Non-SMC Condensin I Complex Subunit D2 Is a Prognostic Factor in Triple-Negative Breast Cancer for the Ability to Promote Cell Cycle and Enhance Invasion
2019; Elsevier BV; Volume: 190; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2019.09.014
ISSN1525-2191
AutoresYajing Zhang, Fangfang Liu, Chengli Zhang, Meijing Ren, Manchao Kuang, Ting Xiao, Xuebing Di, Lin Feng, Li Fu, Shujun Cheng,
Tópico(s)Cancer-related Molecular Pathways
ResumoTriple-negative breast cancer (TNBC) is a heterogeneous disease with an unfavorable prognosis and no specific targeted therapies. The role of non-SMC condensin I complex subunit D2 (NCAPD2), a regulatory subunit of the condensin I complex that mainly participates in chromosome condensation and segregation, has not been reported in cancer. We therefore evaluated the prognostic value and biological function of NCAPD2 in TNBC. The expression of NCAPD2 was studied in 179 TNBC tissues by immunohistochemistry, and associations among NCAPD2 expression, clinicopathologic features, and the prognosis information of patients with TNBC were analyzed. The mRNA expression profiles of 99 TNBC tissues were also studied, and cell biological behaviors were detected when NCAPD2 was altered in three TNBC cell lines. NCAPD2 expression was positively associated with lymph node metastasis (P = 3.84 × 10−06), poor overall survival (P = 0.0033), and worse disease-free survival (P = 0.0013) of patients with TNBC. Moreover, knockdown of NCAPD2 might cause G2/M arrest through the p53 signaling pathway, which led to proliferation inhibition, polyploid cell production, and cell apoptosis and inhibited the invasiveness of TNBC cells. For the first time, we report the close association between NCAPD2 and cancer and demonstrate that NCAPD2 plays an important role in TNBC progression and acts as an independent poor prognostic factor and a potential therapeutic target for TNBC. Triple-negative breast cancer (TNBC) is a heterogeneous disease with an unfavorable prognosis and no specific targeted therapies. The role of non-SMC condensin I complex subunit D2 (NCAPD2), a regulatory subunit of the condensin I complex that mainly participates in chromosome condensation and segregation, has not been reported in cancer. We therefore evaluated the prognostic value and biological function of NCAPD2 in TNBC. The expression of NCAPD2 was studied in 179 TNBC tissues by immunohistochemistry, and associations among NCAPD2 expression, clinicopathologic features, and the prognosis information of patients with TNBC were analyzed. The mRNA expression profiles of 99 TNBC tissues were also studied, and cell biological behaviors were detected when NCAPD2 was altered in three TNBC cell lines. NCAPD2 expression was positively associated with lymph node metastasis (P = 3.84 × 10−06), poor overall survival (P = 0.0033), and worse disease-free survival (P = 0.0013) of patients with TNBC. Moreover, knockdown of NCAPD2 might cause G2/M arrest through the p53 signaling pathway, which led to proliferation inhibition, polyploid cell production, and cell apoptosis and inhibited the invasiveness of TNBC cells. For the first time, we report the close association between NCAPD2 and cancer and demonstrate that NCAPD2 plays an important role in TNBC progression and acts as an independent poor prognostic factor and a potential therapeutic target for TNBC. Triple-negative breast cancer (TNBC), which is the most aggressive subtype of breast cancer, accounts for 15% to 20% of all patients with breast cancer and is characterized by lack of expression of estrogen receptor (ER) and progesterone receptor (PR) and an absence of amplification of human epidermal growth factor receptor 2 (HER2).1Carey L.A. Dees E.C. Sawyer L. Gatti L. Moore D.T. Collichio F. Ollila D.W. Sartor C.I. Graham M.L. Perou C.M. The triple negative paradox: primary tumor chemosensitivity of breast cancer subtypes.Clin Cancer Res. 2007; 13: 2329-2334Crossref PubMed Scopus (1630) Google Scholar, 2Carey L.A. Perou C.M. Livasy C.A. Dressler L.G. Cowan D. Conway K. Karaca G. Troester M.A. Tse C.K. Edmiston S. Deming S.L. Geradts J. Cheang M.C. Nielsen T.O. Moorman P.G. Earp H.S. Millikan R.C. Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study.JAMA. 2006; 295: 2492-2502Crossref PubMed Scopus (2912) Google Scholar, 3Sorlie T. Perou C.M. Tibshirani R. Aas T. Geisler S. Johnsen H. Hastie T. Eisen M.B. van de Rijn M. Jeffrey S.S. Thorsen T. Quist H. Matese J.C. Brown P.O. Botstein D. Lonning P.E. Borresen-Dale A.L. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications.Proc Natl Acad Sci U S A. 2001; 98: 10869-10874Crossref PubMed Scopus (8513) Google Scholar, 4Sorlie T. Tibshirani R. Parker J. Hastie T. Marron J.S. Nobel A. Deng S. Johnsen H. 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Borresen-Dale A.L. Brown P.O. Botstein D. Molecular portraits of human breast tumours.Nature. 2000; 406: 747-752Crossref PubMed Scopus (11662) Google Scholar, 10Mustacchi G. De Laurentiis M. The role of taxanes in triple-negative breast cancer: literature review.Drug Des Devel Ther. 2015; 9: 4303-4318Crossref PubMed Scopus (85) Google Scholar Therefore, exploration of the molecular basis of TNBC and developing effective therapeutic strategies, which will eventually improve the survival rate of patients with TNBC, is urgently needed. The segregation of two copies of replicated genomic DNA into each daughter cell is the major feature of M phase during the cell cycle. During this period, the relaxed chromatin is converted into condensed chromosomes at the very beginning of mitosis, and the entangled sister chromatids resulting from DNA replication are decatenated.11Hirano T. Chromosome cohesion, condensation, and separation.Annu Rev Biochem. 2000; 69: 115-144Crossref PubMed Scopus (226) Google Scholar,12Swedlow J.R. Hirano T. The making of the mitotic chromosome: modern insights into classical questions.Mol Cell. 2003; 11: 557-569Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar Failure to perform this process accurately can lead to detrimental chromosome segregation defects.13Watrin E. Legagneux V. Contribution of hCAP-D2, a non-SMC subunit of condensin I, to chromosome and chromosomal protein dynamics during mitosis.Mol Cell Biol. 2005; 25: 740-750Crossref PubMed Scopus (33) Google Scholar One key factor involved in chromosome condensation and decatenation is a heteropentamer called condensin, which is evolutionarily conserved from bacteria to humans. Non-SMC condensin I complex subunit D2 (NCAPD2) is a subunit of condensin I that is located on chromosome 12p13.3. Transfection and site-directed mutagenesis studies have confirmed that NCAPD2 is regulated by E2F.14Verlinden L. Eelen G. Beullens I. Van Camp M. Van Hummelen P. Engelen K. Van Hellemont R. Marchal K. De Moor B. Foijer F. Te Riele H. Beullens M. Bollen M. Mathieu C. Bouillon R. Verstuyf A. Characterization of the condensin component Cnap1 and protein kinase Melk as novel E2F target genes down-regulated by 1,25-dihydroxyvitamin D3.J Biol Chem. 2005; 280: 37319-37330Crossref PubMed Scopus (40) Google Scholar NCAPD2 contains a bipartite nuclear localization signal in its carboxyl terminus, which is necessary for the nuclear localization and chromosome targeting of condensin I.15Ball Jr., A.R. Schmiesing J.A. Zhou C. Gregson H.C. Okada Y. Doi T. Yokomori K. Identification of a chromosome-targeting domain in the human condensin subunit CNAP1/hCAP-D2/Eg7.Mol Cell Biol. 2002; 22: 5769-5781Crossref PubMed Scopus (47) Google Scholar In addition, the HEAT repeat of NCAPD2, which is a bihelical structure arranged in tandem, may play a role in protein-protein interactions.16Neuwald A.F. Hirano T. HEAT repeats associated with condensins, cohesins, and other complexes involved in chromosome-related functions.Genome Res. 2000; 10: 1445-1452Crossref PubMed Scopus (231) Google Scholar,17Groves M.R. Barford D. Topological characteristics of helical repeat proteins.Curr Opin Struct Biol. 1999; 9: 383-389Crossref PubMed Scopus (277) Google Scholar However, the function of NCAPD2 is not well understood, and previous studies of NCAPD2 have primarily focused on its participation in chromosome condensation and segregation.18Conroy P.C. Saladino C. Dantas T.J. Lalor P. Dockery P. Morrison C.G. C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis.Cell Cycle. 