All the products used in cell culture were obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Antibodies, LATS1 small interfering (si)RNAs and LATS1 negative siRNAs were purchased from Abcam (Cambridge, UK). Simvastatin was purchased from Pfizer, Inc. (New York, NY, USA) and the solvent of simvastatin was dimethyl sulfoxide (DMSO; Xilong Scientific Co., Ltd., Shenzhen, China). Microbubbles used in the present study were obtained from Definity (Lantheus Medical Imaging, Inc., North Billerica, MA, USA). The shell material, gas, mean size, concentration and microbubble half-life were phospholipid, C₃F8, 1.1–3.3 µm, 1.2×10¹⁰ bubbles/ml and 2–10 min, respectively. Based on previous studies, the concentration of microbubbles used in the present study was chosen as 20%; the ratio of microbubble solution: Cell suspension was 2:8 (32,33).

The human breast cancer cell line MCF-7 was purchased from GefanBio (Shanghai, China). Cells were routinely maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). A total of four treatment groups were prepared for the present study as follows: Control group, MCF-7 cells without any treatment or the addition of any solvent; SIM group, MCF-7 cells treated with 12 µM simvastatin for 18 h at 37°C; UM group, MCF-7 cells treated with 0.6 W/cm² ultrasound and 20% microbubbles; and UM+SIM group, MCF-7 cells treated with 0.6 W/cm² ultrasound and 20% microbubbles combined with 12 µM simvastatin. For transfection experiments, the UM+SIM group was used and two additional groups were prepared as follows: UM+SIM+LATS1 siRNAs group, MCF-7 cells treated with 0.6 W/cm² ultrasound and 20% microbubbles, 12 µM simvastatin and 25 nM LATS1 small interfering (si)RNA (GCAATCAGTTAACCGCAA; Invitrogen; Thermo Fisher Scientific, Inc.); and UM+SIM+LATS1 negative siRNAs group, MCF-7 cells treated with 0.6 W/cm² ultrasound and 20% microbubbles, 12 µM simvastatin and 25 nM LATS1 negative siRNA (GCACAGTTAACCGCATAAA; Invitrogen; Thermo Fisher Scientific, Inc.). The transfection regent Lipofectamine^® 3000 was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). Following transfection, the cells were maintained at 37°C for 24 h and then were subject to the subsequent experiments.

Cell viability was measured using an MTT assay. Briefly, MCF-7 cells in the logarithmic phase were seeded (~5×10⁴ cells/ml) in 96-well plates and maintained for 12 h in an incubator at 37°C in an atmosphere containing 5% CO₂. Cells were treated with simvastatin at 1, 3, 6, 12, 25 and 50 µM and ultrasound (Antares ultrasound scanner, Siemens Medical Solutions USA Inc., Malvern, PA, USA) united microbubbles (20%; Lantheus Medical Imaging, Inc.) at 0.1, 0.3, 0.6, 1.0 and 1.5 W/cm². Cells were centrifuged at 224 × g for 1 min at room temperature and the supernatant was removed. Cells were then treated with 100 µl DMSO under low-speed oscillation (400 rpm/min) for 10 min at room temperature. Absorbance was detected at 490 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cell apoptosis was assessed by flow cytometry (FCM). MCF-7 cells were treated as described above. Following washing with PBS (twice, 2–3 min each time), MCF-7 cells were trypsinized using 0.25% trypsin (Beyotime Institute of Biotechnology, Haimen, China) and centrifuged at 224 × g for 1 min at 4°C. The apoptosis detection kit with Annexin V FITC and PI was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The supernatant was removed and cells were suspended in the Annexin-binding buffer provided by the kit at a density of 1×10⁶ cells/ml. The cells were then incubated with Annexin V-fluorescein isothiocyanate and propidium iodide (PI; provided by the kit) at room temperature for 15 min in the dark. Cell apoptosis was assessed using a FACSCalibur flow cytometer with CellQuest analysis software version 3.3 (BD Biosciences, San Diego, CA, USA).

MCF-7 cells in the logarithmic phase were seeded (~6×10⁷ cells/well) in 6-well plates and maintained for 24 h in an incubator at 37°C in an atmosphere containing 5% CO₂, treated as described above and maintained in an incubator at 37°C in an atmosphere containing 5% CO₂ for 48 h. The medium was removed and cells were washed three times with PBS (3 min each time). Following digestion with 0.25% trypsin, the cell suspension was collected and centrifuged for 5 min at 224 × g at 4°C and the supernatant was discarded. Following washing with PBS 3 times (3 min each time), the cells were re-suspended, centrifuged for 5 min at 224 × g at 4°C and the supernatant was discarded once more. Pre-cooled 70% ethanol (1 ml) was added to cells, which were subsequently stored in at 4°C overnight and centrifuged for 5 min at 224 × g. The ethanol was removed and cells were washed three times with PBS (3 min each time). A total 500 µl PBS containing PI (50 µg/ml), RNase A (100 µg/ml) and Triton X-100 (0.2%) was added to the cells for 30 min in the dark. FCM was performed as above and FlowJo software version 7.2 (FlowJo LLC, Ashland, OR, USA) was used for cell cycle analysis according to the manufacturer's protocol.

