The expression of MAGT1 and KLF16 in breast cancer and normal breast samples was retrieved from the Encyclopedia of RNA Interactomes (ENCORI; http://starbase.sysu.edu.cn/index.php), including 1,104 tumor and 113 normal samples. The Human Protein Atlas (HPA; https://www.proteinatlas.org/) was also applied for the analysis of MAGT1 and KLF16 expression by immunohistochemical assay. The binding between MAGT1 promoter and KLF6 was predicted using the JASPAR database (https://jaspar.genereg.net/).

The MCF-10A (cat no. CRL-10317), MCF-7 (cat no. HTB-22), MDA-MB-231 (cat no. HTB-26) and SK-BR-3 (cat no. HTB-30) cell lines were obtained from the American Type Culture Collection (ATCC). The SUM190PT (cat no. YS1334C) cell line was obtained from Shanghai Yaji Biological Technology Co., Ltd. All cells were cultured in DMEM supplemented with 10% FBS (Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.), and maintained at 37°C in a humidified incubator containing 5% CO₂.

For transfection, short hairpin RNAs (shRNA; pGPU6) targeting KLF6 (shRNA-KLF6-1/2) and MAGT1 (shRNA-MAGT1-1/2) and an empty pGPU6 plasmid which was used as the negative control (shRNA-NC) were obtained from Shanghai GenePharma Co., Ltd. In addition, the pcDNA3.1(+) KLF16 overexpression vector (Oe-KLF16) and empty vector NC (Oe-NC) were supplied by Shanghai GenePharma Co., Ltd. The full-length sequence of MAGT1 was amplified and cloned into the pcDNA3.1 plasmid (Shanghai GenePharma Co., Ltd.) to generate pcDNA-MAGT1, and the pcDNA3.1 empty plasmid was used as its negative control (pcDNA-NC). When the cells reached 70-80% confluency, they were transfected with the corresponding aforementioned plasmids (50 nM) using Lipofectamine 3000^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions at 37°C for 48 h. Following 48 h of transfection, subsequent experiments were conducted.

Total RNA was isolated from the cells using the RNeasy Mini kit (Qiagen, Inc.) and then reverse transcribed into complementary DNA (cDNA) using the Strand cDNA Synthesis kit (Takara Bio, Inc.) using the following thermocycling conditions: 70°C for 5 min, 42°C for 1 h and 70°C for 15 min. qPCR was subsequently carried out using a PrimeScript RT-PCR kit (Qiagen, Inc.) in accordance with the manufacturer's instructions. The thermocycling conditions were as follows: Initial denaturation at 94°C for 5 min; followed by 22 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 30 sec. The mRNA levels were analyzed using the 2^−ΔΔCq method (20) and normalized to the reference gene, GAPDH. The primer sequences used were as follows: MAGT1 forward, 5′-CTC AGC CTC TGC CCA AAG AA-3′ and reverse, 5′-CAC AAG GCG ACG GAA CTT GT-3′; KLF16 forward, 5′-CAA GTC CTC GCA CCT AAA GTC-3′ and reverse, 5′-AGC GGG CGA ACT TCT TGT C-3′; GAPDH forward, 5′-CCA TGG GGA AGG TGA AGG TC-3′ and reverse, 5′-AGT GAT GGC ATG GAC TGT GG-3′.

The MCF-7 cells and mouse tissues were collected and lysed with RIPA buffer on ice for 30 min. After determining the protein concentration using a BCA kit (Beyotime Institute of Biotechnology), the same amount of protein (30 µg/lane) was separated on a 12% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes (MilliporeSigma). The membranes were blocked with 5% (w/v) skimmed milk in TBST for 1 h at room temperature, followed by probing with primary antibodies against MAGT1 (dilution 1:600; cat no. 27994-1-AP, ProteinTech Group, Inc.), Ki67 (dilution 1:1,000; cat no. ab16667, Abcam), proliferating cell nuclear antigen (PCNA; dilution 1:1,000; cat no. ab18197, Abcam), MMP2 (dilution 1:1,000; cat no. ab92536, Abcam), MMP9 (dilution 1:1,000; cat no. ab283575, Abcam), KLF16 (dilution 1:1,000; cat no. orb39548, Biorbyt, Ltd.) and GAPDH (dilution 1:2,500; cat no. ab9485, Abcam) at 4°C overnight, followed by incubation with goat anti-rabbit horseradish peroxidase-labeled secondary antibody (dilution 1:2,000; cat no. ab6721, Abcam) at room temperature for 2 h. The bands were visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia). ImageJ software (version 1.8.0; National Institutes of Health) was used for densitometry.

