UNLABELLED: MicroRNAs (miRNAs) are small, noncoding RNA molecules that control expression of target genes. The abnormally expressed miRNAs function as oncogenes or tumor suppressors in human cancer. To evaluate the abundant gene regulation of miR-221 in papillary thyroid carcinoma (PTC), we performed microarray analysis and developed a Gaussia luciferase (Gluc) reporter system regulated by miR-221. METHODS: Total RNAs were isolated from pre-miR-221-treated normal human thyroid cells (HT-ori3) and anti-miR-221-treated papillary thyroid cells (NPA). Microarray analysis was performed with 44,000 probes. The messenger RNA levels of target genes regulated by miR-221 were evaluated using reverse-transcription polymerase chain reaction. Three types of cytomegalovirus (CMV)/Gluc_3' untranslated region (UTR) of homeobox B5 (HOXB5), which included a seed sequence of mature miR-221 in the 3' UTR of HOXB5 after the Gluc stop codon, were transfected into NPA cells, and pre-miR-221 was cotransfected with CMV/Gluc_3' UTR of HOXB5. The Gluc activities in cells were measured by luciferase assay. Mice implanted with PTC-expressing Gluc regulated by miR-221 were monitored with bioluminescence imaging for 6 d. RESULTS: Microarray analysis showed thousands of genes were directly and indirectly regulated by miR-221 and shifted the gene expression pattern of normal thyroid cells toward PTC. Of several genes downregulated more than 2-fold by miR-221, messenger RNA levels of HOXB5 were significantly downregulated by miR-221. Also, in vitro or in vivo Gluc activities using CMV/Gluc_3' UTR of HOXB5 systems were downregulated dose dependently by endogenous or exogenous miR-221. CONCLUSION: MiR-221 overexpressed in PTC drives carcinoma gene expression patterns by directly and indirectly regulating numerous genes, including HOXB5. The bioluminescence imaging system using CMV/Gluc_3' UTR of HOXB5 is a useful tool for noninvasive in vivo long-term monitoring of functional targeting of miR-221.
UNLABELLED: MicroRNAs (miRNAs) are small, noncoding RNA molecules that control expression of target genes. The abnormally expressed miRNAs function as oncogenes or tumor suppressors in humancancer. To evaluate the abundant gene regulation of miR-221 in papillary thyroid carcinoma (PTC), we performed microarray analysis and developed a Gaussia luciferase (Gluc) reporter system regulated by miR-221. METHODS: Total RNAs were isolated from pre-miR-221-treated normal human thyroid cells (HT-ori3) and anti-miR-221-treated papillary thyroid cells (NPA). Microarray analysis was performed with 44,000 probes. The messenger RNA levels of target genes regulated by miR-221 were evaluated using reverse-transcription polymerase chain reaction. Three types of cytomegalovirus (CMV)/Gluc_3' untranslated region (UTR) of homeobox B5 (HOXB5), which included a seed sequence of mature miR-221 in the 3' UTR of HOXB5 after the Gluc stop codon, were transfected into NPA cells, and pre-miR-221 was cotransfected with CMV/Gluc_3' UTR of HOXB5. The Gluc activities in cells were measured by luciferase assay. Mice implanted with PTC-expressing Gluc regulated by miR-221 were monitored with bioluminescence imaging for 6 d. RESULTS: Microarray analysis showed thousands of genes were directly and indirectly regulated by miR-221 and shifted the gene expression pattern of normal thyroid cells toward PTC. Of several genes downregulated more than 2-fold by miR-221, messenger RNA levels of HOXB5 were significantly downregulated by miR-221. Also, in vitro or in vivo Gluc activities using CMV/Gluc_3' UTR of HOXB5 systems were downregulated dose dependently by endogenous or exogenous miR-221. CONCLUSION:MiR-221 overexpressed in PTC drives carcinoma gene expression patterns by directly and indirectly regulating numerous genes, including HOXB5. The bioluminescence imaging system using CMV/Gluc_3' UTR of HOXB5 is a useful tool for noninvasive in vivo long-term monitoring of functional targeting of miR-221.
Authors: Emma V Barker; Nilva K Cervigne; Patricia P Reis; Rashmi S Goswami; Wei Xu; Ilan Weinreb; Jonathan C Irish; Suzanne Kamel-Reid Journal: Mol Cancer Date: 2009-12-23 Impact factor: 27.401