′) were transfected into MCF-7 cells using Lipofectamine 2000 (Invitrogen, Shanghai, China). Transfection efficiency was assessed by quantitative real-time polymerase chain reaction (qPCR) and immunoblotting 48 hours post-transfection.

The cell samples were lysed using RIPA buffer (Beyotime, Beijng, China). Protein concentration was assessed using a Bovine serum albumin (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (30 µg) were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Burlington, MA, USA). Membranes were blocked with 5% dry milk and incubated with primary antibodies against Nucleolar Protein 6 (NOL6) (Abcam, Cambridge, UK, ab228836; 1:500), Twist1 (Abcam, ab50887; 1:1000), galectin-3 (Abcam, ab76245; 1:500), platelet-derived growth factor (PDGF) (Abcam, ab178439; 1:1000), Vascular endothelial growth factor (VEGF) (Abcam, ab291246; 1:500), and beta-actin (Abcam, ab8226; 1:3000). After incubation with Horseradish peroxidase (HRP)-conjugated secondary antibodies (Goat Anti-Rabbit antibody, Abcam, ab6721; 1:2000), the protein bands were visualized using the enhanced chemiluminescence (ECL) detection system (RPN2106, GE, Marlborough, MA, USA).

RNA was extracted from tissue and cell samples using Trizol reagent (TaKaRa, Kusatsu City, Japan), RNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA), total RNA was reverse transcribed into cDNA using the RT reagent Kit (Takara), and quantitative PCR was performed using SYBR Ex Taq™ II (Takara). The primer sequences used were: NOL6: Forward: 5^′-AGGAAGCGTACATTGGCTGAA-3^′, Reverse: 5^′-GTCTCCCGAAGGCGATTAAGC-3^′; TWIST1: Forward: 5^′-GTCCGCAGTCTTACGAGGAG-3^′, Reverse: 5^′-GCTTGAGGGTCTGAATCTTGCT-3^′; Galectin-3: Forward: 5^′-CTTATTAACCTGCCTTTGCCTGG-3^′, Reverse: 5^′-GCAACATCATTCCCTCTTTGGA-3^′; GAPDH: Forward: 5^′-TGTGGGCATCAATGGATTTGG-3^′, Reverse: 5^′-ACACCATGTATTCCGGGTCAAT-3^′. In addition, the relative gene expression was calculated using the 2^{-Δ⁢Δ⁢Ct} method, with GAPDH as the normalization control.

For the cell counting kit-8 (CCK-8) assay, the cells were seeded into 96-well plates and incubated at 37 °C. After 1, 2, 3, 4, 5, and 6 days, the cells were treated with CCK-8 reagent and incubated for 4 hours at 37 °C. OD value at 450 nm was measured using a Bio-Rad spectrophotometer (Berkeley, CA, USA).

For the 5-ethynyl-2^′-deoxyuridine (EdU) assay, the cells were plated in 24-well plates and incubated at 37 °C for 24 hours. Subsequently, the cells were treated with EdU reagent (10 µM; RiboBio, Guangzhou, China) for 2 hours. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained with Apollo® reaction cocktail (RiboBio, China) according to the manufacturer’s instructions. Nuclei were counterstained with DAPI (Sigma-Aldrich, Shanghai, China), and images were captured using a fluorescence microscope (Zeiss, Oberkochen, Germany).

BC cells were fixed with 4% paraformaldehyde (PFA) and blocked with 5% bovine serum albumin (BSA). Cells were then incubated with primary antibodies against CD41 (Abcam, ab134131; 1:200) and subsequently with secondary antibodies conjugated with Alexa 555 (Invitrogen, 1:1000, CA, USA) and phalloidin-FITC (Abcam, ab235137, 1:1000, UK). The nuclei were counterstained with DAPI for 5 minutes. Images were acquired using a fluorescence microscope (Zeiss, Germany).

The MCF-7 cells were washed with PBS and fixed. They were then stained with anti-GPIb-IX (CD42b) antibody (BD Biosciences, Franklin Lake, NJ, USA, Cat# 551061, Lot# 123456) and anti-GP-IIb (CD41) antibody (BD Biosciences, Cat# 551061, Lot# 789012) for 20 minutes, and their expression levels were measured using a BD FACSCalibur (Franklin Lakes, NJ, USA) according to the manufacturer’s instructions.