Cell viability was assessed using MTT assay. The transfected cells were seeded in 96-well plates at 37°C for incubation of 24, 48 and 72 h, respectively. MTT solution (5 mg/ml; MilliporeSigma) was added to each well to incubate the cells at 37°C for a further 4 h. Dimethyl sulfoxide (DMSO) was used to dissolve the formazan. The absorbance at 570 nm was measured using a multi-well scanning spectrophotometer (Bio-Rad Model 2550 EIA Reader).

The MCF-7 cells were plated in six-well plates in DMEM containing 10% FBS at 37°C in a humidified incubator containing 5% CO₂. The medium was refreshed every 3 days. After 2 weeks, the cells were washed with PBS, fixed with 4% formaldehyde (Sigma-Aldrich; Merck KGaA) for 30 min at room temperature and stained with crystal violet (Sigma-Aldrich; Merck KGaA) for 30 min at room temperature for observation.

The MCF-7 cells were plated in six-well plates in DMEM containing 10% FBS at 37°C in a humidified incubator containing 5% CO₂. After 24 h, a straight line was drawn using a 200 µl pipette tip. The cells were then washed with PBS three times and incubated in serum-free medium for 48 h. The healing of the scratches was observed under a light microscope (Olympus Corporation) at 0 and 24 h. The gap distance was quantitatively evaluated using ImageJ software (1.8.0 172 version; National Institutes of Health). Migration (%)=[(0 h average scratch distance-24 h average scratch distance)/0 h average scratch distance] ×100.

A total of 1×10⁵ MCF-7 cells was suspended in 200 µl DMEM without FBS and inoculated into the upper chamber of 24-well Transwell inserts (Corning Falcon; Corning, Inc.) pre-coated with Matrigel (BD Biosciences). Subsequently, 500 µl DMEM containing 10% FBS were added to the lower chamber. The Transwell was incubated at 37°C in a humidified incubator containing 5% CO₂. After 24 h, the non-invaded cells were wiped out using a cotton swab, and the cells that had invaded to the bottom of the insert was fixed with 4% formaldehyde for 10 min and stained with crystal violet for 10 min at room temperature. The stained cells were observed under a light microscope (Olympus Corporation).

KLF16 binding motif and MAGT1 promoter (full length, FL) or serial truncations (E1 Del and E2 Del) were cloned into the pGL3-basic vector (E1761; Promega Corporation). A total of 1×10⁵ MCF-7 cells were seeded in 24-well plates. On the following day, the cells were transfected with the pGL3-based reporter constructs (2 µg) and pRL-SV40 vector (2 µg) using Lipofectamine 3000^® (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, the Firefly luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega Corporation), and normalized to Renilla activity.

The MCF-7 cells were cross-linked with 1% formaldehyde at room temperature for 10 min and quenched in 125 mM glycine for 5 min. The cell lysates were then sonicated into fragments. Subsequently, 100 µl lysates containing chromatin fragments were immunoprecipitated with antibody against KLF16 (5 µg; cat. no. sc-377519; Santa Cruz Biotechnology, Inc.) or IgG (5 µg; cat. no. 2729; Cell Signaling Technology, Inc.) at 4°C overnight. The immune complexes were recovered by the addition of A/G-agarose beads (Santa Cruz Biotechnology, Inc.). The precipitated DNA was purified using the ChIP DNA Clean & Concentrator kits (Zymo Research) and then analyzed using RT-qPCR as described above.

