TCGA/GEO database analysis
TCGA (The Cancer Genome Atlas - Cancer Genome) (https://cancergenome.nih.gov/) was used to identify the differential expression of miR-155-5p in 1234 corresponding breast cancer patients (including 1103 primary solid breast tumor and 104 solid normal breast tissue) and of TP53INP1 in 1123 breast cancer tissues and 111 normal breast tissues derived from the TCGA Data portal. The patients were grouped into percentiles according to different mRNA/miRNA expression profiles. We checked for a relationship with the survival by choosing a cut-off to optimally split the samples into two groups. Optimally was defined as the significant separation in Overall survival (OS) using the optimal P values for TCGA datasets. Log-rank test was employed to determine the association between mRNA/miRNA expression and OS respectively. In addition, we also verified the expression of miR-155-5p in different breast subtype tissues in the GEO (Gene Expression Omnibus) datasets (https://www.ncbi.nlm.nih.gov/geo/) GSE45666 [37] and GSE59246 [38].
Cell culture and treatments
Breast cancer cell lines MCF-7 were purchased from The Chinese Academy of Sciences Shanghai ASTRI Cell Resource Center and cultured under the conditions specified by the manufacturer. Cells were cultured in a model of progression to paclitaxel-resistance for more than 6 months until cells exhibited resistance to cell growth inhibition by 10 µg/ml paclitaxel. pcDNA3.1 plasmids, siRNAs and miRNA mimics/inhibitor were transfected with OMEM (Gibco, Thermo Fisher Scientific, USA) and lip2000 reagent (Life Technologies) following the instructions of the reagent in six-well plates. The control group added equal volumes of OMEM and lip2000. TP53INP1 siRNAs or pcDNA3.1 plasmid (Gene Pharma, Shanghai, China) were used to stabilize silence or overexpression. The sequences of siRNAs for TP53INP1 were shown as follows: si-TP53INP1-1: 5'-CCU GCU UUC UCC AGU UUG ATT UCA AAC UGG AGA AAG CAG GTT-3', si-TP53INP1-2: 5'-CCG UGG GAC UGA UGA AUU ATT UAA UUC AUC AGU CCC ACG GTT-3', si-TP53INP1-3: 5'-GGU GGA UUA ACC ACU AUC ATT UGA UAG UGG UUA AUC CAC CTT-3'. All functional assays were performed at 24 h after transfection.
RNA extraction and real-time PCR. Total RNAs were extracted from cells in each group using the Trizol reagent (Invitrogen, California, USA) and analyzed by a spectrophotometer. The samples should meet the standard of:1.8 < OD260/280<2.0. Then RNA was reversely transcribed into complementary DNA (cDNA) by using the miRNA first strand cDNA synthesis kit (Thermo Fisher Scientific, USA) for miRNA expression, as well as the reverse transcription kit (TaKaRa, Japan) for mRNA expression. Quantitative real-time PCR (qRT-PCR) was carried out using the SYBR premix Ex Taq-Ⅱ kit (Takara, Japan) in an ABI 7500HT fast real-time PCR System (Applied Biosystems, USA). mRNA expression was normalized to GAPDH while miRNA level was normalized to U6 snoRNA. The following primers were used for PCR analysis: GAPDH: forward 5'-CAG CCT CAA GAT CAT CAG CA-3' reverse 5'-TGT GGT CAT GAG TCC TTC CA-3' ; TP53INP1: forward 5'-GAC TTC ATA GAT ACT TGC AC-3' reverse 5'-ATT GGA CAT GAC TCA AAC TG-3' ; Bcl-2: forward 5'-ATG TGT GTG GAG AGC GTC AA-3' reverse 5'-ACA GTT CCA CAA AGG CAT CC-3' ; Bak-1: forward 5'-CCC AGG ACA CAG AGG AGG TTT-3' reverse 5'-GCC TCC TGT TCC TGC TGA TG-3' ; Bax: forward 5'-GGG GACG AAC TGG ACA GTA A-3' reverse 5'-CAG TTG AAG TTG CCG TCA GA-3' ; Caspase-3: forward 5'-ACA AAT GGA CCT GTT GAC CTG A-3' reverse 5'-ACA CCA CTG TCT GTC TCA ATG C-3'. Analysis and fold change were determined according to the the comparative threshold cycle (Ct) method.
Western blot analysis
Cells were washed by cold PBS, the whole-cell lysate was prepared with RIPA (50 mM Tris, PH 8.0), supplemented with 1% protein phosphatase inhibitors and 1 mM PMSF (Beyotime, Shanghai, China), followed by frequent mixing and being placed on ice for 30 min. When the lysate began to become clear, the protein was centrifuged at 4 °C at 12000 rpm for 15 min. The concentration of proteins was measured by the Bio-Rad DC protein assay (Bio-Rad, USA). Where indicated, 10 or 15 mg protein was boiled and mixed well with SDS buffer according to a certain ratio for 15 min. Protein samples were separated and transferred onto the PVDF membranes (Bio-Rad, USA) by 10% SDS-PAGE. Following 100 min of blocking in phosphate-buffered saline with 5% fat-free milk, membranes were incubated with the following antibodies: primary antibody: TP53INP1 (ab154877, Abcam, 1:2000); Bcl-2 (ab196495, Abcam, 1:1500); Caspase-3 (ab13586, Abcam, 1:5000); Bax (23931-1-AP, Proteintech, 1:1000); Bak-1 (24552-1-AP, Proteintech, 1:1000) and β-actin (sc-130065,Santa Cruz, 1:3000) together with secondary antibody: goat anti-rabbit IgG-HRP (sc-2004, Santa Cruz, 1:5000) and goat anti-mouse IgG-HRP (sc-2005,Santa Cruz, 1:5000). Bands were visualized using ChemiDoc XRS + system and Bio-Rad-Image-Lab-Software-5.2.1.