2012; 11: 3769-3778Crossref PubMed Scopus (29) Google Scholar, 19Fang G. Zhang D. Yin H. Zheng L. Bi X. Yuan L. Centlein mediates an interaction between C-Nap1 and Cep68 to maintain centrosome cohesion.J Cell Sci. 2014; 127: 1631-1639Crossref PubMed Scopus (43) Google Scholar, 20Martin C.A. Murray J.E. Carroll P. Leitch A. Mackenzie K.J. Halachev M. Fetit A.E. Keith C. Bicknell L.S. Fluteau A. Gautier P. Hall E.A. Joss S. Soares G. Silva J. Bober M.B. Duker A. Wise C.A. Quigley A.J. Phadke S.R. Deciphering Developmental Disorders Study; Wood AJ, Vagnarelli P, Jackson AP: Mutations in genes encoding condensin complex proteins cause microcephaly through decatenation failure at mitosis.Genes Dev. 2016; 30: 2158-2172Crossref PubMed Scopus (76) Google Scholar These studies found that the recruitment of condensin I onto chromosomes depends on the interaction between the chromosome-targeting domain of NCAPD2 and the phosphorylated histone H3.21Schmiesing J.A. Gregson H.C. Zhou S. Yokomori K. A human condensin complex containing hCAP-C-hCAP-E and CNAP1, a homolog of Xenopus XCAP-D2, colocalizes with phosphorylated histone H3 during the early stage of mitotic chromosome condensation.Mol Cell Biol. 2000; 20: 6996-7006Crossref PubMed Scopus (101) Google Scholar Several observations have revealed the misalignment of chromosomes on the metaphase plate in the absence of NCAPD2. Moreover, depletion of NCAPD2 affects both the individualization of mitotic chromosomes and the resolution of sister chromatids.13Watrin E. Legagneux V. Contribution of hCAP-D2, a non-SMC subunit of condensin I, to chromosome and chromosomal protein dynamics during mitosis.Mol Cell Biol. 2005; 25: 740-750Crossref PubMed Scopus (33) Google Scholar,22Ono T. Fang Y. Spector D.L. Hirano T. Spatial and temporal regulation of Condensins I and II in mitotic chromosome assembly in human cells.Mol Biol Cell. 2004; 15: 3296-3308Crossref PubMed Scopus (274) Google Scholar Recent research has found that NCAPD2 also interacts with rootletin, which functions in centrosome cohesion and restrains DNA damage–induced centriole splitting.23Yang J. Adamian M. Li T. Rootletin interacts with C-Nap1 and may function as a physical linker between the pair of centrioles/basal bodies in cells.Mol Biol Cell. 2006; 17: 1033-1040Crossref PubMed Scopus (115) Google Scholar,24Conroy P.C. Saladino C. Dantas T.J. Lalor P. Dockery P. Morrison C.G. C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis.Cell Cycle. 2012; 11: 3769-3778Crossref PubMed Scopus (35) Google Scholar However, as a pivotal participant in the cell cycle, the role of NCAPD2 in cancer has not yet been reported. In this study, the protein expression level of NCAPD2 was evaluated in 179 primary TNBC samples by immunohistochemistry, and the association among the protein expression, the clinicopathologic features, and the prognosis of these patients was determined. Moreover, mRNA expression profiles of TNBC tissues and NCAPD2 low-expression TNBC cell models were used to investigate the role of NCAPD2 in TNBC. All tissue samples were obtained from 179 female patients who were diagnosed with primary breast cancer and underwent surgery at the Department of Pathology, Tianjin Medical University Cancer Hospital (Tianjin, China) between 2003 and 2006. No patients received chemotherapy or radiotherapy before surgery. Surgically resected tissue samples were fixed in 10% formaldehyde and then embedded in paraffin for 4-μm histologic sectioning. The histopathologic examination was performed by two independent and experienced pathologists (L.Fu and F.L., who work in the Department of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China, and have been engaged in pathologic diagnosis