- The effect of weightlessness on progesterone receptor gene expression changes in Triple negative breast cancer cell line
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Maryam Salavatifar,1,*
1. Space biology and Environment Center, A&S Research institute, Ministry of Science, Research and Technology, ,
- Introduction: Spaceflight and ground-based weightlessness analog experiments have suggested that gravity can affect cell growth, metabolism, morphology, development, and gene expressions (1, 2). Alteration on properties of different cell lines such as cancer cells or stem cells under weightlessness was reported in several studies (3, 4). In this study, we evaluated the alteration of progesterone receptor gene expression in human triple-negative breast cancer cells after 1 and 3 days of exposure to weightlessness conditions.
- Methods: MDA-MB-231 is triple-negative breast cancer (TNBC) cell line as it lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)(5). The cells were cultured in Dulbecco's modified Eagle medium (DMEM), containing 10% sodium bicarbonate, supplemented with 10% fetal bovine serum (FBS), and penicillin (50 IU/ml)/streptomycin (50 µg/ml). All cell lines were incubated at 37°C in a humidified incubator with 5% CO2.in order to simulating microgravity, two-dimensional clinostat was employed. Cells were cultured at culture tube (TPP, Switzerland) and after the cells stick, tubes were completely filled by the medium. The rotation times were 1 and 3 days 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. Melting curve analysis was performed for each gene 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. Relevant diagrams were drawn using the Graphpad Prism 7 programs.
- Results: Results and discussion
Gravity is one of the most important mechanical factors on earth that has immense effects on living organisms. Every change in an organism is affected by changes in its cells. However, information on the effects of no gravity on the gene expression is very low. Thus, simulated microgravity may be used as a novel methodology for manipulating cells along with other biochemical techniques. To investigate the effects of weightlessness, a simulator was used. After 1 and 3 days exposure to microgravity conditions, the RNA was extracted from cells and the cDNA were constructed. After that, a real-time PCR method was employed for investigation progesterone receptor gene alteration. No significant changes in gene expression were observed. On the other hand, microgravity had no effect on the progesterone receptor gene on MDA-MB-231 breast cancer cell lines.
- Conclusion: The result proved that weightlessness has not detectable influence on the gene expression of progesterone receptor in MDA-MB-231 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: weightlessness, triple-negative breast cancer cell line, progesterone receptor