Aberrant promoter hypermethylation of miR-335 and miR-145 is involved in breast cancer PD-L1 overexpression

Clinical tissue samples and cell lines

50 breast cancer patient samples were obtained from Khatam-al-Anbya Hospital in Tehran, Iran. The hospital records included sex, age, lymph node metastases, and clinical grade (Fig. 1c). Radiotherapy or chemotherapy patients before surgery were excluded. A pathologist without patient data diagnosed tumor samples using the WHO’s approach. The Pasteur Institute of Iran authorized this work (Ethical code: IR.PII.REC.1398.023). Subjects gave informed consent. Patient data was analyzed for demographic and clinical information. Three human BC cell lines (MCF-7, BT-549, and MDA-MB-231), and a breast normal cell line (MCF-10a), were used in this investigation. MDA-MB-231 is extremely aggressive, invasive, and poorly differentiated and lacks estrogen receptor (ER), progesterone receptor (PR), and HER2 (human epidermal growth factor receptor 2) expression. BT-549 cells are epithelial cells isolated from a papillary, invasive ductal tumor and are characterized as triple-negative/basal-B breast mammary carcinoma. MCF-7 has estrogen, progesterone, and glucocorticoid receptors. MCF-7 is an adherent epithelial luminal cell line positive for estrogen and progesterone receptors. They are hormone-dependent, yet serum steroid hormones are enough to develop cells. MCF10A is the most often used normal breast cell model. These breast tissue cells spontaneously immortalized. They are non-tumorigenic and estrogen receptor-negative. All cell lines were grown in a 37 °C humidified incubator with 5% CO2 and DMEM with 10% fetal bovine serum and 1% penicillin/streptomycin (Sigma Aldrich, St. Louis, MO, USA).

Kaplan–Meier overall survival analysis

To assess the prognostic value of the PD-L1 gene, miR-335, and miR-145 in breast cancer patients, the Kaplan–Meier Plotter website (Budapest, Hungary) (http://kmplot.com/analysis/) (accessed May 14, 2022) was utilized⁶⁰. For this purpose, PD-L1, miR-335, and 145 Kaplan–Meier plots for breast cancer patients were generated. Log rank p-values of 0.05 were considered statistically significant for the Kaplan–Meier (KM) plots of all genes.

Immunohistochemistry using the PD-L1 antibody

At least three serial sections of formalin-fixed, paraffin-embedded BC tissues were placed on positively charged glass microscope slides. For IHC, Khatam-al-anbia Pathology Lab used a technique refined and validated for research specimen testing with PD-L1 monoclonal antibody (Master Diagnostica, Spain). For IHC, Khatam-al-anbia Pathology Lab used a technique optimized and validated for research specimen testing with PD-L1 monoclonal antibody (Master Diagnostica, Spain). Similarly, 100 μl of rabbit monoclonal anti-PD-L1 antibody at 200 × dilution was incubated for 60 min at room temperature, washed, subsequently incubated with HRP- and anti-rabbit antibody-conjugated polymer for 8 min, washed, incubated with hydrogen peroxide reagent, washed, and finally incubated with DAB substrate for signal development.

Dual Luciferase assay

MiR-335, miR-145, and target gene 3′-UTR interaction was demonstrated using a dual-luciferase assay. The 3′-UTR of PD-L1 and a special scramble sequence (AAGCTTCATAGGGCATAGC) as a negative control were cloned into the psiCHECKTM-2 vector (Promega, C8021) to perform luciferase reporter assays (PCR primers are listed in Table 2). BC cells were transfected with a psiCHECK-2 plasmid containing PD-L1 binding site mutations at positions 1307-1313 and 1318-1324, as well as a negative control or a miR-335 and-145 mimic. In 48-well plates, BT549, MDA-MB231, and MCF7 cells were co-transfected with psiCHECK-2 vectors containing the 3′-UTR of PD-L1 or a scramble sequence and 100 nM of each precursor miR (335, 145, or scramble miRs) using HyperFect (Qiagen, Germany). Renilla luciferase activity was adjusted to firefly luciferase. At 24 h, 48 h, and 72 h after transfection, luciferase activity was assessed (Promega, USA).