The levels of T-box Protein 2 (TXB2) (Abcam, ab133054) and ATP production (Abcam, ab113849) in serum samples were measured using Enzyme-Linked Immunosorbent Assay (ELISA) kits (Abcam) following the manufacturer’s guidelines.

Transwell inserts (BD Falcon) were used with 24-well plates as the lower chambers. For migration assays, the inserts were left uncoated. For invasion assays, inserts were coated with 100 µL of Matrigel (diluted 1:3 with serum-free media). Cells (1 $\times$ 10⁵) were seeded in the upper chamber. After 24 hours, the cells that had migrated or invaded the underside of the membrane were fixed with 4% paraformaldehyde, stained with 0.2% crystal violet, and imaged.

HMEC cells were plated onto 24-well plates pre-coated with 50% Matrigel and cultured in medium from the MCF-7 cells upon the indicated transfections. Tube formation was assessed 4 hours later by capturing images with a fluorescence microscope (Carl Zeiss Inc, Oberkochen, Germany).

Nude mice (6-week-old female BALB/c nu/nu, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were subcutaneously injected with 1 $\times$ 10⁶ MCF-7 cells in 100 µL of PBS into the flanks. The mice were randomly divided into 2 groups (n = 6 per group): control shRNA, NOL6 shRNA. Tumor volume was measured every 4 days using calipers and calculated using the formula: volume = (length $\times$ width²)/2. The tumor should not exceed 2000 mm³ in mice. After 20 days, all mice were euthanized (cervical dislocation), and tumors were excised, weighed, and prepared for further analysis.

Tissue samples were sliced, dehydrated through absolute alcohol, and rehydrated. The sections were fixed with 4% paraformaldehyde for 30 minutes, blocked with 2% BSA and then incubated with primary antibodies against CD41 (Abcam, ab134131; 1:200) and CD31 (Abcam, ab28364; 1:500) for 2 hours. All sections were incubated with HRP antibody for 1.5 h followed by adding diaminobenzidine in 5 min, and all sections were then counterstained with hematoxylin.

Statistical analysis was performed using GraphPad 6.0 (GraphPad Software, Inc., Boston, MA, USA). For measurement data, the data were first assessed for normality using the Shapiro-Wilk test. For data that did not conform to a normal distribution, results are presented as medians with interquartile ranges, non-parametric tests were employed. Specifically, the Mann-Whitney U test was used to compare differences between two independent groups, and the Kruskal-Wallis test was applied for comparisons across multiple groups. Data are presented as mean $\pm$ SD for normally distributed data. Independent t-tests were used to compare the means between two groups. For categorical data, the data are presented as n (%). Statistical significance was determined using p 0.05.

To investigate NOL6 expression in BC, we analyzed transcript per million (TPM) values from the TCGA database. The analysis revealed that NOL6 expression was significantly elevated in BC tissues compared to normal tissues (p 0.001, Fig. 1a). Furthermore, the GEPIA database showed that high NOL6 expression was associated with poorer overall survival rates in BC patients (p = 0.02, Fig. 1b). To validate these findings, we conducted quantitative PCR (qPCR) assays and observed that NOL6 mRNA levels were significantly higher in tumor tissues than in normal tissues (p 0.001, Fig. 1c). Consistent with these results, immunoblotting of representative BC tissues confirmed increased NOL6 expression (p 0.001, Fig. 1d). Additionally, we examined NOL6 mRNA levels in BC patients categorized by blood stasis constitution versus non-blood stasis constitution. The analysis showed that patients with a blood stasis constitution had higher NOL6 mRNA levels compared to those without this constitution (p 0.001, Fig. 1e). Elevated P-selectin levels, which suggest increased platelet aggregation, were also observed in BC patients, particularly in those with blood stasis constitution (p 0.001, Fig. 1f). This observation was supported by clinical pathological analysis, which indicated a correlation between NOL6 expression, P-selectin levels, blood coagulation function, and angiogenesis indicators, suggesting a link between NOL6 expression, platelet aggregation, and angiogenesis (Table 1). Furthermore, we assessed NOL6 expression in various BC cell lines, including MCF-7, MDA-MB-231, and SW527, and found significantly higher expression levels compared to the normal breast cell line HMEC (p 0.001, Fig. 1g).

