Stability of human follicle-stimulating hormone receptor mRNA in stably transfected cells
Zhu, C.; Tian, H.
Journal of Tongji Medical University 21(1): 8-12
In order to assess the impact of mRNA degradation on steady state levels of follicle-stimulating hormone receptor (FSHR) mRNA and on regulation of FSHR gene expression, the stability and half-life of FSHR mRNA were determined in transfected cells expressing recombinant FSHR. Time-dependent changes in FSHR mRNA content were determined by nuclease protection-solution hybridization assay (NPA) or by qualitative reverse transcription-competitive polymerase chain reaction (RT-PCR) in cultured hFSHR-YI cells, cell lines stably transfected with a human FSHR cDNA. FSHR mRNA content remained constant during 8 h control incubations of hFSHR-Y1 cells (NPA, 2.9 +/- 0.3 micrograms/mg RNA; RT-PCR, 2.7 +/- 0.3 micrograms/mg RNA). Actinomycin D (ActD, 5 micrograms/ml) inhibited mRNA synthesis, as assessed by incorporation of [3 H]uridine into total RNA, by 90% within 1 h in hFSHR-Y1 cells. No effect of ActD on cellular morphology or viability was observed. ActD caused a time-dependent decrease in FSHR mRNA content in hFSHR-Y1 cell lines with a lag time of 1 h. There were no significant differences in the rate of FSHR mRNA degradation between the two methods of mRNA quantification. The half-life of hFSHR mRNA was 3.6 +/- 0.2 h by NPA and 3.1 +/- 0.1 h by RT-PCR. The results indicated that degradation of mRNA was an important process in maintenance of steady state expression of the FSHR gene in cells stably expressing recombinant receptor.