- Investigation of progesterone receptor gene expression changes in MCF-7 breast cancer cell line under simulated microgravity
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Maryam Salavatifar,1,*
1. Space biology and Environment Center, A&S Research institute, Ministry of Science, Research and Technology, ,
- Introduction: The living organisms on the surface of the earth are under influence of gravity force (1g) and any alteration on that, as experienced by astronauts during space travel, exhibit effects on cell functions by participation with biochemical pathways and gene expressions (1, 2). In some studies, it has been observed that changes in gravity, reduce the growth of some cancer cells, and alter the expression of some genes and cellular functions (3, 4). However, very little information is available on the effects of microgravity on the related gene expression. The aim of this study was to investigate the effect of simulated microgravity conditions on changes in progesterone receptor gene expression in MCF-7 breast cancer cell lines
- Methods: MCF-7 breast cancer cell line has an oestrogen receptor, progesterone receptor but no human epidermal growth factor receptor 2 (5). The cells were cultured in DMEM medium, containing 10% sodium bicarbonate, supplemented with 10% FBS, and penicillin /streptomycin. All cell lines were incubated at 37°C in a humidified incubator with 5% CO2.
The clinostat was employed for simulating microgravity. Cells were seeded at the culture tube and after the cells stick, tubes were completely filled by the medium. The rotation times were 24 and 72 hours and the device rotated by 30 rpm.
Total RNA was isolated from the lysed cells and was subjected to reverse transcription using the qScript™cDNA Synthesis kit. To analysis the gene expression using Quantitative real-time RT-PCR, primer sequences were as follows: PR-total F (5'AGCCCACAATACAGCTTCGAG3'), PR-total R (5'TTTCGACCTCCAAGGACCAT3') (6). As a positive control, glyceraldehyde-3-phosphate dehydrogenase mRNA (GAPDH) was amplified in parallel with the primers: GAPDH F (5'ACGACCACTTTGTCAAGCTCAT3') and GAPDH R 5'TCCACCACCCTGTTGCTGTA3'). Cycling conditions were: 95º C for 30 sec; followed by 40 cycles of 95 ºC for 20 sec and 95 ºC for 40 sec. PCR melts curves were performed and changes in the fold number were calculated by using the 2ΔΔCt method.
A statistical correlation was performed using independent samples t-test and p<0.05 was considered statistically significant.
- Results: Results and discussion
The concept of space cell biology is the study of the physical effects of space conditions such as microgravity on the cells and the answer to the question of whether gravity has direct effects on the cell and its function. It has been shown that in microgravity, the expression of some genes and protein levels produced in cultured cells or laboratory animals have been altered. However, very little information is available on the effects of simulated microgravity on gene expression. The study of these alterations would be beneficial to aid astronauts and improving the quality of human life.
In order to apply microgravity on breast cancer cells, clinostat was applied and microgravity was simulated by rotating the system around the horizontal axis. After 24 and 72 hours of exposure to microgravity conditions, total RNA was extracted from cells and RNA integrity was proved on the agarose gel. After that, the cDNA was constructed and a real-time PCR method was employed for investigation progesterone receptor gene alteration. The results have shown that no significant changes in gene expression were occurring. On the other hand, microgravity had no effect on the progesterone receptor gene on MCF-7 breast cancer cell lines.
- Conclusion: Our results showed that simulated microgravity have not any effect on the progesterone receptor gene expression in MCF-7 cells at transcriptional levels, suggesting that there is a strong transcriptional control of this gene or optimization of microgravity application time may yield better results. Collectively, these results demonstrate that further studies may be necessary and investigation on protein level may yield other interesting results.
- Keywords: progesterone receptor, microgravity, MCF-7 breast cancer cell lines