Fig. 1.

NOL6 is highly expressed in BC. (a) TCGA database analysis of TPM values for NOL6 in 1097 tumor tissues and 114 normal tissues. ***p 0.001. (b) Prognostic analysis from the GEPIA database showing overall survival rates based on NOL6 expression levels in BC patients (p = 0.021). (c) qPCR results demonstrating NOL6 mRNA levels in tumor versus normal tissues. ***p 0.001, BC vs healthy. (d) Immunoblot analysis of NOL6 expression in 3 representative BC tissues and 3 normal tissues (n = 3 per group). ***p 0.001, vs healthy. (e) qPCR data showing NOL6 mRNA levels in serum from BC patients with blood stasis constitution versus those with non-blood stasis constitution (n = 40 per group). *p 0.05, ***p 0.001, vs healthy; ###p 0.001 vs BC (blood stasis constitution). (f) P-selectin levels in serum from BC patients with blood stasis constitution compared to healthy controls. ***p 0.001, vs healthy; ###p 0.001 vs BC (blood stasis constitution). (g) Immunoblot demonstrating NOL6 expression in normal breast cell line HMEC and BC cell lines MCF-7, MDA-MB-231, and SW527. ***p 0.001 vs HMEC. NOL6, GEPIA, Gene Expression Profiling Interactive Analysis; Nucleolar Protein 6; BC, Breast cancer; BRCA, Breast cancer; TCGA, The Cancer Genome Atlas; TPM, transcript per million; qPCR, quantitative PCR.

To elucidate the role of NOL6 in BC cell proliferation, we used shRNA and overexpression plasmids to modulate NOL6 expression in MCF-7 cells. We found that transfection with NOL6 overexpression plasmids resulted in a significant increase in NOL6 expression, while transfection with NOL6 shRNA plasmids led to a marked decrease in its expression (p 0.01, Fig. 2a). Subsequent CCK-8 assays demonstrated that silencing NOL6 resulted in reduced viability of MCF-7 cells, whereas NOL6 overexpression significantly enhanced cell viability (p 0.01, Fig. 2b). Similarly, Edu assays revealed that NOL6 knockdown impeded cell growth, as evidenced by a reduced percentage of Edu-positive cells, while NOL6 overexpression promoted an increase in Edu-positive cells (p 0.001, Fig. 2c,d). These results collectively indicate that the knockdown of NOL6 effectively inhibits the growth of BC cells.

Fig. 2.

Knockdown of NOL6 inhibits the growth of BC cells. (a) Immunoblot analysis showing NOL6 expression levels in MCF-7 cells following the indicated transfections. (b) CCK-8 assay results depicting the growth of MCF-7 cells with different NOL6 expression levels over 6 days, measured by OD450. (c) Edu assay images showing MCF-7 cell proliferation following the indicated transfections, with (d) quantification of Edu-positive cells per field. Scale bar, 100 µm. Each experiment was conducted in triplicate. **p 0.01, ***p 0.001, pcDNA NOL6 vs plasmid containing DNA (pcDNA) Vector; ##p 0.01, ###p 0.001, shNOL6 vs shNC. NC, negative control.

