br Materials and methods br Human tissue
2. Materials and methods
2.1. Human tissue specimens and cells
Formaldehyde-fixed paraffin-embedded (FFPE) BC tissues and un-paired mammary hyperplasia (non-tumor tissues) were randomly col-lected from patients who had undergone surgery at the Shaanxi Provincial People's Hospital in China. Clinicopathological data such as age and gender, as well as histological data, tumor size, lymph node me-tastasis status, ER status, PR status, and AR status were obtained by reviewing their pathology records. Specimens were collected after obtaining written informed consent from the patients as well as approval of the ethical committees. Patient anonymity was maintained throughout the study. Human BC cell lines MCF-7, HCC1937, MDA-MB-231 and MDA-MB-435, human breast epithelium cells HBL-100  and human embryonic kidney (HEK) 293T cells were obtained from the Cell Bank (Shanghai Institute of Biochemistry and Cell Biology, CAS, Shang-hai, China).Cells were maintained in Dulbecco's modified Eagle's me-dium (DMEM) supplemented with 10% fetal bovine serum (Biological Industries) and 1% CH 223191 (100 U/mL penicillin and 100 mg/mL streptomycin sulfate). Cells were grown in 5% CO2 at 37 °C. The cell line was tested for mycoplasma contamination using the Mycoplasma Detection Kit (Beyotime, Haimen, China) and was found to be negative.
2.2. Plasmid construction and transfection
Human miR-3614 precursor (pre-miR-3614) was synthesized by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd. (Shanghai, China). The pre-miR-3614 coding region was cloned into the pcDNA™6.2-GW/EmGFP (Invitrogen). We constructed pcDNA™6.2-GW/EmGFP-pre-miR-3614. The miR-3614 mimics, anti-miR-3614, small interfering RNAs (siRNAs) and their respective negative control RNAs were purchased from Gima (Shanghai, China). The information of all the sequences are provided in Supplementary Table 2. Transfection was performed using Polyplus transfection kit (Jetprime, France) according to the manufacturer's instructions.
2.3. Lentivirus infection
The plasmid shRNA-IGF2BP3 (sc-60846-SH) was purchased from Santa Cruz Biotechnology. The packaged lentivirus of pre-miR-3614 and si-IGF2BP3 were constructed by GeneChem (Shanghai, China) and named LV-miR-3614 and LV-si-IGF2BP3, respectively. The scramble lentiviral vector LV-Ctrl (or LV-si-Ctrl) was used as a control. The lentiviral vector is expressed green fluorescent protein (GFP) tag. For
infection, the MCF-7 and MDA-MB-231 cells were seeded in a 6-well plate and infected with 1 ml of viral stock containing 5 μg/ml polybrene for 12 h, then this medium was replaced by normal culture medium.
The MCF-7 and MDA-MB-231 cells were plated in 6-well plates at a density of 1.5 × 105 cells per well. The next day, these cells were transfected with siRNAs, plasmids, or control scrambler RNA. Untransfected cells were used as controls. After transfection of 24 h, total cellular RNA was extracted with TRIzol (Invitrogen Carlsbad, USA) according to the manufacturer's protocol. The FFPE tissue samples (10 sections) were deparaffinized by incubating in xylene for 10 min and in 100% ethanol for 5 min, and washed with distilled water for 30 s, followed by RNA extraction using the Qiagen FFPE RNeasy Kit (Valencia, CA, USA) according to manufacturer's instructions. The RNA was quantified with a NanoDrop spectrophotometer (Thermo, USA). The PrimeScript RT Reagent Kit (Takara, Japan) and the SYBR Premix Ex Taq II Kit (Takara, Japan) were used to detect the expression of mature miRNAs and mRNA. The primers used are listed in Table 2. qRT-PCR was performed using an IQ5 Multicolor qRT-PCR Detection System (Bio-Rad, USA). β-actin and U6 expression were used as a con-trol to quantify mRNA and miRNA expression, respectively. The 2− Ct method was used for the qRT-PCR analysis.
Total protein was harvested from BC cells using RIPA buffer (CST, Boston, China) after 48 h of transfection, and 20–30 μg of protein lysate was separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). The membranes were probed with the following pri-mary antibodies: TRIM25 (1:2000, Abcam, MA, USA), IGF2BP3(1:1000, Abcam), CyclinD1 (1:1000, Cell Signaling Technology, Danvers, MA), CDK4 (11,000, Cell Signaling) for overnight. After washing with TBST buffer, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Next, the membranes were incubated with ECL (Pierce, Rockford, IL, USA) for chemiluminescence detection.
2.6. Immunohistochemistry (IHC)
IHC was performed as described previously . Tissue sections (4 μm) were deparaffinized with xylene and hydrated using an alcohol gradient. Endogenous peroxidase-blocking and antigen retrieval were performed sequentially. The sections were incubated with polyclonal rabbit anti-TRIM25 (diluted 1:200, Abcam, MA, USA) and anti-IGF2BP3 (diluted 1:200; Abcam, MA, USA) followed by incubation with a secondary antibody IgG (ZSGB-BIO, China). Histological exami-nation was performed using 3,3′-diaminobenzidine kit (DAB, OriGene, China) and hematoxylin. If the proportion of positive cells was N50% in 5 random fields, the specimen was considered to show high TRIM25/ IGF2BP3 expression.