Study design and participants

This descriptive cross-sectional study was conducted in 2022 on 50 patients with high suspicion of breast cancer who were candidates for mastectomy and lumpectomy in a learning hospital. The sample volume was calculated based on reference number [13].

Data collection

From each of the 50 patients with breast cancer who were finally included in the study, two tissue samples were prepared (one from the tumoral part of the tissue and the other from the adjacent healthy tissue), which totaled 100 samples. The studied samples were fresh samples of breast cancer along with its healthy (normal) control from the breast tissue itself. Surgical biopsy samples were collected after obtaining informed consent from the patients in Afzalipour Hospital, Kerman, Iran, and then were prepared for H&E (hematoxylin & eosin) and IHC (immunohistochemistry) staining. H&E slides were reviewed by two pathologists double-blindly.

IHC staining

Dehydrated, deparaffinized sections along with retrieval buffer were microwaved for 20 min (3 min at 850 W; 17 min at 180 W), and then endogenous peroxidase was blocked for 10 min with 0.5% H202. The sections were incubated for 1 h at room temperature with monoclonal antibodies, in this way: HER2-neu (1:100; DAKO), PR (1:100; DAKO, Clone PgR 636), and ER (1:50; DAKO, Clone 1D5): ready to use. Slides were rinsed with wash buffer for 5 min; this step was repeated twice between all stages. Envision polymer (30 min) was added using 3,3′-diaminobenzidine (DAB) as the chromogen (10 min) after these steps hematoxylin staining for 2 min, dehydration, and mounting the slides. Then, the obtained slides were scored by two pathologists according to the standard scores for ER, PR, and c-erb-B2as defined by WHO.

Extraction of RNA from the samples

Biopsy samples were immediately placed in a sterile RNase-free microtube in a nitrogen tank (temperature − 186 °C). After being transferred to the laboratory, the tissue samples were kept at − 80 °C until the RNA extraction stage. To extract RNA, the tissue sample was powdered in a completely sterile mortar containing liquid nitrogen and transferred to a 1.5-µl microtube. One milliliter of TRIzol lysis solution was added to the sample and incubated for 5 min at room temperature. Then 200 µl of chloroform was added to the solution, mixed, and homogenized by screwing the microtube. Then it was centrifuged at 13,000 rpm at 4 °C for 15 min to create three phases. The supernatant phase containing RNA was transferred to another microtube, and 500 µl of isopropanol was added to the solution, shaken back and forth, and centrifuged for 10 min at 13,000 rpm. Then, the supernatant solution was emptied so that the sediment remained at the bottom of the microtube, and after that, 70% ethanol was added to the sediment and centrifuged at 10,000 rpm for 5 min. Then, the supernatant solution was emptied, and the sediment was allowed to dry for 10 min at room temperature. After those 30 µl of water, DNase-RNase free was added to it. After RNA extraction, its quantity and quality were checked by UV spectrum and agarose gel electrophoresis photometric methods.

Before performing the reverse transcription reaction, it was necessary to treat the extracted RNA with DNase 1 enzyme to remove the residual DNA in the medium. For this, 1 µg of RNA, 1 µl of buffer, 1 µl of DNase enzyme, and up to 9 μl of water were added to it and incubated for half an hour at 37°. Then 1 μl of EDTA was added to it and incubated for 10 min at 65 °C.

cDNA synthesis

The treated RNA was used as template for reverse transcription reaction. The reverse transcription reaction was performed in by adding 1 μl of oligo primer, 1 μl of random hexamer on RNA with concentration of 1 µg and up to 12 µl of water at 65 °C for 5 min, and then 4 µl of 5 × buffer, 1 μl of RNase inhibitor (RI),1 μl RT enzyme, and 2 μl of dNTPs mixture in a temperature program of 60 min at a temperature of 42 °C in Applied Biosystem StepOne (ABI).

Quantitative real-time PCR

After performing the reverse transcription reaction, in order to proliferate the desired fragment, the PCR reaction was performed on the product of the reverse transcription reaction. In this study, with emphasis on VDR gene, primer design was done using vector NIT and oligo program bioinformatics software. In addition, sequences were compared and aligned with BLAST Internet software on NCBI website and GeneBank database. Finally, the primer was designed so that gene expression can be checked by real-time PCR method using Cyber Green method.

To perform the real-time PCR reaction, the following materials were added in a sterile microtube:

Cyber Green mix, 12.5 μl

cDNA, 2 μl

Upstream primer, 0.5 μl

Downstream primer, 0.5 μl

Sterile deionized water was added to the final volume of 25 μl according to the temperature program of Master Mix Cyber Green protocol.

Then gene expression was checked by real-time PCR method. CT (cycle threshold) sigmoid diagram of each group of biopsy samples (with HER2, PR, ER index) was compared with VDR primer and beta-actin (housekeeping gene).

Vitamin D receptor (VDR) mRNA expression was used to measure tissue vitamin D levels. This expression level of vitamin D receptor was measured by real-time-PCR method in tumoral breast tissue and healthy parts of the same person. Finally, the level of vitamin D in tumoral and healthy tissues, the degree of difference, and their relationship with the prognostic indicators of people were compared, and the results were reported.

Demographic information of the patients, size of the mass, definitive histological diagnosis of the mass, the presence of metastasis to lymphatic and vascular structures, the presence of lymphatic metastasis, and tumor grade were determined and recorded.

Statistical analysis

Descriptive statistics (frequency, relative frequency, mean, and standard deviation), analytical statistics (independent t-test, chi-square, or Fisher), and SPSS software version 22 software used to analyze the data. A significance level of 0.05 was considered.