Furthermore, to determine the time-course inhibitory effect of miR-206 on CALR expression, human breast cancer stem cells were transfected with miR-206 mimics (50nM) at different time point (0, 24, 48 and 74 hours), and the mRNA and protein expression level of CALR was determined by RT-PCR and western blot analysis, respectively. The gene discussed is CALR; the disease is breast cancer.