Building on the observation that NOL6 promotes platelet aggregation in BC patients (Fig. 1f), we investigated how NOL6 influences platelet aggregation induced by BC cells. Initially, MCF-7 cells were co-cultured with human platelets, and immunostaining assays were performed to assess the expression of the platelet aggregation marker CD41. The results showed that NOL6 overexpression in MCF-7 cells resulted in elevated CD41 levels (p 0.001), whereas CD41 expression was significantly reduced in NOL6-depleted cells (p 0.01, Fig. 3a). FCM assays further confirmed these findings by measuring the expression of GPIb-IX and GP-IIb, which are key markers of platelet aggregation. Overexpression of NOL6 led to increased levels of both GPIb-IX and GP-IIb (p 0.001), while NOL6 knockdown reduced their expression, indicating a suppression of platelet aggregation (p 0.05, Fig. 3b). In addition, enzyme-linked immunosorbent assays (ELISA) demonstrated that the levels of TXB2, a marker associated with platelet aggregation, were higher in MCF-7 cells with NOL6 overexpression and lower in cells with NOL6 knockdown (p 0.01), thus validating the role of NOL6 in platelet aggregation (p 0.05, Fig. 3c). Moreover, ATP levels, which are indicative of platelet activation, were found to be increased in MCF-7 cells with NOL6 overexpression (p 0.001) and decreased in cells with NOL6 knockdown (p 0.05, Fig. 3d). Overall, these results indicate that the knockdown of NOL6 effectively inhibits platelet aggregation induced by BC cells.

Fig. 3.

Knockdown of NOL6 inhibits platelet aggregation induced by BC cells. (a) Immunostaining of CD41 in MCF-7 cells with the indicated transfections. Red panel indicates CD41, green panel indicates actin, and blue panel indicates DAPI. Scale bar, 30 µm. (b) Flow cytometry (FCM) analysis of GPIb-IX and GP-IIb expression levels in MCF-7 cells following different transfections. (c) Enzyme-Linked Immunosorbent Assay (ELISA) results showing TXB2 levels in MCF-7 cells with altered NOL6 expression. (d) Measurement of ATP levels in MCF-7 cells upon indicated transfections. Each experiment was conducted in triplicate. **p 0.01, ***p 0.001, pcDNA NOL6 vs pcDNA Vector; #p 0.05, ##p 0.01, shNOL6 vs shNC. NC, negative control. ELISA, Enzyme-Linked Immunosorbent Assay.

To evaluate the impact of NOL6 on BC cell motility and angiogenesis, we performed several assays. Transwell assays revealed that NOL6 overexpression significantly enhanced the migration and invasion of MCF-7 cells (p 0.001), while its depletion markedly reduced these processes (p 0.01, Fig. 4a,b). Subsequent tube formation assays demonstrated that NOL6 overexpression in MCF-7 cells increased the angiogenic potential of HMECs, as evidenced by a higher number of branch points (p 0.001, Fig. 4c,d). In contrast, NOL6 depletion led to a significant reduction in angiogenesis by Human Umbilical Vein Endothelial Cells (HUVECs) (p 0.001, Fig. 4c,d). Additionally, immunoblotting analysis showed that NOL6 overexpression upregulated the expression of angiogenesis-related factors, such as PDGF and VEGF (p 0.05), whereas NOL6 depletion resulted in decreased levels of these factors (p 0.01, Fig. 4e). Taken together, these findings collectively indicate that the knockdown of NOL6 inhibits both the angiogenic capacity and motility of BC cells.

Fig. 4.

Knockdown of NOL6 inhibits angiogenesis and motility of BC cells. (a) Transwell assays assessing the migration and invasion of MCF-7 cells following NOL6 modulation. Scale bar, 50 µm. (b) Quantification of migration and invasion cell counts from panel (a). (c) Tube formation assays illustrating the effects of NOL6 expression of MCF-7 cells and its culture medium on human umbilical vein endothelial cell (HUVEC) angiogenesis. Scale bar, 200 µm. CM, culture medium. (d) Quantification of branch points from panel (c). (e) Immunoblot analysis showing platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) expression levels in HUVECs in response to NOL6 modulation in MCF-7 cells. Each experiment was conducted in triplicate. *p 0.05, **p 0.01, ***p 0.001, pcDNA NOL6 vs pcDNA Vector; ##p 0.01, ###p 0.001, shNOL6 vs shNC. NC, negative control. HUVEC,Human Umbilical Vein Endothelial Cell. PDGF, platelet-derived growth factor; VEGF, Vascular endothelial growth factor.

Next, we explored the underlying mechanism by which NOL6 influences BC through immunoblot analyses. We observed that overexpression of NOL6 resulted in increased levels of Twist1 and galectin-3 in MCF-7 cells, while NOL6 depletion led to reduced expression of these proteins, suggesting a regulatory role of NOL6 in the Twist1/galectin-3 axis (p 0.01, Fig. 5a). This observation was further supported by qPCR assays, which confirmed that NOL6 modulates the mRNA levels of Twist1 and galectin-3 in MCF-7 cells (p 0.01, Fig. 5b).

