Patients and samples
This study was a descriptional cross sectional study. All samples were obtained from 70 women who underwent biopsy or mastectomy surgical operation at Tabriz Emam Reza Hospital from July 2009 until May 2010. The whole project carried out in Tabriz Drug Applied Research Center. The samples were examined histologically for the presence of tumor cells by a pathologist. The patients met the following criteria: primary unilateral non metastatic breast carcinoma for which complete clinical, histological and biological data were available; and no radiotherapy or chemotherapy before surgery. After the interview, during surgery tumor tissue samples were taken into liquid nitrogen in sterile tubes, then as soon as possible aliquated and stored at −70 °C until subsequent analysis. In order to reduce the influence of treatment on measurements, questionnaire data was obtained prior surgery.
Body mass index (BMI)
Accordingly WHO Rep 2000 (World Health Organization, 2000), BMI was calculated as kg/m2 using information from clinical notes at time of diagnosis. In the analyses, BMI was divided into two categories: BMI < 25 kg/m2 (non-obese) and BMI ≥ 25 kg/m2 (obese).
Collection of questionnaire data-tumor tissues
After patients were approved to participate, written informed consent was obtained from all subjects. All participants undertook to fill in questionnaire. It took information on social demographic characteristics, age, age of menark, menstrual history, body mass index and menopause status. The study was blind and data collectors were unaware of the study hypothesis. All participants were also measured for height and weight via standardized techniques. BMI, as an indicator of generalized obesity, was calculated as weight in kilograms divided by the square of height in meter.
Immediately after the interview, a 10 ml blood sample was drawn into coded EDTA-treated tubes and centrifuged at 3000 rpm for 10 min at room temperature within 1 h of collection. Plasma, Buffy coat and red blood cells were separated and stored at −70 °C until subsequent analysis. To avoid the influence of treatment on measurements, questionnaire data and blood specimens were obtained prior to initiation of definitive breast cancer therapy including surgery and/or radio- or chemotherapy.
Steroid hormones level (plasma estrogen, androgen and progesterone levels)
Plasma concentrations of estrogen, androgen and progesterone were determined by the use of commercially available quantitative sandwich enzyme-linked immunosorbant assay (ELISA) kits (DRG Estradiol ELISA, EIA 2693, DRG Progesterone ELISA, EIA1561 and DRG Androgen ELISA, EIA-1559 Germany). The sensitivity of this assay was 9.714 pg/ml for estradiol, for both progesterone and androgen was 0.083 ng/ml. Masked split specimens included within each batch were used to calculate the coefficient of variation (CV) within and between batches: the intra- and inter-assay CVs of estaradiol, progesterone and androgen were below 6.81% and 7.25%, 5.4% and 9.96% and 4.16% and 9.94%, respectively. All matched blood samples were handled identically and assayed in the same analytical run. The blood samples were labeled by number only and ordered randomly within each case–control pair.
Preparation of total RNA
Approximately 100 mg of tissue from each tumor was quick frozen in liquid nitrogen and pulverized manually with a hammer to a fine powder. The cells powder were harvested and resuspended in 1 ml of RNX plus reagent in a clean RNase-free tube. After incubation for 5 minutes at room temperature the sample was done pipetteing and subsequently treated with adding 200 μl of chloroform and was taken at room temperature for 5 minutes after shaking rigorously for 15 seconds. The mixture was centrifuged at 12,000 × g for 15 minutes and then the aqueous phase containing the RNA was transferred to a clean RNase-free tube. The total RNA was precipitated by adding 0.5 ml of isopropyl alcohol and incubated at room temperature for 15 minutes. The pellet including total RNA was washed by using 75% ethanol and centrifuge at 7,500 × g for 8 minutes. After drying ethanol, the RNA pellet re-suspended in TE buffer. The concentration of total RNA was calculated based on OD260/280 ratio measurements as a means to address purity of RNA.
RNA was converted to cDNA after treating with DNase I. Reverse transcription of RNA was done in a final volume of 20 μl by using of cDNA first strand synthesis kit (Fermentase) random hexamere and 1 μg of total RNA. The samples were incubated at 65 °C for 10 min and 42 °C for 60 min, and reverse transcriptase was inactivated by heating at 70 °C for 5 min and cooling at 4 °C for 5 min.
Real-time RT- PCR
Principle: reactions are characterized by detection of cycling amplification of the PCR product, rather than the amount of PCR product accumulated after a fixed number of cycles. If the amount of the target molecule was larger, the earlier a significant increase in fluorescence is observed. The parameter Ct (threshold cycle) is defined as the cycle number at which the fluorescence generated by cleavage of the probe passes a fixed threshold above baseline. We used the Delta;Delta;CT method for determination of relative ERs gene expression. The Ct of each target gene compared to the Ct of it’s internal control (beta actin gene).
Final results, expressed as N-fold differences in tested gene (obese) relative to control gene (none obese), termed “N fold changes,” were determined as follows:
where Delta;Ct values of the sample and control are determined by subtracting the average Ct value of the target gene from the average Ct value of the beta actin gene.
qRT-PCR assay: generation of standards for target gene
To be sure of equal efficiency of PCR assay, serial dilutions of one sample that expressed target gene in high levels selected and standard curve for both ERs and control gene were prepared. PCR efficiency for both control and target gene was acceptable. Moreover, Melting curve analysis showed that there was only one segment (one pick) and this is an indication for amplification of desired specific PCR product in our study.
After determination of RNA concentration based on absorbance and cDNA synthesis, the precise amount of cDNA was added to each reaction mix. Quantitative detection of mRNA molecules was determined by quantitative real-time RT-PCR technique using the Syber Green-I (Fermentase Co. according to the instructions) by the Rotor-GeneTM 6000 system (Corbett Research, Australia) according to the manufacturer’s instructions. After cDNA synthesis, specific primers were used to amplify ERs and steroid hormone receptors progesterone and androgen receptor. (Primer sequences shown in ). Human beta actin mRNA was selected as the housekeeping gene (reference gene) and amplified by using the specific forward and reverse primers. The Beta-actin mRNA measured as an internal control (a mRNA that does not vary in abundance respective to cell type) was included in each analysis. Briefly, each sample was normalized to the number of cells harvested. The protocol for detection of RNA consisted started by denatured at 95 °C for 10 minutes. This step was followed by 40 cycles of denaturing at 95 °C for 15 second; annealing at 60 °C for 30 seconds; extending at 72 °C for 30 seconds, which was then followed by melting curve analysis of 70 °C to 95 °C for conformity assessment.
Primer sequences for RT-real Time PCR.
Differences between obese and non-obese groups in age at enrollment, age at menarche, age at menopause were tested using the chi-square test. In addition, Student’s t-test was used to evaluate differences in categorical breast cancer risk factors between obese and non-obese. Pearson correlation coefficients were used to examine cross-sectional relationships between steroid receptors, gene expression and BMI among subjects. Chi-square test was performed to test the relation between steroid receptors mRNA level and clinicopathological parameters (For this purpose, all steroid receptors according to their mRNA content divided in two subgroups as low and high mRNA level). All statistical analysis was conducted using SPSS statistical software (version 16). P values below the conventional level of statistical significance (P < 0.05) were considered statistically significant.