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Updated: Jun 11, 2026
In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice
Published on: August 23, 2019
Effects of silencing RET/PTC1 junction oncogene in human papillary thyroid carcinoma cells
Marie Gilbert-Sirieix1, Hugues Ripoche, Claude Malvy
1UMR8203 CNRS, Institut Gustave Roussy, 114 rue Edouard Vaillant, Villejuif Cedex, France.
Background:
RET/PTC1 rearrangement is the most common genetic alteration identified to date in papillary thyroid carcinomas (PTC) and represents an interesting target for small interfering RNA (siRNA) strategies because it is present only in the tumor cells and not in the normal cells. Our aims were (i) to target the RET/PTC1 oncogene by siRNAs, (ii) to assess the knockdown effects on cell growth and cell cycle regulation, and (iii) to identify genes affected by the RET/PTC1 silencing.
Methods:
Three efficient siRNAs previously designed in our laboratory in a model of murine PTC (RP-1 cells) were used to knockdown RET/PTC1 in the TPC-1 cells. By reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative RT-PCR (Q-RT-PCR) they were found unable to silence RET/PTC1. After sequencing, we redesigned an siRNA against RET/PTC1 (siRNARET/PTC1) and compared it for its efficiency and specificity with an siRNA against RET (siRNARET) in the TPC-1 cells, in human cell lines that expressed RET (MCF-7 and BT-474 cells), and in the murine RP-1 cells. The effects on cell cycle growth (MTT tests), cell cycle (flow cytometry), and apoptosis (TUNEL method) were studied. Genes affected by the RET/PTC1 knockdown were identified by microarray analysis followed by Q-RT-PCR validation.
Results:
A mutation was found by sequencing within the H4 part of the RET/PTC1 junction leading to a ²⁹⁷T→G substitution. The redesigned siRNARET/PTC1 inhibits about 85% of the oncogene expression in the human TCP-1 cells. The specificity of the siRNARET/PTC1 was confirmed by the absence of a silencing effect on the human breast MCF-7 and BT-474 cells without RET/PTC1 and the murine RP-1 with ²⁹⁷G→T mutation. The downregulation of RET/PTC1 modified the cell cycle and induced an apoptotic response. Microarray analysis revealed an inhibition of E2F2 transcription factor known to be involved in the cell cycle regulation.
Conclusions:
This study shows the impact of a point mutation within a junction oncogene on the siRNA design. In the case of a therapeutic approach by siRNA, the junction oncogene must be systematically sequenced. The E2F2 gene regulation would have a biological significance and seems to be directly mediated by RET/PTC1.
Insights
A point mutation in the RET/PTC1 oncogene impacted siRNA design for papillary thyroid cancer (PTC) therapy. Sequencing the oncogene is crucial for effective siRNA strategies targeting cancer cells.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Papillary thyroid carcinoma (PTC) frequently exhibits RET/PTC1 rearrangement, a tumor-specific genetic alteration.
- Small interfering RNA (siRNA) strategies offer a targeted approach for PTC treatment due to the oncogene's presence only in cancer cells.
Purpose of the Study:
- To develop and validate siRNA targeting the RET/PTC1 oncogene in PTC.
- To evaluate the impact of RET/PTC1 knockdown on cancer cell proliferation, cell cycle, and apoptosis.
- To identify genes regulated by RET/PTC1 signaling.
Main Methods:
- Redesigned and tested siRNA targeting RET/PTC1 in TPC-1 cells, comparing its efficacy and specificity against a control siRNA.
- Assessed effects on cell growth (MTT), cell cycle progression (flow cytometry), and apoptosis (TUNEL assay).
- Utilized microarray analysis followed by quantitative RT-PCR (Q-RT-PCR) to identify genes affected by RET/PTC1 knockdown.
Main Results:
- A point mutation (²⁹⁷T→G) within the RET/PTC1 junction was identified, necessitating siRNA redesign.
- The optimized siRNARET/PTC1 achieved ~85% inhibition of RET/PTC1 expression in TPC-1 cells, with confirmed specificity.
- Downregulation of RET/PTC1 induced cell cycle arrest and apoptosis, and inhibited E2F2 transcription, a key cell cycle regulator.
Conclusions:
- Systematic sequencing of junction oncogenes is essential for successful siRNA-based therapeutic design.
- RET/PTC1 directly mediates the regulation of the E2F2 gene, highlighting its role in cell cycle control.
- This study underscores the importance of considering genetic variations in oncogenes for targeted cancer therapies.
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