Fig. 5.

NOL6 promotes the Twist1/galectin-3 axis in BC. (a) Immunoblot analysis of Twist1 and Galectin-3 expression in MCF-7 cells following NOL6 modulation. (b) qPCR assays assessing the mRNA levels of Twist1 and galectin-3 in MCF-7 cells upon NOL6 modulation. Each experiment was conducted in triplicate. **p 0.01, ***p 0.001, pcDNA NOL6 vs pcDNA Vector; #p 0.05, ##p 0.01, ###p 0.001, shNOL6 vs shNC. NC, negative control.

Additionally, we investigated whether the effects of NOL6 depletion could be reversed by Twist1 overexpression. The results from both immunoblot and qPCR assays demonstrated that Twist1 overexpression rescued the expression levels of Twist1 and galectin-3 reduced by NOL6 depletion (p 0.01, Supplementary Fig. 1a,b). Furthermore, CCK-8 assays showed that Twist1 overexpression mitigated the inhibition of MCF-7 cell growth induced by NOL6 depletion (p 0.001, Supplementary Fig. 1c). Flow cytometry (FCM) assays revealed that Twist1 overexpression restored the levels of GPIb-IX and GP-IIb in MCF-7 cells following NOL6 depletion, suggesting a recovery in platelet aggregation potential (p 0.01, Supplementary Fig. 1d). Similarly, ELISA showed that Twist1 overexpression counteracted the decrease in TXB2 levels caused by NOL6 ablation (p 0.01, Supplementary Fig. 1e). Finally, Twist1 overexpression reversed the suppression of tube formation in MCF-7 cells induced by NOL6 depletion, as observed in tube formation assays (p 0.001, Supplementary Fig. 1f). In summary, these results collectively demonstrate that NOL6 promotes the Twist1/galectin-3 axis in BC, influencing both cellular growth and platelet aggregation.

To assess the impact of NOL6 on BC tumor growth in vivo, we utilized shRNAs targeting NOL6 to decrease its expression in MCF-7 cells. These NOL6-depleted MCF-7 cells were then injected subcutaneously into the abdomen of nude mice. After 20 days, the results demonstrated that NOL6 ablation led to a significant suppression of tumor growth, as evidenced by reduced tumor volume and weight (p 0.05, Fig. 6a). The silencing efficiency of NOL6 in the tumors was confirmed by immunoblot analysis, which also showed decreased levels of Twist1 and galectin-3 in the tumor tissues from NOL6-depleted mice (p 0.001, Fig. 6b). Immunohistochemical (IHC) assays further revealed that NOL6 depletion resulted in reduced CD41 levels in tumor tissues, indicating suppression of platelet aggregation (p 0.001, Fig. 6c). Additionally, a decrease in CD31 levels was observed in the tumor tissues from NOL6-depleted mice, suggesting an inhibition of angiogenesis (p 0.001, Fig. 6d). These findings collectively suggest that NOL6 plays a role in promoting BC tumor growth in vivo, and its depletion leads to reduced tumor progression through mechanisms involving platelet aggregation and angiogenesis.

Fig. 6.

NOL6 inhibits the growth of BC in vivo. (a) Tumor growth assays illustrating the effects of NOL6 on BC progression in vivo. Representative images of tumors are shown (left), with tumor volume and weight measured every 4 days over 20 days (n = 6 per group). (b) Immunoblot analysis displaying the expression of NOL6 (left), Twist1, and Galectin-3 (right) in tumor tissues from the indicated groups. (c) Immunohistochemical (IHC) assays showing CD41 expression in tumor tissues, with quantitative analysis included. Scale bar, 200 µm. (d) IHC assays showing CD31 expression in tumor tissues, with quantitative analysis included. Scale bar, 200 µm. Each experiment was performed in triplicate. *p 0.05, ***p 0.001, shNOL6 vs shNC. NC, negative control. IHC, Immunohistochemical.