Two human breast cancer cell lines, MCF-7 and MDA-MB-231, were used. The cells were obtained from the Department of Pathology, Shandong University School of Medicine, and cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Beyotime, Shanghai, China) at 37°C in a humidified atmosphere with 5% CO₂. Upon reaching 80-90% confluence, the cells were passaged by trypsinization. Both the MCF-7 and MDA-MB-231 cells were authenticated by KeyGEN Biotech (Nanjing, China) using short tandem repeat (STR) analysis before purchase. All experiments were conducted using mycoplasma-free cells.

Cells in the logarithmic phase were seeded onto six-well plates, and transfected with siRNA against SR-B1 (si-SR-B1; 5’-CCT GAA TGA TGT GTC TCT GCA-3’) purchased from RiboBio (Guangzhou, China) using Lipofectamine 2000 (Invitrogen, CA, USA). Cells transfected with scrambled siRNA were used as controls. Transfection efficiency was evaluated using real-time PCR and Western blotting.

Approximately 24 h after transfection, the cells (1×10²) were subjected to a CCK-8 assay using a commercial kit (RiboBiotech, Guangzhou, China). Cellular proliferation in each group was measured every 24 h according to the manufacturer’s instructions. For the colony formation assay, cells (1 × 10²) were seeded into 60 mm culture dishes 24 h after transfection. After 14 days of culture, the colonies were fixed and stained with 1% crystal violet (Solarbio, Beijing, China). The number of colonies was counted and normalized for further analysis.

After siRNA transfection, a uniform scratch was made across the cell monolayer when the cells reached 100% confluence. Detached cells and debris were removed by washing with PBS, and the culture medium was replaced with medium containing 2% FBS. Images of the wound area were captured every 12 h, and the migration distance was quantified after 24 h using ImageJ software (version 1.53).

Cells (1×10²) suspended in serum-free medium were seeded into the upper chamber of a Transwell insert (Thermo Fisher, Lenexa, KS, USA). The lower chamber was filled with 600µL of DMEM containing 10% FBS. After incubation at 37°C in a CO₂ incubator overnight, the cells that had invaded and adhered to the lower surface of the membrane were fixed and stained with 1% crystal violet. The number of invading cells in five randomly selected fields was counted under a microscope.

After transfection, apoptosis was induced by serum starvation for 24 h. Harvested cells were centrifuged for 5 min and resuspended in PBS. Subsequently, approximately 1 × 10² cells were stained with 5μL Annexin V-FITC and 10μL PI in the dark at room temperature. Apoptosis was analyzed using a flow cytometer (NovoCyte, Agilent, Palo Alto, CA, USA), and data were processed using FlowJo software.

Total RNA was extracted from cells using TRIzol reagent (Thermo Fisher, Lenexa, KS, USA). Complementary DNA (cDNA) was synthesized using a commercial kit (Qiagen). Amplification was performed using SYBR Green on a Real-Time PCR system (Bio-Rad, Hercules, CA, USA). The specific primers for SR-B1 were as follows: 5’-CAT CAA GCA GCA GGT CCT TA-3’; 5’-CGT CAA AGA AGT AGA CGG AGAG-3’. The amplification conditions were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative gene expression levels were calculated using the 2^CT method.

Total protein was extracted from both cell lines and its concentration was determined using a BCA assay kit (Kangwei Biotech, China). Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies against SR-B1 (1:1000; Novus, USA), Bcl-2 (1:1000, PTG, USA), Bax (1:1000, PTG, USA), Caspase-3 (1:1000, PTG, USA), PTEN (1:1000, PTG, USA), cyclin D1 (1:1000, PTG, USA), and P70 (1:1000, PTG, USA), AKT (1:1000, CST, USA), p-AKT (1:1000, CST, USA), mTOR (1:1000, CST, USA) and p-mTOR (1:1000, CST, USA). After washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000; ZSGB-BIO, Beijing, China). Membranes probed with GAPDH were used as the internal standard. All bands were visualized using Image Lab software (Bio-Rad, Hercules, CA, USA), and band intensities were quantified using ImageJ software.

Data are presented as mean ± standard deviation. Statistical analyses were performed with SPSS 19.0 software. All experiments were performed at least in triplicates. Student’s t-test was used for comparisons between two groups, whereas one-way analysis of variance (ANOVA) was used for comparisons among multiple groups. Statistical significance was considered in the presence of a p-value of less than 0.05.

The transfection efficiency of the siRNA was validated using real-time PCR and Western blot analysis (Figure 1A-C). SR-B1 knockdown significantly reduced the proliferation of MDA-MB-231 and MCF-7 cells at 48 and 72 h compared to that in the Control Group (p<0.05, Figure 2A). Similarly, the knockdown of SR-B1 markedly decreased colony formation compared to the Control Group (p<0.05, Figure 2B and C).

Figure 1 *p<0.05 versus; **p<0.01 versus NC.

Figure 2 *p<0.05 versus NC.

Compared with the Control Group, SR-B1 knockdown significantly suppressed the migration of MCF-7 and MDA-MB-231 cells at 48 h (p<0.05, Figure 3A and B). Transwell assays showed that SR-B1 silencing markedly inhibited the invasiveness of both cell lines (p<0.05, Figure 4A and B).

Figure 3 *p<0.05 versus NC.

Figure 4 *p<0.05 versus NC.

Compared with the Control Group, SR-B1 knockdown significantly increased apoptosis in MCF-7 and MDA-MB-231 cells (p<0.05, Figure 5A and B). In addition, silencing of SR-B1 markedly upregulated the expression of pro-apoptotic proteins, including Bax and active Caspase-3, while downregulating the anti-apoptotic protein Bcl-2, compared to the Control Group (Figure 5C and D).

Figure 5 *p<0.05 versus NC; ***p<0.001 versus NC.

Knockdown of SR-B1 inhibits activation of the PI3K/AKT signaling pathway. The expression of PTEN, a key molecular switch in the PI3K/AKT signaling pathway, was significantly upregulated in the si-SR-B1 group compared to the Control Group. The phosphorylation of AKT and mTOR was markedly reduced following SR-B1 knockdown (p<0.05). Additionally, the expression of downstream target genes, including cyclin D1 and P70, was downregulated following SR-B1 knockdown (p<0.05; Figure 6A and B). These results indicate that SR-B1 knockdown regulates the PI3K/AKT signaling pathway, which may be related to the regulation of cellular proliferation and migration in breast cancer.

Figure 6 *p<0.05 versus NC; ***p<0.001 versus NC.