for the last 5 years) who were blinded to the groups in this study. All samples were identified as TNBC by ER, PR, and HER2 staining. The study was reviewed and approved by the Institutional Ethics Committee of Tianjin Medical University Cancer Institute and Hospital, and all patients provided written informed consent. All methods were performed in accordance with the approved guidelines. After routine deparaffinization in xylene and rehydration in a graded ethanol of decreasing concentrations, the tissue sections were incubated in 3% H2O2 for 10 minutes to block endogenous peroxidase activity. Then the sections were subjected to antigen retrieval by heating in citrate buffer pH 6.0 at 121°C for 150 seconds. After cooling to room temperature, the sections were treated with 5% normal goat serum to block nonspecific binding and incubated with a mouse monoclonal antibody against NCAPD2 at a 1:100 dilution (catalog number ab56885; Abcam, Cambridge, UK) overnight at 4°C. Then a biotin-conjugated secondary antibody was applied, and NCAPD2 expression was detected using a streptavidin-peroxidase complex (catalog number SP-9000; Zhongshan Golden Bridge Biotechnology Company, Zhongshan, China). The chromogenic reaction was performed using a commercial diaminobenzidine kit (catalog number ZLI-9550; Zhongshan Golden Bridge Biotechnology Company). Finally, the sections were counterstained in hematoxylin. The immunohistochemistry-stained sections were analyzed and scored independently by two experienced pathologists (L.Fu and F.L.) who were blinded to the patients' clinicopathologic data. The staining intensity was scored as 0, 1, 2, or 3 based on color (no color, ecru, brown, and puce, respectively), and the percentage of positively stained tumor cells was determined, with a staining percentage ≥1% considered positive. The staining intensity scores and the percentage of the positive tumor cells were used to calculate H-scores with the following formula: H-score = Σ (percentage of positively stained tumor cells) × (staining intensity) H-scores were then used to categorize the samples into four groups as follows: negative (−), score 0; mild (+), score of >0 to 1; moderate (++), score of >1 to 2; and marked (+++), score of >2 to 3. The human TNBC cell lines MDA-MB-231, MDA-MB-453, and MDA-MB-468 were obtained from the National Infrastructure of Cell Line Resource (Beijing, China). MDA-MB-231 and MDA-MB-453 cells were maintained in RPMI 1640 medium (catalog number 21875109; Gibco, Waltham, MA) supplemented with 10% fetal bovine serum (DMEM; catalog number 12484-028; Gibco). MDA-MB-468 cells were maintained in Dulbecco’s modified Eagle’s medium (catalog number 11966025; Gibco) supplemented with 10% fetal bovine serum and 2 mmol/mL of l-glutamine. All the cell lines were cultured in a humidified incubator at 37°C with 5% CO2. For the knockdown experiments, cells were transfected with siRNA targeting NCAPD2 (siNCAPD2; catalog number HSS115010; Invitrogen, Carlsbad, CA) or nontargeting control (NC; catalog number 12935100; Invitrogen) using the Lipofectamine RNAiMAX transfection reagent (catalog number 13778150; Invitrogen) according to the manufacturer's instructions. Total RNA was extracted using TRIzol reagent (catalog number 15596018; Thermo Fisher Scientific, Waltham, MA) and reverse transcribed using a SuperScript II reverse transcription kit according to the manufacturer's protocol (catalog number 18064014; Thermo Fisher Scientific). Then real-time RT-PCR was conducted as described in our previous report.25Tsai C.Y. Wang C.S. Tsai M.M. Chi H.C. Cheng W.L. Tseng Y.H. Chen C.Y. Lin C.D. Wu J.I. Wang L.H. Lin K.H. Interleukin-32 increases human gastric cancer cell invasion associated with tumor progression and metastasis.Clin Cancer Res. 2014; 20: 2276-2288Crossref PubMed Scopus (81) Google Scholar Melting curves were generated, and the relative mRNA expression level of the target gene was normalized to that of 18S ribosomal RNA. All data are presented as the means of three