2.1 Cell lines

Cell lines (Manassas, VA, USA), including MCF-10A (human BC epithelial cell), MCF-7 and MDA-MB-231 (BC cell lines) were cultured in RMIP-1640 medium containing glutamine, 1% penicillin/streptomycin, and 10% FBS. All the media were provided by Thermo Fisher Scientific. Cell culture conditions were maintained at 37 °C with 5% CO2.

2.2 Preparation of AuNPs and AuNPs-miR-875-5p

The Frence method was employed to synthesize AuNPs. miR-875-5p mimic and miR-NC were synthesized by Ribobio (Guangzhou, China). To prepare AuNPs-miR-875-5p, thiolated miR-875-5p was mixed with 10 nM AuNP at RNA (1 nmol); AuNP (500 μL) was supplemented with 0.5% Tween-20. After 5 min, NaCl was gradually added until the concentration was 0.5 M [30]. Then, RNAs were centrifuged to collect functionalized AuNPs and added with PBS at 4 °C. miR-875-5p concentration in AuNP-miR-875-5p solution was determined after miRNA release from NP with dithiothreitol [31]. AuNP and AuNP-miR-875-5p were characterized by UV–Vis spectrophotometry, dynamic light scattering (DLS) and transmission electron microscope (TEM) according to the manufacturer's instructions.

2.3 Cell culture and treatment

BC cells were treated with AuNP-miR-875-5p (equal to 100 nM miR-875-5p, unless noted otherwise), or equal concentration of AuNP-miR-NC as a negative group, or media control, and then harvested after 48 h. Si-MTDH (GenePharma, Suzhou, China) was transfected into BC cells at 100 nM using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA). Si-MTDH transfection efficiency was about 70–80%, and gene intervention lasted for at least 48 h. BC cells were first transfected with AuNP-miR-875-5p (100 nM) for combination assay of MTDH cDNA plasmids without 3′-UTR (Addgene,www.addgene.org) and AuNP-miR-875-5p. After 48 h, cells were co-transfected with MTDH cDNA plasmid (2 μg) and AuNP-miR-875-5p (100 nM) for 72 h.

2.4 Luciferase activity assay

After amplification, MTDH 3′-UTR containing miR-875-5p binding site was inserted into the pGL3 vector (Life technologies). The complementary site of miR-875-5p with AGGUAU sequence in MTDH 3′-UTR was mutated. BC cells were cotransfected with MTDH 3′-UTR constructs (100 ng) and 100 nM miRNA, and tested on a dual luciferase reporter analyzer (Promega Corporation, Wisconsin, USA).

2.5 RadioImmunoPrecipitation assay RIP assay

RadioImmunoPrecipitation (RIP) was detected according to the Magna RIP Kit (Millipore Corp, Bedford, MA). The cell lysate was collected with RIP lysis buffer and combined with magnetic beads bound to Ago2 or IgG (6 h at 4 °C). After elution, the RNA products were purified and tested the enrichment level of miR-875-5p by RT-qPCR.

2.6 RT-qPCR

Total RNA was extracted with TRIzol reagent (Tiangen, Beijing, China) to generare cDNA using the Superscript II-Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA), and PCR was done with Premix Ex Taq Kit (Takara) in the 7300 real-time PCR system (Life Technologies). The thermal cycling reaction was set with 95 ℃ for 30 s, followed by 40 cycles of 95 ℃ for 5 s, 65 ℃ for 31 s, 95 ℃ for 15 s, 65 ℃ for 1 min, and 95 ℃ for 15 s. PCR primers (Table 1) were provided by Invitrogen.

Table 1 Primer sequences used for RT-qPCR2.7 Cell uptake test

BC cells were grown in a 96-well plate at 8 × 103 cells/well for 24 h and added with AuNP-miR-875-5p (50 nM miR-875-5p) for 1, 3, or 6 h. At each time point, cells after 4% PFA treatment for 15 min were dyed with 100 mM DAPI for 3 min, observed under an Olympus SZX12 fluorescence microscope, and analyzed by PC running MagnaFire 2.0 camera software (Optronics, CA, USA).

2.8 Flow cytometry analysis of cell apoptosis

The apoptosis rate was assessed by Fluorescein Isothiocyanate (FITC) Annexin V Apoptosis Detection Kit (BD Biosciences, USA), Cells in 12-well plates at 1 × 105 cells/well were incubated with AuNP-miR-875-5p (50 nM miR-875-5p). BC cells were washed twice with pre-cooled PBS, re-suspended in 500 μl 1 × binding buffer, and stained with 5 μl Annexin V-FITC and 5 μl PI solutions, respectively. After incubation at room temperature for 15 min, the proportion of apoptotic cells was measured on FACScan flow cytometer (BD Biosciences, USA).

