In vivo visualization of expression and function of miR221 in papillary thyroid carcinoma

MicroRNA 221 has been known to be one of up-regulated miRNAs in papillary thyroid carcinoma. To evaluate the expression and function of miR221, we measured the quantities of primary or mature miR221 in normal thyroid or papillary cancer cells and examined the gene expression of Gaussian luciferase (Glue) regulated by miR221. Total RNA and small RNA were isolated from normal thyroid cells (HT-ori3) and papillary thyroid cancer cells (NPA, TPC-1). The quantities of primary miR221 or mature miR221 in cells were measured by qRT-PCR. We constructed a CMV/Gluc-3xPTmiR221 including 3 times repeated perfect target sequences of miR221 in the 3'UTR of Gluc. CMV/Gluc-3xPTmiR221 was transfected into HT-ori3, NPA, and TPC-1, CMV/Gluc was used as a control. Also, precursor miR221 or anti-miR221 were co-transfected with CMV/Gluc-3xPTmiR221 into each cells and compared the Gluc activities by luciferase assay and in vivo bioluminescence image. The quantities of primary miR221 of NPA or TPC-1 were 2.24 or 1.5 times more than that of HT-ori3, and quantities of mature miR221 in NPA or TPC-1 were 17 or 7 times more than that of HT-ori3, respectively. Gluc activities in NPA or TPC-1 transfected with CMV/Gluc-3xPTmiR221 were repressed 2 times or more than those of CMV/Gluc, respectively. Also, Gluc activities in NPA or TPC-1 co-transfected with pre-miR221 and CMV/Gluc-3xPTmiR221 were repressed 5 times or more down-regulated than those of CMV/Gluc, respectively. The other hand, Gluc activities in NPA or TPC-1 co-transfected with anti-miR221 and CMV/Gluc-3xPTmiR221 were retrieved as those of CMV/Gluc. In vivo bioluminescence images also showed that Gluc activities were repressed by pre.miR221 and retrieved by anti-miR221. These results suggest that CMV/Gluc-3xPTmiR221 system may be a useful tool for monitoring the quantities of miR221 and gene regulation by miR221 in a living animal