A total of 36 male BALB/c nude mice weighing 18-22 g were obtained from HFK Bioscience Co, Ltd. and housed in a standard environment with a controlled temperature (20±2°C), humidity (50±5%), a 12/12-h light/dark cycle and free access to water and food. The mice were allowed to acclimatize to their environment for 1 week prior to the experiments. All animal experiment procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animals of The Second Clinical Medical College of North Sichuan Medical College and were approved by the Ethics Committee of The Second Clinical Medical College of North Sichuan Medical College (Nanchong, China; approval no. NSMC-2021-94). Mice (6 mice per group) were randomly divided into three groups in the first section as follows: The control group, shRNA-NC group and shRNA-MAGT1 group. In subsequent experiments, mice (6 mice per group) were randomly divided into three groups as follows: The control group, sh-KLF16 group and sh-KLF16s+pcDNA-MAGT1 group. Approximately 1×10⁷ transfected MCF-7 cells were subcutaneously injected into the right axillary of the mice. After the tumor was formed, the body weight and tumor size of the mice were recorded every 3 days, and the tumor size was calculated using the following equation: 1/2 × length × width². At the end of the 21st day, the 36 mice were euthanized by an intraperitoneal injection of 120 mg/kg sodium pentobarbital, and the tumors were removed after the heartbeat cessation and respiratory arrest of the nude mice were confirmed. The mice were monitored every day, and the humane endpoints were the following: A marked reduction in food or water intake, labored breathing, inability to stand and no response to external stimuli. No abnormal signs that signified the humane endpoints of the experiment were observed in any of the mice during the experiment. The tumors were frozen at −80°C for further analysis.

The mean ± standard deviation (SD) for each independent assay was used to present the experimental data. The association of survival with MAGT1 expression was estimated using the Kaplan-Meier analysis with the log-rank test. An unpaired student's t-test or one-way ANOVA assay followed by Tukey's post hoc test were applied for comparisons between two groups or among more than two groups, respectively. A value of P<0.05 was considered to indicate a statistically significant difference.

Firstly, bioinformatics analysis was conducted to examine the expression level of MAGT1 in breast cancer. As shown in Fig. 1A, the analysis of the ENCORI database (http://starbase.sysu.edu.cn/index.php) revealed a relatively high expression of MAGT1 in breast cancer samples in comparison to normal samples (P<0.01). The immunohistochemical data obtained from the HPA database (https://www.proteinatlas.org/) further verified the upregulated expression level of MAGT1 in breast tumor tissues (Fig. 1B). Further analysis revealed that there was no obvious association between MAGT1 expression and the tumor stage of breast cancer (Fig. 1C). The overall survival assay revealed that the high expression of MAGT1 was significantly associated with a poor survival time (Fig. 1D). These data suggested that MAGT1 expression was upregulated in breast cancer, and the upregulated expression of MAGT1 predicted a poor outcome of patients.

To explore the specific role of MAGT1 in breast cancer, the MCF-7 cells were selected for use in further experiments, as these cells exhibited a significantly high expression of MAGT1 in comparison to the MCF-10A cells, and exhibited the highest expression level of MAGT1 among several breast cancer cell lines (MCF-7, MDA-MB-231, SK-BR-3 and SUM190PT cells) (Fig. 2A). Due to the high expression level of MAGT1 in breast cancer, the MCF-7 cells were transfected with shRNA-MAGT1-1 or shRNA-MAGT1-2 to achieve MAGT1 knockdown. As shown in Fig. 2B and C, transfection with both shRNA-MAGT1-1 and shRNA-MAGT1-2 resulted in the significantly decreased mRNA and protein expression of MAGT1; however, as shRNA-MAGT1-2 decreased MAGT1 to a greater degree, it was thus applied for use in subsequent experiments. A series of cellular biological experiments were then carried out to evaluate the effects of MAGT1 on the biological behaviors of breast cancer cells. MTT and colony formation assays revealed that MAGT1 knockdown markedly restricted cell viability and the formation of cell colonies, suggesting that the cell proliferative ability was suppressed upon MAGT1 knockdown in MCF-7 cells. This was also verified by the downregulated protein expression of Ki67 and PCNA in the shRNA-MAGT1 group, compared to the shRNA-NC group (Fig. 2D-F). In addition, the decreased wound healing and invasive abilities of the cells in the shRNA-MAGT1 group, accompanied by the decreased protein expression of MMP2 and MMP9, demonstrated that MAGT1 knockdown also restricted the cell migratory and invasive abilities of the MCF-7 cells (Fig. 2G-J).

To further validate the aforementioned findings in vitro, and in vivo experiment was conducted. Male BALB/c nude mice were subcutaneously injected with MCF-7 cells transfected with shRNA-NC or shRNA-MAGT1. As shown in Fig. 3A-C, the tumor size and tumor weight were markedly decreased when MAGT1 was knocked down. In particular, the mouse body weight and tumor size were monitored every 3 days before the mice were sacrificed. No evident differences in body weight were observed; however, the tumor volume was significantly decreased upon MAGT1 knockdown (Fig. 3D and E). Furthermore, the protein expression level of Ki67, PCNA, MMP2 and MMP9 exhibited a notable decrease in the tumor tissues of mice injected with cells in which was MAGT1 knocked down (Fig. 3F). This finding was consistent with the in vitro findings.