2.9 Wound-healing assay

BC cells were plated in 24-well plates at 5 × 105 cells/well overnight. Vertical wounds were made with a 200 μL pipette tip, and a fresh medium containing PBS, AuNP-miR-875-5p (80 nM miR-875-5p) or Au-miR-NC was added, respectively. Images were taken at 0, 24 and 48 h using a Nikon microscope.

2.10 Cell migration and invasion assays

BC cells (5 × 104) were plated in the upper chamber pre-filled with serum-free medium supplemented with PBS, AuNPmiR-875-5p (80 nM miR-875-5p), or Au-miR-NC. The lower chamber was added with a medium containing 10% FBS and the same concentration of drug. After 24 h, migrating cells were fixed with 4% PFA for 15 min and stained with 1% crystal violet solution. Cells were counted under a Nikon microscope. The invasive activity was assayed with the transwell chamber precoated with diluted Matrigel.

2.11 Colony formation assay

BC cells (1 × 103 cells/well) treated with AuNP-miR-875-5p (80 nM miR-875-5p) or Au-miR-NC were inoculated into a six-well plate and cultured in DMEM containing 10% FBS. Two weeks later, the colonies were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, and stained with 0.1% crystal violet for 30 min. Finally, the number of colonies was calculated.

2.12 Cell viability and proliferation assay

Cell Counting Kit-8 (CCK8, Dojindo, Japan) was used to detect cell viability according to manufacturer's protocol. BC cell suspension (100 μl) was inoculated into a 96-well plate at 3 × 103 cells per well. At each time point, 10 μl CCK-8 solution (Dojindo) was added to each well and incubated at 37℃ for 2 h. The absorbance at 450 nm was then measured on a microplate reader (Bio-Rad, CA, USA).

2.13 Determination of Caspase-3 and caspase-9 activity

Cell proteins were extracted using RIPA buffer (Beyotime) and quantified using the BCA Protein Assay Kit (EMD Millipore). The protein (50 µg) was incubated with caspase-9 (C1158) and caspase-3 (C1116) (Beyotime) for 2 h at 37 °C, and the absorbance was measured at 405 nm using an Orion II microplate photometer (Berthold Technologies GmbH & Co. KG).

2.14 Western Blot

Cells were dissolved in a radioimmunoprecipitation assay buffer with protease inhibitors and quantified by Bradford assay (Bio-Rad). Proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore). The samples were blocked with 5% skim milk powder for 1 h and incubated with primary antibodies MTDH (Abcam; 1:10,000); GAPDH (CST; 1:1000), p-AKT (CST; 1:1000), Bax (CST, 1:1000), and PTEN (CST; 1:1000) overnight at 4 °C. After three washes with TBST, the secondary antibody conjugated with horseradish peroxidase (CST) was incubated for 1 h and developed using an ECL kit (ultrassignal, China).

2.15 Experiments on animals

BALB/c nude mice (males, 5–6 weeks old, 18–20 g) were purchased from HFK Company (Beijing, China) and propagated under pathogen-free conditions. All animal experiments were conducted complying with the Guide for the Care and Use of Laboratory Animals of Jining Medical University. For xenograft tumor models, 5 × 106 MCF-7 cells were inoculated subcutaneously in 5 nude mice, respectively. At days 7, 10, 14, and 17 after MCF-7 cell inoculation, tumor size reached 100 mm3, and AuNPs were injected subcutaneously at 4 nmol/kg miRNA, a total of 4 times. Tumor size (length and width) and mouse body weight were measured. Tumor volume = (length × width2) × 0.5. Finally, tumors were harvested from euthanized mice and photographed.

2.16 Immunohistochemical staining

Tumor paraffin tissue (4 μm) was heated at 65 °C for 2 h, dewaxed, and microwaved in EDTA buffer. Sections were placed in 3% H2O2 for 10 min and blocked with 5% BSA for 20 min. The diluted primary antibody (50 μl) was added overnight and 50–100 μl secondary antibody was incubated for 50 min. Then, DAB-developed sections were counterstained with hematoxylin solution, differentiated with 1% hydrochloric acid alcohol, soaked in ammonia, dehydrated with gradient alcohol, cleared with xylene, and sealed with neutral glue [32].

2.17 Data analysis

Data were expressed as mean ± standard deviation (SD) and collected from experiments with at least three replicates. Data comparison was carried out using Student's t-test or one-way ANOVA. p < 0.05 was considered statistically significant.