Materials and methods
Cell culture
The murine mammary carcinoma cell line 4T1 was obtained from the cell bank of Pasteur Institute of Iran (C604) and cultivated in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS (fetal bovine serum) and 2% Penicillin–Streptomycin (all from Gibco, USA). The cells incubated in 37 °C with 95% air and 5% of carbon dioxide (CO2).
Mammary tumor induction and isolation of primary and highly metastatic breast cancer cells
Induction of mammary tumor in BALB/c mice and isolation of primary and metastatic tumor cells, were performed as described in our previous work [19, 20]. The Ethics Committee of Shahroud University of Medical Sciences approved this study for ethics in animal research (registration number: IR.SHMU.REC.1400.265). The isolated cells were cultured in a DMEM with 10% FBS, 100 U/ml Penicillin, and 100 ug/ml Streptomycin (all from Gibco, USA). Ultimately, the cells were incubated at 37 °C in 5% CO2 and passaged two times.
Real-time qRT-PCR assay
Primary and metastatic tumor cells (1 × 104) were seeded in a 24-well plate. Total RNA extraction, cDNA synthesis and Real-time PCR procedure were preformed similar to our previous works [20]. Briefly after 48 h, total RNA was extracted from the seeded cells using Trizol reagent. The extracted RNA's quality, yield, and size were analyzed using spectrophotometry and electrophoresis. The first-strand cDNA synthesis was performed using a reverse transcription system (Easy cDNA Synthesis Kit for RNA or mRNA to cDNA—pars tous). A Real-time PCR procedure was executed based on the 1 ul cDNA in all samples. According to the manufacturer's instruction, quantization of all gene transcripts was done by SYBR Green Real-time PCR Master Mix (Amplicon A/S, Denmark) using StepOnePlus™ Real-Time PCR System. The amplification procedure was as follows: 1 cycle of 95 °C for 15 °min, 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. The exact mRNA expression was normalized to the expression level of GAPDH. Gene expression of each target was calculated by using the 1/ΔCT method. GAPDH amplification was used as normalization controls for OPN transcription level evaluation and the RT-qPCR. The delta CT value was calculated by applying the following calculation: (CT of OPN (tOPN or OPN5)) − (CT of housekeeping gene).
The used primers are as follows:
For tOPN, Forward 5′-GGATGAATCTGACGAATCTCAC-3′, Reverse 5′- CCTTAGACTCACCGCTCTTC-3′;
For OPN5, Forward 5′-TGGTGGTGATCTAGTGGTG-3′, Reverse 5′- CATGGTCGTAGTTAGTCCTG-3′;
For GADPH, Forward 5′-CCTGGAGAAACCTGCCAAGTA-3′, Reverse 5′-GGCATCGAAGGTGGAAGAGT-3′.
Statistical analysis
Results are expressed as the mean ± standard deviation. Data were analyzed by GraphPad Prism statistical software 6.0 (GraphPad Software, La Jolla, CA, USA) using Paired Samples t-test. P < 0.05 was considered statistically significant.
Results
Isolation of primary and metastatic tumor cells
Due to multiple passages and manipulations, most breast cancer cell lines have changed their function and genomes [21]. So, we decided to use the primary and highly metastatic tumor cells that were isolated from cancerous mice tissues. We properly extracted primary and metastatic tumor cells from subcutaneous primary tumor and lung of cancerous mice, respectively. The metastatic tumor cells in the lung after primary isolation, form colonies in the culture medium. Due to the high rate of growth and proliferation, the tumor cells in these colonies are purified after three passages. These tumor cells are called lung metastatic tumor cells or 4T1L (Fig. 1A) while tumor cells that are obtained in the same way, from the original tissue of the tumor, are primary tumor cells called 4T1T (Fig. 1B).
OPN has five splice variants and four isoforms in mouse
Although OPN has five splice variants (Fig. 2) and four isoforms in mice, total OPN (tOPN) is the main focus of most studies of OPN in cancer. Hence, in this study, we used two primer sets; the first primer set covers all splice variants of OPN, and the second set only covers OPN5.
Expression of OPN5 was confirmed in primary and metastatic tumor cells
The expression of tOPN and OPN5 was analyzed in the primary and metastatic tumor cells. As shown in Fig. 3, both in 4T1T and 4T1L OPN5 were expressed. In 4T1T 80% of tOPN expression belongs to OPN5 but in in 4T1L OPN5 constitute only 10 of tOPN expression.