A total of 30 patients with breast cancer treated at the Affiliated Hospital of Binzhou Medical University (Yantai, China) were enrolled in the present study. The mean age of the patients was 55 years (age range, 50–65 years). Following the receipt of written informed consent, paired tumor and adjacent normal tissues were collected from the patients between June 6 and December 15, 2016 and immediately frozen and stored in liquid nitrogen at −196°C prior to use. Following the extraction of total RNA and proteins from these samples, the expression of TRIM11 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. All experiments conducted in the present study were approved by the Ethics Committee of Binzhou Medical University.

Human breast cancer MCF-7 and MDA-MB-231 cell lines were obtained from Type Culture Collection of the Chinese Academy of Science (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% antibiotic (a mixture of penicillin and streptomycin; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) in a 5% CO₂ incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C. During the period of incubation, the medium was changed every two days.

TRIM11 short hairpin RNA (shRNA) (3′-CAGAAGTTGTGCCTATGGA-5′) and a negative control shRNA (3′-CCTAAGGTTAAGTCGCCCTCG-5′) were applied to construct RNA interference lentiviruses. The coding DNA sequence region of TRIM11 synthesized by Genewiz, Inc. (South Plainfield, NJ, USA) was inserted into EcoRI/BamHI restriction sites of a pLVX-AcGFP1-C1 plasmid and confirmed by DNA sequencing (Shanghai Majorbio Pharmaceutical Technology Co., Ltd., Shanghai, China). Lentiviral constructs of pLKO.1-shTRIM11 (1,000 ng), pLVX-AcGFP1-C1-TRIM11 (1,000 ng) or pLVX-AcGFP1-C1, and pLKO.1 (Addgene, Inc., Cambridge, MA, USA) empty vectors were co-transfected with psPAX2 and pMD2.G (Addgene, Inc.) viral packaging plasmids into 293T cells by Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following transfection for 48 h, the viral supernatant that was used to infect MCF-7 and MDA-MB-231 cells was collected (17).

To regulate TRIM11 expression in the MCF-7 and MDA-MB-231 cells, lentivirus-mediated RNA interference and overexpression were used, and four groups of cultured cells were determined: The first group consisted of wild-type cells cultured with DMEM (Control); the second group consisted of cells infected with the negative control (NC) lentivirus; the third group consisted of cells infected with the TRIM11 shRNA lentivirus (shTRIM11); and the fourth group consisted of cells infected with the TRIM11 recombinant lentivirus (oeTRIM11). Following infection for 0, 24, 48 or 72 h, proliferation assays were performed, and RT-qPCR and western blot analyses were performed, as well as apoptosis assays 48 h later.

To further investigate the impact of TRIM11 on breast cancer, following downregulation of TRIM11, the cells were treated with 5 µg/ml cisplatin, paclitaxel, adriamycin, 5-FU or gemcitabine individually for 24 h. In addition, the apoptosis rates of MCF-7 and MDA-MB-231 cells were detected using flow cytometric analysis.

Cells in the logarithmic growth phase were digested by 0.25% trypsin (Beijing Solarbio Science & Technology Co., Ltd.), inoculated in 96-well culture plates at a density of 3×10³ cells/well and then cultured at 37°C in a 5% CO₂ humidified incubator overnight. On the next day, according to the aforementioned experimental grouping, the cells were infected with lentivirus for 0, 24, 48 and 72 h. Subsequently, 100 µl 10% Cell Counting Kit-8 (CCK-8; SAB Biotherapeutics, Inc., Sioux Falls, SD, USA) solution in serum-free DMEM was added to each well and incubated for 1 h. Finally, the optical density of the absorbance at 450 nm was measured using a microplate reader (Perlong Medical Equipment Co., Ltd., Beijing, China).

Following extraction from tissue samples or lentivirus-infected MCF-7 cells by TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), total RNA was quantified and then 1% agarose gel electrophoresis was used to confirm the integrity of the isolated RNA. Subsequently, isolated RNA was reverse transcribed into cDNA with a procedure of 37°C for 30 min, 85°C for 5 min and 4°C for 5 min using a reverse transcriptase kit (Fermentas; Thermo Fisher Scientific, Inc.). RT-qPCR reactions were conducted on an ABI Prism 7300 instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.) with a SYBR-Green PCR kit (Thermo Fisher Scientific, Inc.). Subsequently, the mRNA level of TRIM11 normalized to GAPDH was analyzed by the 2^−∆∆Cq method (23). The primer sequences used were as follows: TRIM11 forward, 5′-GAGAACGTGAACAGGAAGGAG-3′ and reverse, 5′-CCATCGGTGGCACTGTAGAA-3′; GAPDH forward, 5′-CACCCACTCCTCCACCTTTG-3′ and reverse, 5′-CCACCACCCTGTTGCTGTAG-3′. In addition, the thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 45 sec; 95°C for 15 sec and 60°C for 1 min for one cycle; and 95°C for 15 sec and 60°C for 15 sec for one cycle (24).

