Human TNBC BT-549, MDA-MB-231, MDA-MB-436 and MDA-MB-468 cells were obtained from the American Type Culture Collection. 293T cells were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. TNBC cells were cultured in RPMI 1640 medium (HyClone; Cytiva) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin and streptomycin solution (Corning, Inc.). 293T cells were cultured in DMEM (HyClone; Cytiva) supplemented with 10% FBS and 1% penicillin and streptomycin solution. Cell culture was maintained in a 37°C incubator with 5% CO₂.

To silence SOX9 in MDA-MB-231 and MDA-MB-468 cells, the lentivirus vector pGCSIL-GFP was constructed, which contained a short hairpin (sh)RNA sequence targeting SOX9. shRNA sequences were as follow: Negative control (shNC), 5′-TTCTCCGAACGTGTCACGT-3′; shSOX9-1, 5′-GCATCCTTCAATTTCTGTATA-3′; shSOX9-2, 5′-GCGGAGGAAGTCGGTGAAGAA-3′; and shSOX9-3, 5′-CTCCACCTTCACCTACATGAA-3′. For lentivirus packaging, the expression plasmid (pGCSIL-GFP; 20 mg; Shanghai GeneChem Co., Ltd.) with two helper plasmids (pHelper 1, 15 mg and pHelper 2, 10 mg; Shanghai GeneChem Co., Ltd.) were co-transfected into 293T cells using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in 10-cm plates. After 48 and 72 h of transfection, lentiviruses were harvested. Following purification and titration, the harvested virus particles were used to infect MDA-MB-231 and MDA-MB-468 cells using a multiplicity of infection of 20. After 72 h, cells were harvested for knockdown efficiency examination when green fluorescence was visible.

CCK-8 reagent was purchased from Sigma-Aldrich (Merck KGaA) and used according to the manufacturer's instructions. MDA-MB-231 and MDA-MB-468 cells were trypsinized and seeded in triplicate into 96-well plates at a concentration of 2,000 cells/well. Each well contained 100 µl culture medium. After 6, 12, 24, 36, 48 and 72 h, 10 µl CCK-8 reagent was added to each well. The plates were maintained in a 37°C incubator for 3 h. The absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, Inc.) after agitation for 30 sec.

shNC- and shSOX9-transfected MDA-MB-231 and MDA-MB-468 cells were seeded into 6-well plates at concentrations of 50, 100, 200, 250, 500 and 1,000 cells/well. The culture medium was replaced by fresh medium every 3 days. The colonies were formed after culturing for 14 days at 37°C. The colonies were washed three times with PBS, fixed with 4% paraformaldehyde (Wuhan Servicebio Technology Co., Ltd.) for 20 min at room temperature and stained with Giemsa (Sigma-Aldrich; Merck KGaA) staining solution for 20 min at room temperature. A colony was defined as >50 cells. The colony formation rate was calculated by colony number/seeding number.

Transwell assay was performed to assess migration and invasion. For invasion, Matrigel^® (Corning, Inc.) was mixed with serum-free medium in a 1:8 ratio. A total of 100 µl of the mixture was added onto the upper surface of the migration chambers (Corning, Inc.; 8.0-µm filter) and incubated at 37°C for 30 min. MDA-MB-231 cells were trypsinized and washed with serum-free medium. A total of 1×10⁴ cells in 100 µl serum-free medium were seeded onto the upper surface of the chambers, while the lower chambers were filled with medium with 10% FBS. After 24 h at 37°C, the culture medium and the cells attached on the upper surface were removed. Cells attached on the lower surface were fixed with methanol for 30 min at room temperature and stained with 0.1% crystal violet for 30 min at room temperature. The images of migratory and invasive cells were collected using a light microscope (Olympus Corporation; magnification, ×100).

MDA-MB-231 cells were seeded into 6-well plates and cultured overnight. The cells were grown to ~100% confluence in the wells. Linear scratch wounds were developed using 10-µl pipette tips. The floating cells were washed five times with PBS. Serum-free medium was used to maintain the cells. The images were captured at 0, 6, 12, 18 and 24 h using an inverted light microscope (Olympus Corporation; magnification, ×100). The wound-healing percentage was calculated using the following formula: (Wound area at 0 h-wound area of indicated time)/(wound area at 0 h) ×100. Wound area was analyzed using ImageJ (v4.0; National Institute of Health).

Total RNA in MDA-MB-231 and MDA-MB-468 cells was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was reverse transcribed to cDNA using the Reverse transcription system kit (Promega Corporation; cat. no. A3500), according to the manufacturer's instructions. qPCR analysis was performed using SYBR Master Mixture (Takara Bio, Inc.) on the Agilent MX3000p Real Time PCR system (Agilent Technologies, Inc.) as follows: 1 cycle at 95°C for 30 sec; 40 cycles at 95°C for 5 sec and 60°C for 20 sec; 1 cycle at 65°C for 15 sec. qPCR primers were as follows: SOX9 forward, 5′-AGCGAACGCACATCAAGAC-3′ and reverse, 5′-CTGTAGGCGATCTGTTGGGG-3′; and GAPDH forward, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′. mRNA expression was analyzed using the 2^−ΔΔCq method (14).