RNA isolation, cDNA synthesis, and quantitative real-time PCR

RiboPureTM kit extracted total RNA (Life Technology, MA, and USA). The RNA concentration was quantified by a spectrophotometer (IMPLEN, Munich, Germany), and quality was determined by the 260/A280 absorbance ratio. Using 1 g of RNA as a template and a cDNA synthesis kit, the M-MuLV reverse transcriptase enzyme generated cDNA (Fermentas, MA, USA). In each experiment, two RNA samples without reverse transcriptase were used as negative controls for DNA contamination. Real-time PCR used 2 µl of 1:5 diluted reverse transcriptase. Real-time PCR reactions were performed in a mixture containing 140 ng of specific primers (Table 2) and 10 μl of SYBR green master mix (Life Technology, MA, USA) in a total volume of 20 μl under the following conditions: initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturing at 95 °C for 20 s, annealing at 59 °C for 30 s, and extension at 72 °C the reference gene was GAPDH. Statistical analysis for relative mRNA expression was performed by the Relative Expression Software Tool (REST) proposed by Pfaffl⁶¹. REST estimated the fold change and P-value (0.05). Each primer pair examined amplification from serially diluted cDNA. They generated a 5-point standard curve in triplicate. All PCR assays displayed efficiencies of between 1.8 and 2.0.

Primer design for HRM, real-time PCR analysis, and stem-loop

MethylPrimer Express V1.0 (ABI) and Methprimer (http://www.urogene.org/methprimer/index.html) were used to design miR-335 and miR-145 primers. MethBLAST (http://www.medgen.ugent.be/methBLAST) was used to perform BLAST. Allele ID 6 was used to create PD-L1 primers. The NCBI Nucleotide Program (http://blast.nlm.nih.gov/) was used to determine each primer’s specificity. Chen et al.’s were changed to boost stem-loop flexibility and sensitivity. Adding 14 nucleotides to the original sequence lengthened the loop and enabled the design of a universal reverse primer and TaqMan probe inside it. Substitutions decreased the melting temperature of the stem section. This newly designed structure detects each miR by adding complimentary nucleotides to each stem-loop. Real-time PCR primers employed practically complete sequences of miR-335 and miR-145 (Table 2). Referencing U6.

DNA Extraction and Bisulfite Modification

Per the manufacturer’s instructions, genomic DNA was isolated from BC tissues and adjacent non-tumor tissues using the Universal Genomic DNA Extraction Kit (Qiagen, Germany). The DNA from these tissues was tested for quality and integrity using electrophoresis on a 1% agarose gel and quantified spectrophotometrically. Genomic DNA (2 µg) was then subjected to bisulfite conversion using an EZ DNA Methylation Kit (Qiagen, Germany). The bisulfite conversion reaction was incubated in a PCR thermocycler at 98 °C for 5 min, followed by 60 °C for 25 min, 95 °C for 5 min, 60 °C for 85 min, 95 °C for 5 min, and 60 °C for 175 min, with a final incubation at 4 °C for up to 20 h. The modified DNA samples were dissolved in ddH2O and stored at − 20 °C.

Methylation studies by high-resolution melting analysis

Bisulfite-modified DNA was amplified using primers that targeted methylated regions of miR-335 and -145 promoters (Table 2). Diluting 100% methylated DNA with unmethylated bisulfite-modified DNA produced 25%, 50%, and 75% standard controls (Qiagen, Hilden, Germany). Each experiment had these standards. The reaction mixture contained 10 μl of Meltdoctor master mix, 10 ng of bisulfite-treated DNA, 200 nM of each primer, and 20 μl of PCR-grade water. Step one PCR and high-resolution melting (HRM) were performed (ABI)⁶².

All reactions were carried out at 95 °C for 15 min, 60 °C for 30 s, 72 °C for 20 s, and 65–90 °C at 0.1 °C/s (ramps). Step One: The program performed HRM-curve analysis, then compared fluorescence at the melting point to unmethylated DNA fluorescence. Triplicate experiments were done. After the fluorescence drop, melting curves were normalized using HRM software.

MicroRNA transfections

Pre-miR precursors of pre-miR-335, pre-miR-145, and control pre-miR precursor (scramble) from Ambion (Life Technology, AM17102). Each well of a 12-well plate was seeded with 0.8 × 10⁶ cells one day before transfection. When the confluence of seeded cells reached 80%, they were transfected with 100 nM of synthetic pre-miR-335 or scramble oligonucleotides using HiPerFect (Qiagen, Germany), according to the company protocol. After six hours, the transfection medium was replaced with a fresh medium containing 10% FBS. Transfected cell lines were harvested at 24, 48, and 72 h post-transfection.