Radioimmunoprecipitation assay lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) containing protease and phosphatase inhibitors was used to homogenize the cells or tissue samples by incubation at 4°C for 30 min. Next, cells or tissue lysates were centrifuged at 12,000 × g at 4°C for 10 min, and the proteins in the supernatant were acquired and later quantified by a bicinchoninic acid protein quantification kit (Thermo Fisher Scientific, Inc.). SDS-PAGE (10%; JRDUN Biotechnology Co., Ltd., Shanghai, China) was applied to separate the proteins (25 µg), followed by semi-dry transfer onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA) by electroblotting. Subsequently, 5% skimmed milk (BD Biosciences, Franklin Lakes, NJ, USA) was used to block the membranes for 1 h at room temperature. Next, the membranes were incubated with primary antibodies against TRIM11 (1:2,000; cat. no. Ab111694; Abcam, Cambridge, UK), tumor protein p53 [p53; 1:1,000; cat. no. 2524, Cell Signaling Technology (CST), Inc., Danvers, MA, USA], phosphatase and tensin homolog (PTEN; 1:1,000; cat. no. 9552; CST, Inc.), B-cell lymphoma 2 (Bcl-2; 1:200; cat. no. Sc-492; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Bcl-2-associated X protein (Bax; 1:300; cat. no. Sc-493; Santa Cruz Biotechnology, Inc.), phosphorylated c-Jun N-terminal kinase 1/2 (p-JNK1/2; 1:2,000; cat. no. 9255; CST, Inc.), JNK1/2 (1:1,000; cat. no. 9252; CST, Inc.), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2; 1:1,000; cat. no. 9101; CST, Inc.), ERK1/2 (1:1,000; cat. no. 9102; CST, Inc.) or GAPDH (1:2,000; cat. no. 5174; CST, Inc.) at 4°C overnight with gentle agitating. Following 5–6 washes, the membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies (1:1,000; Beyotime Institute of Biotechnology, Haimen, China) of goat anti-rabbit (cat. no. A0208) and goat anti-mouse (cat. no. A0216) at 37°C for 1 h. Finally, the membranes were incubated in the dark with immobilon western chemiluminescent horseradish peroxidase substrate (EMD Millipore) for 5 min and the protein bands were visualized under an enhanced chemiluminescence imaging system (Tanon-5200; Tanon Science and Technology Co., Ltd., Shanghai, China). GAPDH as an endogenous reference gene, the relative protein expression levels were calculated by ImageJ software 1.47v (National Institutes of Health, Bethesda, MD, USA).

A flow cytometer (BD Biosciences) was used to detect the proportion of apoptotic cells. Treated cells were digested, resuspended and counted. Resuspended cells (5,000,000–10,000,000) were centrifuged at 1,000 × g for 5 min to obtain the cell precipitants. Subsequently, the cells were resuspended gently with 195 µl binding buffer for Annexin V-fluorescein isothiocyanate (FITC) staining. Next, following incubation with 5 µl Annexin V-FITC (Beyotime Institute of Biotechnology) for 15 min in the dark at 4°C, 5 µl propidium iodide (PI) was added and cells were incubated for a further 5 min at 4°C. Simultaneously, a tube without Annexin V-FITC or PI was used as a negative control. Subsequently, the cell apoptosis rates were determined by flow cytometry (FCM) and analyzed using BD Accuri™ C6 Software (Version 1.0.264.21; BD Biosciences).

All the statistical analyses were performed using GraphPad Prism 7.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Student's t-tests were performed to assess the quantitative data compared between two groups, and one-way analysis of variance was used for multiple comparisons. All data were based on at least three independent experiments and values are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

A total of 30 paired tumor and normal tissues of patients with breast cancer were collected, and RT-qPCR and western blot analysis were applied to quantify TRIM11 expression levels in these tissues. As demonstrated in Fig. 1, statistical analysis by Student's t-tests revealed that mRNA (Fig. 1A) and protein (Fig. 1B) levels of TRIM11 were significantly higher in the breast cancer tissues than in the normal tissues, which indicated that TRIM11 may serve a pivotal role in the progression of breast cancer.