Western blotting was used to determine the protein expression levels in TNBC cells. Total protein (40 µg) was extracted from MDA-MB-231 and MDA-MB-468 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology), supplemented with protease and phosphatase inhibitors cocktail (Roche Diagnostics). The protein concentration was detected using a BCA Protein Assay kit. A total of 40 µg of protein was loaded and separated via 12% SDS-PAGE, followed by transfer to PVDF membranes (EMD Millipore). Membranes were blocked with 5% skimmed milk dissolved in PBS-Tween (0.1% Tween) at room temperature for 1.5 h and incubated with primary antibodies overnight at 4°C. After incubating with peroxidase-conjugated secondary antibodies for 2 h at room temperature, the protein abundance was analyzed using an enhanced chemiluminescence system (EMD Millipore). Primary antibodies against SOX9 (1:1,000; cat. no. ab185966) and GAPDH (1:1,000; cat. no. ab8245) were purchased from Abcam. HRP-conjugated anti-mouse (1:5,000; cat. no. sc-2005) or anti-rabbit (1:5,000; cat. no. sc-2004) secondary antibodies were from Santa Cruz Biotechnology, Inc.

Apoptosis was analyzed using an annexin V/PI apoptosis detection kit according to the manufacturer's protocol (cat. no. 88-8007-72; Invitrogen; Thermo Fisher Scientific, Inc.). MDA-MB-231 and MDA-MB-468 cells were trypsinized with EDTA-free 0.25% Trypsin (Corning, Inc.). The cells and cell supernatants were collected by centrifugation at 1,000 × g for 5 min at room temperature. The cell pellet was washed using iced D-Hanks (pH, 7.2–7.4) buffer and 1X binding buffer. The cell pellet was resuspended in 200 µl 1X binding buffer. After incubating with 10 µl Annexin V-APC for 10–15 min in the dark at room temperature, cells were stained with PI staining buffer (5 µl) for 10–15 min on ice at room temperature. A total of 400–800 µl 1X binding buffer was added and apoptosis was analyzed on a Guava easyCyte HT system (EMD Millipore). The data was analyzed using the guavasoft software (v2.7; Merck KGaA).

PI staining was used to analyze the cell cycle. When MDA-MB-231 and MDA-MB-468 cells reached 80% confluence, they were trypsinized and centrifuged at 1,000 × g for 5 min at room temperature. The cell pellet was washed using iced D-Hanks (pH 7.2–7.4) buffer and fixed with iced 75% ethanol for ≥1 h at −20°C. The cells were centrifuged at 1,000 × g for 5 min at room temperature and the pellet was washed twice by D-Hanks buffer. Finally, the cell pellet was resuspended in 0.6–1 ml staining buffer [40X PI solution (2 mg/ml):100X RNase solution (10 mg/ml):1X D-Hanks; 25:10:1,000) at room temperature for 30 min and subjected to cell cycle analysis using a Guava easyCyte HT system (EMD Millipore). The data was analyzed using the guavasoft software (v2.7; Merck KGaA).

Firstly, total RNA was extracted from shNC- and shSOX9-transfected MDA-MB-231 cells using TRIzol reagent. Subsequently, RNA sequencing was performed by Beijing Aoweisen Gene Technology Co., Ltd. Briefly, the total RNA integrity was evaluated using an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc.). The RNA Clean XP kit (cat. no. A63987; Beckman Coulter, Inc.) and RNase-free DNase Set (cat. no. 79254; Qiagen GmbH) were used for RNA clean-up and DNA removing, respectively. A total of 1 µg purified RNA was used for cDNA library generation using a VAHTSTM mRNA-seq v2 library Prep kit (cat. no. NR612-01; Vazyme Biotech Co., Ltd.). The quality of constructed cDNA libraries were evaluated with an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) according to the manufacturer's protocol. The products were sequenced using an Illumina NovaSeq 6000 SP Reagent kit v1.5 (300 cycles; cat. no. 20028400; Illumina, Inc.) with a paired-end read length of 150 bp. A total of 1 nM of the final library was used for sequencing. For data analysis, TopHat was used to map reads to a reference genome (hg38) (15). Gene expression was measured using the fragments per kilobase of transcript per million mapped reads (FPKM) normalization method. FPKM quantification was performed using Cufflinks v2.2.1 (http://cole-trapnell-lab.github.io/cufflinks/install/) (16). The significantly differentially expressed genes were obtained using a threshold of fold-change >1.5 and P<0.05.

For the analysis using TCGA data, 1,104 breast invasion cancer tissues and 113 normal tissues were analyzed in http://starbase.sysu.edu.cn/panCancer.php.

The data were presented as the mean ± SEM of three technical repeats and were analyzed using GraphPad Prism 6.0 (GraphPad Software, Inc.). The difference between 2 groups was analyzed by unpaired Student's t-test. One-way ANOVA followed by Tukey's post-hoc test was used to compare the differences among >2 groups. P<0.05 was considered to indicate a statistically significant difference.