Flow cytometry using cultured cell lines

For flow cytometric analysis, cells were first incubated with Fc receptor block (BD Biosciences, for human cells) or anti-CD16/32 (eBioscience, for mouse cells) with a LIVE/DEAD viability dye (Thermo Fisher Scientific) for 10 min at 4 °C in FACS buffer (PBS/0.5% BSA/2 mM EDTA), followed by staining with antibody panels for 30 min at 4 °C. The following human antibodies with corresponding isotype controls were used: the PD-L1 monoclonal antibody (Cat. No. 329705) was purchased from BioLegend. The results of flow cytometry were analyzed using FlowJo software.

Western blot

Total protein was extracted from transfected and non-transfected cells using RIPA buffer (Beyotime, China) containing a protease inhibitor cocktail (Roche, USA). Protein concentration was quantified by a BCA protein assay kit (Beyotime, China). Lysates were combined with equal volumes of 2× Laemmli sample buffer. After 5 minutes of boiling, 15 μg of lysates were SDS-PAGEd and transferred to a 0.2 μm immune-BlotTM PVDF membrane (Cat. No. 162-017777; Bio-Rad Laboratories, CA, USA). 5% BSA (Cat. No. A-7888; Sigma Aldrich, MO, USA) in 0.1% Tween 20 blocked the membranes for 1 h. The primary antibodies for Western blotting were anti-PD-L1 (Cat. No. ab233482; Abcam) and anti-GAPDH (Cat. No. ab9485; Abcam). After the washing step, the membrane was incubated with secondary antibodies (goat anti-rabbit IgG H&L (HRP) (Cat No. ab6721; Abcam). Then, an ECL chromogenic substrate (BIO-RAD, USA) was applied to detect the signals. Densitometry of protein bands was done using Quantity One software to divide the percentage area under the curve of each band by its corresponding GAPDH band, and then calculated values were compared between them.

Cell proliferation analysis

Cell proliferation was assessed using the MTT kit (Sigma, St. Louis, MO, USA). Briefly, MDA-MB231, BT549, and MCF7 (3 × 10⁴ cells/well in 96-well microplates) were transfected with pre-miR-335, miR-145, or negative control. Cells in the logarithmic growth phase were harvested and seeded on a 96-well plate. At 24, 48, and 72 h after seeding, 10 μl of MTT was added to each well, and the cells were incubated for 4 h. We supplemented each well with 150 μl of DMSO, and the optical density (OD) was recorded at 540 nm. Viability = 100 × (absorbance of the treated sample) / (absorbance of control). All experiments were repeated three times, and the average results were calculated. The experiments were repeated three times, and average results were calculated.

Cell cycle and apoptosis assay

To determine the effects of miR-335 and -145 on apoptosis and cell cycle in BC cells, MDA-MB231, BT549, and MCF7 cells were seeded in 24-well plates with a density of 1 × 10⁶ cells/mL in growth medium one day before transfection. The cells were transfected with scramble oligonucleotides or pre-miR-335 and pre-miR-145 by HiPerFect (Qiagen, Germany) according to the manufacturer’s protocol. A cold 70% ethanol solution was applied to the cells for 24 hours at − 20 °C. The cells were stained with a propidium iodide (PI) solution after a washing step and left to dry for 30 min at room temperature. Flow cytometry was used to analyze the cell cycle (BD Biosciences, San Jose, CA, USA). According to the manufacturer’s instructions, cells were stained twice with FITC-labeled Annexin V and PI using the Annexin V-FITC/PI Apoptosis Detection Kit (Solarbio, CA1020). We repeated this test three times, and the data it produced was analyzed by flow cytometry in FL1 and FL3 channels in the Partec Flow cytometer (Supplementary file).

Statistical analysis

At least three real-time PCR, cell proliferation, cell cycle, and apoptosis tests were run. The data is mean ± SEM. Each test’s data was entered into GraphPad Prism V.8 for statistical analysis using one-way ANOVA. The t-test was used to compare cancerous and non-cancerous breast cells and samples. All real-time PCR results were analyzed using the CT technique with REST 2009 software (Qiagen, Hilden, Germany) and normalized against GAPDH for mRNA and U6 for miRNA. Pearson’s correlation coefficient tested the two groups’ correlation. P < 0.05 signifies statistical significance. Log-rank was used to compare Kaplan–Meier survival curves.

Ethics approval and consent to participate

The present study was conducted under the instructions accepted by the Ethics Committee of Pasteur Institute of Iran (Ethical code: IR.PII.REC.1398.023), written informed consent to participate, and consent to publish forms was obtained from all participants involved in the present study.

Informed consent

Written informed consent was obtained from all enrolled subjects.