Cultured cells were lysed on ice in radioimmunoprecipitation assay lysis buffer (1 mM MgCl₂, 1% Triton X-100, 10 mM Tris-HCl and 0.1% SDS, pH 7.4) and fragments using an ultrasonic cell disruptor (Branson Ultrasonics Corp., Danbury, CT, USA). The fragments were removed and the supernatant liquor was collected by centrifugation at 224 × g for 5 min at 4°C. The concentration of proteins was established using a Bradford protein assay kit (Bio-Rad Laboratories, Inc.) following the manufacturer's protocol. Equal quantities of proteins (50 µg) were separated by 5% SDS-PAGE and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). Following washing with PBS for 3 times (5 min each time), nonspecific binding blocked with 5% low fat dried milk at room temperature for 2 h. Membranes were incubated overnight with the following primary antibodies: Anti-phosphorylated (p-)ERK (ab65142; 1:800), anti-p-AKT (ab18206; 1:1,000), anti-p-mTOR (ab84400; 1:1,000), anti-β-actin (ab8227; 1:2,000), anti-LATS1 (ab70561; 1:8,000), anti-YAP (ab39361; 1:1,000), anti-p-YAP (ab153077; 1:500), anti-RHAMM (ab143134; 1:1,000), anti-Kruppel-like factor 5 (KLF5; ab137676; 1:2,000) at 4°C. Then, horseradish peroxidase-conjugated secondary antibodies (bs-0293M; 1:1,000; BIOSS, Beijing, China) were added at room temperature for 1 h. The results were quantified using EMD Millipore™ Immobilon™ Western Chemiluminescent HRP Substrate (EMD Millipore). The data were analyzed by ImageJ version 1.48 (National Institutes of Health, Bethesda, MD, USA).

Data are presented as the mean ± standard deviation. The results were analyzed by one-way analysis of variance followed by Turkey's test. P<0.05 was considered to indicate a statistically significant difference.

MTT results revealed that simvastatin reduced the viability of MCF-7 cells in a dose-dependent manner (Fig. 1A). In addition, it was indicated that simvastatin decreased the viability of MCF-7 cells treated with different intensities and durations of LFU and microbubbles (Fig. 1B). Thus, it was confirmed that simvastatin and LFU combined with microbubbles is able to suppress the viability and growth of MCF-7 cells.

Cell cycle arrest and apoptosis of MCF-7 cells treated with LFU and microbubbles combined with simvastatin was evaluated. The results demonstrated that the percentage of cells in G1 phase in the UM+SIM group (82.47%) was higher compared with other groups (CON, 58.32%; SIM, 78.34%; UM, 65.90%; Fig. 2). This indicates that LFU and microbubbles combined with simvastatin induced cell cycle arrest at G1 phase. FCM data (Fig. 3) revealed that the percentage of apoptotic cells in the UM+SIM group (35.63%) was significantly higher compared with the CON (2.30%), SIM (23.64%) and UM groups (9.61%). This suggests that LFU and microbubbles combined with simvastatin induces apoptosis in MCF-7 cells.

The expression of LATS1, YAP and RHAMM, together with their downstream proteins, was investigated in MCF-7 cells. In the UM+SIM group, the levels of YAP and RHAMM were significantly lower compared with the control (Fig. 4A). However, an increase in LATS1 and p-YAP expression was observed in MCF-7 cells treated with LFU and microbubbles combined with simvastatin compared with control cells (Fig. 4A). These results indicate that LFU and microbubbles combined with simvastatin downregulates the expression of YAP and RHAMM, upregulates LATS1 expression and promotes the phosphorylation of YAP in MCF-7 cells. KLF5, a downstream protein of YAP, had the lowest expression in the UM+SIM group (Fig. 4A). In addition, the expression of downstream proteins associated with LATS1/YAP/RHAMM pathway in MCF-7 cells is presented in Fig. 4. Compared with other groups, the expression of phosphorylated ERK, AKT and mTOR was lowest in MCF-7 cells treated with LFU and microbubbles combined with simvastatin (Fig. 4B). However, no significant difference was observed ERK, AKT and mTOR expression between groups. It was demonstrated that LFU and microbubbles combined with simvastatin enhanced LATS1 expression and reduced YAP and RHAMM expression in MCF-7 cells. In addition, LFU and microbubbles combined with simvastatin further downregulated the expression of downstream proteins associated with LATS1/YAP/RHAMM, involving ERK, AKT and mTOR. These data suggest that LFU and microbubbles combined with simvastatin affects the LATS1/YAP/RHAMM pathway in MCF-7 cells.

MTT results (Fig. 5) revealed that the viability of MCF-7 cells in the UM+SIM+LATS1 siRNA group was increased compared with other groups. Furthermore, it was identified that the viability of MCF-7 cells in the UM+SIM and UM+SIM+LATS1 negative siRNA groups was very similar, which suggests that LATS1 suppresses the growth of MCF-7 cells. It was also identified that, compared with the other groups, the apoptosis of MCF-7 cells was suppressed in the UM+SIM+LATS1 siRNA group (Fig. 5B and C). These data indicate that LATS1 promotes the apoptosis of MCF-7 cells. In order to verify this, the expression of proteins associated with the LATS1/YAP/RHAMM pathway was further assessed. The results demonstrated that, in the UM+SIM+LATS1 siRNAs group, the expression of LATS1 and p-YAP in was lower compared with other groups. However, YAP, RHAMM and KLF5 expression in MCF-7 cells was upregulated in the UM+SIM+LATS1 siRNA group (Fig. 6A). The expression of p-ERK, AKT and mTOR in the UM+SIM+LATS1 siRNA group was higher compared with other groups (Fig. 6B). As the upregulation of LATS1 expression was accompanied by a decrease in YAP and RHAMM expression, it was confirmed that LATS1 negatively regulates the expression of YAP and RHAMM in MCF-7 cells. These data suggest that LATS1 acts as a negative regulator in the LATS1/YAP/RHAMM pathway in MCF-7 cells. Furthermore, a hypothetical model of the pathway and indirect role of LATS1 in MCF-7 cells was established (Fig. 7).