replicates. The primers used for the amplifications are as follows: NCAPD2 forward, 5′-TGGAGGGGTGAATCAGTATGT-3′; NCAPD2 reverse, 5′-GCGGGATACCACTTTTATCAGG-3′; 18S forward, 5′-CTGAGAAACGGCTACCACATCC-3′; 18S reverse, 5′-GCACCAGACTTGCCCTCCA-3′. The proliferation abilities of three TNBC cell lines were measured using the Cell Counting Kit 8 (CCK-8) assay (catalog number CK04; Dojindo, Kumamoto, Japan). Briefly, cells were seeded in 96-well plates at a density of 2000 (MDA-MB-231 and MDA-MB-453) or 4000 (MDA-MB-468) cells per well. After adhesion for 24 hours, the cells were transfected with siNCAPD2 or NC. After incubation for 0 to 120 hours at an interval of 24 hours, the cells were stained with 10 μL of CCK-8 solution in 90 μL of culture medium for 2.5 hours at 37°C at each time point. Cell proliferation was measured as the absorbance at 450 nm using a microplate reader (BioTek, Winooski, VT). Corning BioCoat Matrigel Invasion Chambers (24 wells, 8.0-μm pore size, Corning, Corning, NY) were used for Transwell invasion assays according to the manufacturer's instructions. Briefly, after 48 hours of transfection, 1 × 105 cells suspended in 500 μL of serum-free medium were seeded into the upper chambers, whereas 750 μL of complete medium with 10% fetal bovine serum was injected into the lower chambers as a chemoattractant. After incubation for 24 hours, the cells that had invaded through the membrane were fixed in cold methanol followed by staining with 0.1% crystal violet, and then cells were counted in at least five fields under a light microscope. Cells were harvested and total protein was extracted using radioimmunoprecipitation assay lysis buffer (catalog number C1053; Applygen, Beijing, China) supplemented with protease inhibitor cocktail (catalog number 78410; Thermo Fisher Scientific). The Pierce BCA Protein Assay Kit was used to quantify the protein concentration (catalog number 23225; Thermo Fisher Scientific). Subsequently, equal amounts of the proteins were separated by SDS-PAGE and transferred onto a 0.25-μm polyvinylidene difluoride membrane (catalog number IPFL00010; Millipore, Burlington, MA) via a wet trans-blot system (BioRad, Hercules, CA). The membranes were then blocked and incubated with an antibody against NCAPD2 (catalog number ab56885; Abcam, Cambridge, UK) overnight at 4°C, followed by incubation with a horseradish peroxidase–conjugated secondary antibody (catalog number ZDR-5307; Zhongshan Golden Bridge Biotechnology Company) and analyzed using an enhanced chemiluminescence detection kit (catalog number P1050; Applygen). Finally, images were obtained with an ImageQuant LAS 4000 mini system (GE Healthcare, Chicago, IL). β-actin (catalog number sc-58673; Santa Cruz Biotechnology, Santa Cruz, CA) was used as an internal control. The effect of NCAPD2 on cell cycle distribution was determined by flow cytometry. Briefly, after transfection with siNCAPD2 or NC for 96 hours, cells were harvested, fixed in 70% ice-cold ethanol, and incubated overnight at −20°C. Cells were washed with phosphate-buffered saline and resuspended in 500 μL of propidium iodide (PI)/RNase Staining Buffer (catalog number 550825; BD, Franklin Lakes, NJ). After incubation for 15 minutes in the dark at room temperature, cells were analyzed by flow cytometry using a LSRII flow cytometer (BD). Each experiment was performed at least three times. The fractions of cells in G0/G1, S, and G2/M phases and the ratio of polyploid cells were analyzed using ModFit LT software version 3.2 (BD). Cell apoptosis was detected using the fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (catalog number 556547; BD) according to the supplier's protocols. Ninety-six hours after transfection, cells were collected, centrifuged, and resuspended in 500 μL of 1× binding buffer. Then 5 μL of Annexin V–FITC and 10 μL of PI were added to each tube. The tubes were incubated in the dark at room temperature for 15 minutes. Cell apoptosis assays were performed immediately on an LSRII flow cytometer (BD). Normalized