Characterization and vectorization of siRNA targeting RET / PTC 1 in human papillary thyroid carcinoma cells

RET/PTC1 fusion oncogene is the most common genetic alteration identified to date in thyroid papillary carcinomas (PTC) and represents a good target for small interfering RNA (siRNA). Our aim was: 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 vectorize it in order to protect it from degradation. Methods. Human cell lines expressing RET/PTC1 were transfected by siRNA RET/PTC1, inhibition of the oncogene expression was assessed by qRT-PCR and by Western blot. Conjugation of siRNA RET/PTC1 to squalene was performed by coupling it to squalene. In vivo studies are performed in nude mice. Conclusion. In this short communication, we report the main published results obtained during last years.


Introduction.
Cancers due to chromosomal translocations are considered to represent around 20 % of all cancers [1].Genomic rearrangements leading to intragenic fusion are mainly found in some types of haematopoietic malignancies and sarcomas.They have recently been described also in carcinomas [2,3].The mechanism of formation of most of these translocations is still unclear, except for RET oncogene in papillary thyroid carcinomas (PTC) where ionizing radiation is described as an important factor [4].Thus, thousands of people developed thyroid cancers after the Chernobyl catastrophe [5,6].The RET/PTC oncogene was isolated almost twenty years ago [7,8].RET/PTC is an early event in the process of thyroid carcinogenesis and plays a critical role in the generation of papillary carcinoma [9].The RET protooncogene codes for a cell membrane receptor tyro sine kinase and has a role in the regulation of cell growth, survival, differentiation and migration [10].
Rearrangement involving the chromosome 10 between RET and a ubiquitous gene leads to the abnormal expression of a chimeric constitutively activated RET protein in follicular cells [11].To date, 12 different fusion pattern genes have been reported to form at least 17 different RET hybrid oncogenes.The spatial proximity of Ret gene with H4 during thyrocyte interphase can explain the RET/PTC1 formation [12].This fusion oncogene is essentially restricted to the papillary histotype (60-70 %) [13] and to the Hurthle thyroid tumours (58 %) [14] and its incidence increases after radiation exposure.
Moreover, the RET/PTC1 fusion oncogene is present only in the tumour cells and not in the surrounding normal cells, this constitutes an area of important research on emerging therapies such as using small interfering RNA (siRNA) to target.Therefore, we targeted the RET/PTC1 oncogene by siRNAs and assessed the knockdown effects on cell growth and cell cycle regulation, then we vectorized it because the biological efficacy of the siRNAs is hampered by their short plasmatic half-life due to poor stability in biological fluids and by their low intracellular penetration due to their highly hydrophilic character [15,16].
Material and methods.The human TPC-1 cell line that harbours the RET/PTC1 rearrangement was used.A siRNA targeted to H4/RET termed siRNA RET/PTC1 was designed to knockdown RET/PTC1 in the TPC-1 cells.It was transiently transfected in TPC-1 cells using Lipofectamine 2000 transfection reagent.Then, total RNA was extracted and first-strand cDNA was generated with M-MLV (Reverse Transcription), real time PCR Q-RT-PCR was used to assess the knockdown efficiency.
Western blot was performed using Ret antibody and b-actin was used as loading control.The siRNA RET/ PTC1 effects on cell cycle growth (MTT tests), cell cycle (flow cytometry) and apoptosis (TUNEL method) were studied (Fig. 1).For vectorization, a non cationic new approach for the delivery of siRNA targeted toward the RET/PTC1 fusion oncogene; it is based on the concept of «squalenoylation», consisting in the bio-conjugation of a drug substance to squalene (SQ), a non ionic and biocompatible natural lipid [17,18].The acyclic isoprenoid chain of squalene has been covalently coupled with siRNA RET/PTC1 at the 3'-terminus of the sense strand via maleimide-sulfhydryl chemistry.Nanoparticles were obtained by precipitation in H 2 O.These nanoparticles were tested in vivo, on a mice xenografted RET/ PTC1 experimental model (Fig. 2).

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Results and discussion.By Q-RT-PCR we found that both siRET/PTC1 significantly reduced the RET/ PTC1 mRNA levels by approximately 80 %.MTT assay showed a significant decrease in the TPC-1 growth rate in siRNA RET/PTC1-treated cells when compared to untreated cells.Cell cycle progression analysis showed a significant increase of the S-phase in siRNAstreated cells when compared with the untreated cells.Apoptotic cells were detected using TUNEL staining and a fluorescence microscope.Comprehensive counts showed a modest increase in apoptotic index of about 8.4 ± 1.5 % [19].
Conclusions.The siRNA RET/PTC1 designed is efficient and specific to RET/PTC1 oncogene, which should open new prospects in treatments by siRNAs for PTC or Hurthle thyroid tumours with RET/PTC1 junction.The «squalenoylation» offers a new non cationic plate-form for the siRNA delivery.Further pharmacological assays are needed both on cell cultures and in vivo before to generalize this concept to other nucleic targets.
Acknowledgments.This paper summarizes a part of the PhD thesis work of Marie Gilbert-Sirieix (biological part) and of Mouna Raouane (chemical part).The chemical part of the study was done by the staff of Professor Patrick Couvreur and Doctor Didier Desmaele, UMR 8612 CNRS, laboratoire «Physico-chimie, pharmacotechnie & Biopharmacie», faculte de Pharmacie, Chatenay-Malabry, France.

Fig. 1 .Fig. 2 .
Fig. 1.Gene silencing efficiency of siRNA RET/PTC1 in TPC-1 cells: A -the RET/PTC1 mRNA expression was analyzed by Q-RT-PCR for treated cells by siRNA RET/PTC1 (black) or siRNA CT (grey) and compared to untreated cells (white); the bars represent mean ± SD of at least three independent experiments; ***significant change compared to untreated cells using ANOVA followed by Bonferroni's test (p < < 0.001); B -RET/PTC-1 protein level in untransfected and transfected cells with siRNA RET/PTC1 or siRNA CT at 50 nM.Proteins were extracted 24 h after transfection and analyzed by Western blot using Ret antibody; b-actin was used as loading control for Western blot; experiments were performed in triplicate.1 -untreated cells; 2control siRNA; 3 -RET/PTC1 siRNA.