MiR-155-3p target prediction
Prediction and analysis of the target genes of miRNA were performed by TargetScan v7.1 (www.targetscan.org/), miRanda (www.microrn-a.org/), miRTarBase (mirtarbase.mbc.nctu.edu.tw/php/index.php), miRmap (https://mirmap.ezlab.org/), and PITA (genie.weizmann.ac.il/pubs/mir07/mir07data.html), respectively.
Luciferase Reporter Assay
MCF-7 cells were seeded into the 6-well plates (BD, USA) and cultured until the MCF-7 cells reached 75%-90% confluence. Dual-luciferase assays were performed according to instructions by using a site-directed mutagenesis kit (Takara, Kusatsu, Japan). The activities of firefly and renilla luciferases were assayed by the dual-luciferase reporter assay system (Promega, USA) according to the reagent instructions. The luciferase activity was examined using Dual-Luciferase Reporter assay kit (Promega, USA) after transfection for 48 h.
Migration assay
Cell migration was determined through the wound healing assay, which was also called the “scratch” assay. In brief, cells were digested with 0.25% trypsin, centrifuged, and prepared into the single cell suspension. Cell suspension was cultured into 6-well plate until the cell density reached 80%-90%. A wound track was introduced by scraping the cell monolayer with the germ-free pipette tip(10 µl). Attention should be paid to keep the same width of these cuts, then, cells were washed twice with PBS, and treated with related dose. The average extent of wound closure was quantified in 3 random fields with × 50 magnification at 0 and 24 h, respectively. All microscope images were obtained with the Olympus IX73 (Olympus Corporation, Japan).
Invasion assay
For the invasion assay, cells were digested with 0.25% trypsin, prepared into single cell suspension with serum-free medium and the cell density of each group was maintained same. According to the experimental requirements, each group of cells (5 × 103 cells per well) were seeded into the upper chamber (8 µm pore polycarbonate membrane filters, with Matrigel (BD Biosciences, San Jose, CA, USA)), and 0.5 ml DMEM containing 10% FBS was added to the inferior chamber. The cells were cultured for 24 h at 37 °C in 5% CO2. Later, the Transwell cell culture medium was removed the membranes were fixed with 4% polyformaldehyde for 30 min, stained with Giemsa for 30 min, and counted (5 random fields under 200×) using light microscopy. All microscope images were obtained with Olympus IX73 (Olympus Corporation, Japan).
Apoptosis assay and Cell cycle analysis
According to the instructions of the Muse Cell Cycle Assay Kit (Merck Millipore, USA), whole cells were washed with cold PBS, centrifugated and fixed with 70% ethyl alcohol, treated with 100 mg/ml RNase A and labeled with 50 mg/ml propidium iodide (PI) for 1 h at 4 °C. The experiment results were analyzed by a Muse Cell Analyzer (Merck Millipore, USA). For Annexin V staining (BD, Biosciences), whole cells were transfected and then trypsinized, washed, and resuspended in cold PBS containing with 25 mg/ml Annexin-V-FITC and 50 mg/ml 7-AAD prior to FACS analysis following the instructions of Muse Annexin V and Dead Cell kit (Merck Millipore, USA).
Determination of cell viability
The MCF-7 or MCF-7/PR cells were seeded in six-well plates at a density of 1*105/ml and transfected with miR-155-5p mimics/inhibitor or pc-TP53INP1/si-TP53INP1 in three independent replicates using SRB Cell Proliferation and cytotoxicity assay kit (Boyao,Shanghai༌China) in accordance with the manufacturer’s protocol. 24 hours after transfection, cells were reseeded in 96-well plates in the presence of different treatment at density of 1 × 104 per well and treated with paclitaxel at a range of concentration of 5 to 80 µg/ml (5,10,20,30,50,80) in the medium for 48 h. The OD value was read in a microplate reader (Bio-Rad, USA) at 515 nm. The % cell viability was calculated with the following formula:
% Cell viability = (OD control/OD treatment) × 100
% Cell inhibition = 1- % Cell viability
In order to test the cell's resistance to paclitaxel, we tested the cell viability at different concentrations, using these data to plot XY and fit the data with a straight line (linear regression). IC50 value was then estimated using the fitted line. All these analyses and the production of pictures are done in Graphpad Prism.
Statistical analysis
All experiments were performed for at least three times, and datas were statistically analyzed using the unpaired, two-tailed Student’s t-tests followed by Newman-Keuls test built into GraphPad Prism (GraphPad Software 8.0.1; San Diego, CA). All data were expressed as mean ± SE. Furthermore, all statistical differences included a 95% confidence interval (CI).