mRNA expression data of The Cancer Genome Atlas (TCGA), including 99 TNBC samples and six normal breast tissues from patients with TNBC, were obtained from the cBioPortal for Cancer Genomics database (http://www.cbioportal.org, last accessed April 28, 2019). Pearson's product-moment correlation test was performed to identify genes that were significantly correlated with NCAPD2 (P < 0.001 and |cor| > 0.3), and DAVID version 6.8 and R package clusterProfiler was applied to analyze the Gene Ontology (GO) enrichment of gene functions and KEGG pathways (https://david.ncifcrf.gov, last accessed April 28, 2019). All results are the means of at least three independent experiments with separately treated and transfected cells. Data are expressed as the means ± SEM. Statistical analyses were performed using SPSS software version 17.0 (SPSS, Chicago, IL). The correlations between NCAPD2 expression and the clinicopathologic parameters were evaluated using the χ2 test. Survival curves were obtained by the Kaplan-Meier method, and the differences in survival were analyzed by the log-rank test. Multivariate analyses were conducted using a Cox proportional hazards model. Differences were considered statistically significant at P < 0.05. In TNBC tissues, the staining pattern of NCAPD2 was intense and clear in the cytoplasm. In general, the rates of negative, mild, moderate, and marked expression were 25.69% (n = 46/179), 60.33% (n = 108/179), 8.93% (n = 16/179), and 5.11% (n = 9/179), respectively (Figure 1, A–D ). According to the staining intensity, the samples were divided into two groups, with negative staining defined as negative NCAPD2 expression group (NCAPD2 negative) and mild, moderate, and marked staining defined as positive NCAPD2 expression group (NCAPD2 positive). The correlations between NCAPD2 expression and a variety of clinical features were further analyzed. NCAPD2 expression was frequently associated with lymph node involvement (P = 3.84 × 10−06) (Table 1). However, the expression of NCAPD2 was not associated with patient age (P = 0.3042), tumor size (P = 0.2358), or pathologic grade (P = 0.3018).Table 1Association between Non-SMC Condensin I Complex Subunit D2 (NCAPD2) Expression and the Clinicopathologic Characteristics of 179 Patients with TNBCCharacteristicNCAPD2 negative, n (total = 46)NCAPD2 positive, n (total = 133)PAge, years (median = 52 years)0.3042 ≤522380 >522353Tumor size0.2358 T1617 T23482 T3/T4425Pathologic grade0.3018 105 22170 31844Lymph node involvement3.84 × 10−06 N03643 N1647 N2428 N3010Two cases in the NCAPD2-negative group and nine cases in NCAPD2-positive group have missing tumor size characteristics. Seven cases in the NCAPD2-negative group and 14 cases in NCAPD2-positive group have missing pathologic grade characteristics. Five cases in the NCAPD2-positive group have missing lymph node involvement characteristics. Open table in a new tab Two cases in the NCAPD2-negative group and nine cases in NCAPD2-positive group have missing tumor size characteristics. Seven cases in the NCAPD2-negative group and 14 cases in NCAPD2-positive group have missing pathologic grade characteristics. Five cases in the NCAPD2-positive group have missing lymph node involvement characteristics. Statistical analysis revealed that positive NCAPD2 expression was significantly correlated with poor overall survival (Figure 1E). A Cox proportional hazards model was applied to estimate the effect of NCAPD2 expression on overall survival. Both univariate and multivariate Cox regression analyses found that positive NCAPD2 expression increased the risk of TNBC-related death compared with negative NCAPD2 expression (Supplemental Table S1). Moreover, patients with positive NCAPD2 expression had significantly worse disease-free survival than those with negative expression (Figure 1F). Both univariate and multivariate Cox regression analyses found that positive NCAPD2 expression could increase the risk of TNBC progression (Supplemental Table S2). Taken together, these results