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Updated: Jul 1, 2026
In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice
Published on: August 23, 2019
In vivo imaging of functional targeting of miR-221 in papillary thyroid carcinoma
Hyun Joo Kim1, Young Ha Kim, Dong Soo Lee
1Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, Korea.
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.
Insights
MicroRNA-221 (miR-221) drives papillary thyroid carcinoma (PTC) by regulating genes like HOXB5. A novel bioluminescence imaging system allows for noninvasive monitoring of miR-221 activity in vivo.
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Background:
- MicroRNAs (miRNAs) are key regulators of gene expression.
- Aberrant miRNA expression is implicated in human cancers, acting as oncogenes or tumor suppressors.
- Papillary thyroid carcinoma (PTC) exhibits altered miRNA profiles, necessitating investigation into specific miRNA roles.
Purpose of the Study:
- To investigate the role of miR-221 in papillary thyroid carcinoma (PTC).
- To identify genes regulated by miR-221 in PTC.
- To develop and validate a reporter system for monitoring miR-221 activity.
Main Methods:
- Microarray analysis to identify genes regulated by miR-221.
- Development of a Gaussia luciferase (Gluc) reporter system to assess miR-221 activity.
- Reverse-transcription polymerase chain reaction (RT-PCR) to quantify target gene mRNA levels.
- In vitro and in vivo experiments using transfected cells and mice.
Main Results:
- Microarray analysis revealed thousands of genes regulated by miR-221, altering normal thyroid cell gene expression towards a PTC pattern.
- HOXB5 mRNA levels were significantly downregulated by miR-221.
- In vitro and in vivo luciferase assays demonstrated dose-dependent downregulation of Gluc activity by miR-221.
Conclusions:
- Overexpressed miR-221 in PTC promotes carcinoma gene expression patterns through direct and indirect regulation of numerous genes, including HOXB5.
- The CMV/Gluc_3' UTR of HOXB5 bioluminescence imaging system provides a valuable tool for noninvasive, long-term in vivo monitoring of miR-221 function.