Subsequently, to identify the role of KLF16 in breast cancer, its expression in breast cancer was examined using bioinformatics analysis. As exhibited in Fig. 4A and B, the expression of KLF16 in tumor samples was higher than that in normal samples, according to the ENCORI and HPA databases. Even though KLF16 expression was not associated with tumor stage (Fig. 4C), these results also indicated a potential involvement of KLF16 in breast cancer. Of note, it was predicted using the JASPAR database that there were two potential KLF16 response elements (E1 and E2) binding to the MAGT1 promoter (Fig. 5A). To further ensure the association between KLF16 and MAGT1, the MCF-7 cells were first transfected with sh-KLF16-1/2 and sh-NC, respectively. The results revealed that the expression level of KLF16 was markedly decreased by transfection with sh-KLF16-1/2 (Fig. 5B and C). Due to a higher transfection efficacy, sh-KLF16-1 was selected for use in further experiments. KLF16 knockdown was then found to exert an inhibitory effect on the expression level of MAGT1 (Fig. 5D). Subsequently, luciferase reporter assay demonstrated that KLF16 knockdown reduced the transcriptional activity of MAGT1 (Fig. 5E). To determine which responsive elements were mainly responsible for the regulatory effects of KLF16 on MAGT1, the MCF-7 cells were co-transfected with serial truncations (E1 Del and E2 Del) of the MAGT1 promoter and Oe-NC/Oe-KLF16. As shown in in Fig. 5F, the transcriptional activity of MAGT1 was strictly limited in the E1 Del group upon KLF16 overexpression, indicating that E1 in the MAGT1 promoter was the main response element for this binding association between KLF16 and the MAGT1 promoter. This result was then verified by ChIP assay, as KLF16 was enriched at the ZNF217 promoter within the E2 region (Fig. 5G).

As was expected, KLF16 was also highly expressed in breast cancer cell lines, particularly in the MCF-7 cells (Fig. 6A). Subsequently, through gain- and loss-of-function experiments, the effects of KLF16 and MAGT1 on breast cancer progression were investigated. Firstly, transfection with pcDNA-MAGT1 successfully overexpressed MAGT1 expression at the mRNA and protein level in MCF-7 cells (Fig. 6B and C). The MCF-7 cells were then transfected with shRNA-NC or shRNA-KLF16 alone, or co-transfected with shRNA-KLF16 and pcDNA-NC/pcDNA-MAGT1. The mRNA and protein expression level of MAGT1 was decreased by KLF16 knockdown, followed by a restoration by a simultaneous transfection with pcDNA-MAGT1 (Fig. 6D and E).

In addition, a series of cellular biological behaviors were assessed using MTT, colony formation, wound healing and Transwell assays, as aforementioned. The results revealed that KLF16 knockdown markedly reduced cell viability and cell colonies, and also decreased the protein expression of Ki67 and PCNA, suggesting that KLF16 knockdown suppressed MCF-7 cell proliferation (Fig. 6F-H). In addition, the suppressive effects of KLF16 knockdown on cell migration and invasion were also evidenced by the hindered wound healing and decreased number of invasive cells, accompanied by the downregulated protein expression of MMP2 and MMP9 (Fig. 6I-L). Nevertheless, simultaneous transfection with pcDNA-MAGT1 and sh-KLF16 partly abolished these suppressive effects of KLF16 knockdown on cell proliferation, migration and invasion compared with transfection with sh-KLF16 alone (Fig. 6F-L). Eventually, these in vitro findings were also verified in vivo. Male BALB/c nude mice were subcutaneously injected with MCF-7 cells transfected with shKLF16 or co-transfected with sh-KLF16 and pcDNA-MAGT1. As illustrated in Fig. 7A-C, the reduced tumor size and tumor weight induced by KLF16 knockdown were partly reversed by MAGT1 overexpression. The mouse body weight continued to increase with time prolonging before, without notable differences among the groups (Fig. 7D). The speed of tumor growth during this period was hindered by KLF16 knockdown, which was partly abolished by MAGT1 overexpression (Fig. 7E). Furthermore, the reduced protein expression of Ki67, PCNA, MMP2 and MMP9 by KLF16 knockdown was markedly elevated by MAGT1 overexpression (Fig. 7F).