Effects of silencing RET/PTC1 junction oncogene in human papillary thyroid carcinoma cells.
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.
Gilbert-Sirieix
Marie
M
UMR8203 CNRS, Institut Gustave Roussy, 114 rue Edouard Vaillant, Villejuif Cedex, France.
Ripoche
Hugues
H
Malvy
Claude
C
Massaad-Massade
Liliane
L
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002291:Carcinoma, Papillary / Q000188:drug therapy / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D020868:Gene Silencing; D006801:Humans; D051379:Mice; D046228:Microarray Analysis; D041681:NIH 3T3 Cells; D015514:Oncogene Proteins, Fusion / Q000235:genetics* / Q000627:therapeutic use; D017354:Point Mutation; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000627:therapeutic use*; D013964:Thyroid Neoplasms / Q000188:drug therapy / Q000235:genetics*
D015514:Oncogene Proteins, Fusion; D034741:RNA, Small Interfering; D051096:Proto-Oncogene Proteins c-ret