For further study, breast cancer MCF-7 and MDA-MB-231 cells were infected with TRIM11 interference and overexpression lentiviruses to downregulate and upregulate TRIM11 expression, respectively. After 48 h of infection, mRNA and protein expression levels of TRIM11 were quantified by RT-qPCR and western blot analysis, respectively. As demonstrated in Fig. 2, in MCF-7 (Fig. 2A and B) and MDA-MB-231 (Fig. 2C and D) cells, TRIM11 expression was significantly reduced when infected with the shTRIM11 lentivirus, whereas significantly increased expression was observed following infection with the oeTRIM11 lentivirus. Therefore, the TRIM11 lentiviruses were applied in the subsequent experiments due to their effective regulation of TRIM11 expression.

Following downregulation and upregulation of TRIM11 expression in MCF-7 and MDA-MB-231 cells, cell viability was evaluated by CCK-8 assay, and FCM with Annexin V-FITC/PI staining was performed to detect cell apoptosis rates. As demonstrated in Fig. 3, TRIM11 silencing significantly decreased the cell proliferation rates of MCF-7 cells, whereas there was a significant increase in cell proliferation when TRIM11 was overexpressed (Fig. 3A). In addition, the effect of TRIM11 on MCF-7 cell apoptosis was opposite to its effect on the cell proliferation (Fig. 3B). Additionally, downregulation and upregulation of TRIM11 also had the same effect on the proliferation and apoptosis of MDA-MB-231 cells (Fig. 3C and D). These data demonstrated that TRIM11 has an effect on the growth and survival of breast cancer cells by promoting cell proliferation and inhibiting cell apoptosis.

The present study also further investigated the mechanism underlying the anti-apoptotic effect of TRIM11 downregulation on breast cancer cells. Following TRIM11 downregulation and upregulation, the protein levels of several associated genes were examined by western blot analysis. As demonstrated in Fig. 4, in MCF-7 (Fig. 4A) and MDA-MB-231 (Fig. 4B) cells, the expression of apoptosis-associated genes PTEN, p53 and Bax was significantly increased when TRIM11 was downregulated. By contrast, PTEN, p53 and Bax expression levels were markedly reduced following TRIM11 upregulation. In addition, the levels of Bcl-2, p-ERK1/2 and p-JNK1/2 were positively associated with the alterations in TRIM11 expression, while ERK1/2 and JNK1/2 expression levels were unchanged. PTEN, p53 and Bax are pro-apoptotic proteins (25), and the ratio of Bax/Bcl-2 was reported to determine whether or not a cell was committed to apoptosis (26). All data revealed that TRIM11 may facilitate the proliferation of breast cancer cells through activating ERK1/2 and JNK1/2 signaling, which further regulates the expression of apoptosis-associated genes.

Previous studies have demonstrated that the chemotherapeutic drugs cisplatin, paclitaxel, adriamycin, 5-FU and gemcitabine are widely used in the clinical treatment of breast cancer and other cancer types (5,9–13). Additionally, it was reported that 5 µM cisplatin significantly inhibited the proliferation rate of breast cancer cells (27). To further investigate the effects of TRIM11, the aforementioned drugs were utilized and the cell apoptosis rates were measured after 24 h of treatment. As demonstrated in Fig. 5, in MCF-7 (Fig. 5A) and MDA-MB-231 (Fig. 5B) cells, cisplatin and paclitaxel had a more significant pro-apoptotic effect on cell apoptosis. Furthermore, following TRIM11 downregulation, the pro-apoptotic effects of these chemotherapeutic drugs were markedly stronger. Therefore, the results suggested that downregulation of TRIM11 can enhance the pro-apoptotic effects of chemotherapeutic drugs on breast cancer cells.

A total of 4 pairs of cisplatin- and paclitaxel-sensitive and -resistant breast cancer tissues were collected to examine TRIM11 levels. As demonstrated in Fig. 6, a significant increase in the protein expression of TRIM11 was noted in the resistant tissues, whereas TRIM11 expression was much lower in the sensitive tissues. These data further suggested that TRIM11 downregulation enhanced the pro-apoptotic effects of chemotherapeutic drugs, which may be beneficial for the treatment of breast cancer.