indicate that NCAPD2 is an independent predictor of poor survival for patients with TNBC. To determine the associations between NCAPD2 expression patterns and the biological features of cancer, the mRNA expression profiles of 99 TNBC tissue samples from TCGA program were analyzed. Pearson's product-moment correlation test was performed to identify genes that were significantly correlated with NCAPD2. Overall, 1728 genes were recognized to be closely correlated with NCAPD2 (P < 0.001 and |cor| > 0.3), including 956 positively correlated genes and 772 negatively correlated genes. Through GO enrichment analysis, it was found that these positively correlated genes were associated with cell proliferation (cell proliferation, cell division, cell cycle), RNA processing (mRNA processing, mRNA splicing, rRNA processing, mRNA and tRNA export from the nucleus), and DNA replication and repair (ontology: biological process) (Figure 2A). These positively correlated genes were mainly located in the nucleoplasm and cytoplasm (ontology: cellular component) (Figure 2C). On the contrary, the negatively correlated genes were associated with angiogenesis, cell migration, cell proliferation, and signal transduction (ontology: biological process) (Figure 2B) and mainly located in extracellular exosomes and extracellular space (ontology: cellular component) (Figure 2D). KEGG pathway analysis revealed that multiple cellular pathways, including DNA replication, cell cycle, spliceosome, RNA transport, complement and coagulation cascades, and lysosome, were significantly enriched (Figure 2, E and F). These data indicated that NCAPD2 participates in multiple aspects of TNBC tumorigenesis. Therefore, the differential expression of NCAPD2 may correspond to the different prognostic states of patients. The NCAPD2 mRNA expression of six normal and 99 tumor breast tissues from TCGA patients with TNBC was analyzed. The mRNA expression of NCAPD2 in TNBC tissues was significantly higher than in the normal breast tissues that from patients with TNBC (Supplemental Figure S1). To further investigate the biological roles of NCAPD2 in TNBC tumorigenesis and to uncover the veil of the reason why NCAPD2 differential expression caused different prognostic states of patients with TNBC, TNBC cell models were constructed in which NCAPD2 was knocked down. NCAPD2 expression levels were first examined in the three human TNBC cell lines MDA-MB-231, MDA-MB-468, and MDA-MB-453. The results indicate that the expression levels of NCAPD2 were varied in these three TNBC cell lines and that the expression levels were consistent at the RNA and protein levels (Supplemental Figure S2, A and C). TNBC cells were transfected with either siNCAPD2 or NC to deplete NCAPD2. The efficiency of siRNA-mediated knockdown of NCAPD2 was validated by real-time PCR and Western blotting. TNBC cells transfected with siNCAPD2 showed a noteworthy decrease in the NCAPD2 mRNA and protein expression levels compared with the NC group (Supplemental Figure S2, B and D). The effect of siRNAs on cell proliferation was evaluated by CCK-8 assays. The results indicate that the proliferation of TNBC cells was significantly inhibited at 96 and 120 hours after NCAPD2 knockdown compared with that of the NC group (Figure 3A). Moreover, Transwell assays revealed that down-regulation of NCAPD2 resulted in a significant decrease in the invasive capabilities of TNBC cells (Figure 3B), which fits better with the finding that low expression of NCAPD2 is associated with less lymph node metastasis and better overall survival. To address how NCAPD2 contributes to cell proliferation, the cell cycle of TNBC cells was analyzed by flow cytometry after siNCAPD2 or NC transfection. Down-regulation of NCAPD2 caused an accumulation of cells in G2/M phase compared with the NC group of TNBC cells (Figure 4A). In addition, a significant increase of polyploid cells was observed after NCAPD2 depletion (Figure 4B). Furthermore, th
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