SOX9 is a pivotal modulator of stemness and contributes to cancer development (17). To explore the relevance of SOX9 in TNBC, SOX9 expression was detected in BT-549, MDA-MB-231, MDA-MB-436 and MDA-MB-468 cells. SOX9 mRNA expression was lowest in BT-549 cells, while MDA-MB-436 had moderate SOX9 mRNA abundance, and MDA-MB-231 and MDA-MB-468 exhibited the highest SOX9 mRNA expression (Fig. 1A). Western blotting results revealed that SOX9 was highly expressed in MDA-MB-231, MDA-MB-468 and MDA-MB-436 cells, while a marginal signal band was observed in BT-549 cells (Fig. 1B).

Due to SOX9 was higher expression in both MDA-MB-231 and MDA-MB-468 cells than other TNBC cells MDA-MB-436 and BT-549. MDA-MB-231 and MDA-MB-468 cells were chosen for subsequent experiments to study the role of SOX9 in TNBC. Three shRNA sequences were used to inhibit SOX9 expression in the cells. Compared with shNC, shSOX9-1 and shSOX9-2 significantly decreased the SOX9 mRNA expression in MDA-MB-231 cells, while shSOX9-3 did not significantly inhibit SOX9 mRNA expression (Fig. 2A). Western blotting results revealed that all of these three shRNAs downregulated SOX9 expression (Fig. 2A). In MDA-MB-468 cells, all three shRNAs significantly suppressed SOX9 mRNA expression, with shSOX9-2 being the most efficient (Fig. 2B). Western blotting results were consistent with the RT-qPCR results (Fig. 2B). Since shSOX9-2 exhibited the highest knockdown efficiency, it was used for subsequent experiments.

Since SOX9 was efficiently silenced by shSOX9-2, shNC- and shSOX9-2-transfected MDA-MB-231 and MDA-MB-468 cells were subjected to cell proliferation analysis. CCK-8 assay was performed to analyze cell proliferation. The results indicated that, compared with the shNC, SOX9-knockdown significantly decreased the proliferation of MDA-MB-231 and MDA-MB-468 cells (Fig. 3A). To validate the results, colony formation was evaluated in shNC- and shSOX9-2-transfected MDA-MB-231 and MDA-MB-468 cells. Compared with shNC cells, shSOX9-2 cells exhibited significantly decreased colony formation (Fig. 3B). The current results suggested that SOX9-knockdown suppressed the proliferation and colony formation of TNBC cells.

Decreased apoptosis and accelerated cell cycle progression are common features of cancer cells (18). Whether SOX9 regulated apoptosis and the cell cycle was explored by staining the cells with PI/Annexin V-APC and PI, respectively. In MDA-MB-231 and MDA-MB-468 cells, SOX9-knockdown significantly increased apoptosis (Fig. 4A and B). Additionally, SOX9-knockdown resulted in significantly increased G₁ cell cycle phase in both cell lines, compared with the shNC (Fig. 4C and D). However, the S phase in MDA-MB-231 cells remained unchanged, while it was significantly decreased in MDA-MB-468 cells after SOX9-knockdown, compared with that in the shNC-transfected cells (Fig. 4C and D). The G₂ phase was significantly decreased in shSOX9-2-transfected MDA-MB-231 cells compared with that in the shNC-transfected cells, but unchanged in shSOX9-2-transfected MDA-MB-468 cells (Fig. 4C and D). Overall, SOX9 silencing caused cell cycle arrest at the G₁ phase.

TNBC is a malignant tumor with a high metastasis rate (19). It has been reported that SOX9 contributes to the migration and invasion of various cancer cells (17,20,21). Therefore, the present study explored whether SOX9-knockdown regulated the migration of TNBC cells using wound-healing assays. The results revealed that cell migration was significantly repressed by SOX9-knockdown at 12, 18 and 24 h (Fig. 5A and B). To validate the effect of SOX9 on TNBC cell migration and invasion, Transwell assays without or with Matrigel-coated chambers, respectively, were performed. The results indicated that, compared with the shNC, SOX9-knockdown significantly decreased the migration and invasion of TNBC cells (Fig. 5C and D). The results suggested that SOX9 may be essential for TNBC cell migration and invasion.

To elucidate the downstream factors regulated by SOX9, RNA sequencing was performed in control and SOX9-knockdown cells. The results revealed that SOX9-knockdown led to the upregulation and downregulation of numerous genes (Table SI). The top three upregulated genes included nucleophosmin (NPM1), thioredoxin reductase 1 (TXNRD1) and succinate dehydrogenase complex subunit D (SDHD), while the most downregulated genes were nuclear receptor binding SET domain protein 2 (NSD2), eukaryotic translation initiation factor 4γ1 (EIF4G1) and glycogen phosphorylase L (PYGL) (Table SII). The expression levels of these genes in breast invasive carcinoma (BRCA) tissues were analyzed based on data from TCGA database. The results indicated that the expression levels of NPM1, TXNRD1, NSD2 and EIF4G1 were upregulated in BRCA tissues, while PYGL and SDHD was downregulated in BRCA tissues compared with in normal tissues (Fig. 6).