Papillary Thyroid Carcinoma (PTC) is the most common thyroid malignancy, with well-established genetic factors in sporadic cases, but poorly-understood mechanisms in familial forms. This project of thesis investigates a family affected by PTC, focusing on a 199,034 bp deletion in the ECT2 gene (Epithelial Cell Transforming 2), which is involved in cell transformation and cancer progression. This deletion affects the miR-223 binding site, a microRNA that normally downregulates ECT2 expression. The inability of miR-223 to bind could lead to increased ECT2 levels, promoting tumour progression. To assess the impact of this ECT2 deletion, CRISPR-Cas9 was used to create ECT2-mutated models in a thyroid cell line by transfecting Nthy-ori-3-1 cells with specific sgRNAs. Stable clones were then selected and ECT2 expression was measured at both mRNA and protein levels. Finally, the significantly altered mutated clones are further analysed trough functional cellular assays. Surprisingly, heterozygous clones showed a significant reduction in ECT2 expression compared to controls, contradicting the expectation of overexpression. This unexpected result highlights the need for further investigation into ECT2 expression dynamics and its role in cellular behaviour, with the aim of better understanding the role of ECT2 in PTC and contributing to deeper insights into familial thyroid cancer.

Papillary Thyroid Carcinoma (PTC) is the most common thyroid malignancy, with well-established genetic factors in sporadic cases, but poorly-understood mechanisms in familial forms. This project of thesis investigates a family affected by PTC, focusing on a 199,034 bp deletion in the ECT2 gene (Epithelial Cell Transforming 2), which is involved in cell transformation and cancer progression. This deletion affects the miR-223 binding site, a microRNA that normally downregulates ECT2 expression. The inability of miR-223 to bind could lead to increased ECT2 levels, promoting tumour progression. To assess the impact of this ECT2 deletion, CRISPR-Cas9 was used to create ECT2-mutated models in a thyroid cell line by transfecting Nthy-ori-3-1 cells with specific sgRNAs. Stable clones were then selected and ECT2 expression was measured at both mRNA and protein levels. Finally, the significantly altered mutated clones are further analysed trough functional cellular assays. Surprisingly, heterozygous clones showed a significant reduction in ECT2 expression compared to controls, contradicting the expectation of overexpression. This unexpected result highlights the need for further investigation into ECT2 expression dynamics and its role in cellular behaviour, with the aim of better understanding the role of ECT2 in PTC and contributing to deeper insights into familial thyroid cancer.

Genesis and functional analysis of CRISPR-Cas9 engineered cellular models to study a genomic rearrangement involving the ECT2 gene in Familial Papillary Thyroid Carcinoma predisposition

GARBUGGIO, AURORA
2023/2024

Abstract

Papillary Thyroid Carcinoma (PTC) is the most common thyroid malignancy, with well-established genetic factors in sporadic cases, but poorly-understood mechanisms in familial forms. This project of thesis investigates a family affected by PTC, focusing on a 199,034 bp deletion in the ECT2 gene (Epithelial Cell Transforming 2), which is involved in cell transformation and cancer progression. This deletion affects the miR-223 binding site, a microRNA that normally downregulates ECT2 expression. The inability of miR-223 to bind could lead to increased ECT2 levels, promoting tumour progression. To assess the impact of this ECT2 deletion, CRISPR-Cas9 was used to create ECT2-mutated models in a thyroid cell line by transfecting Nthy-ori-3-1 cells with specific sgRNAs. Stable clones were then selected and ECT2 expression was measured at both mRNA and protein levels. Finally, the significantly altered mutated clones are further analysed trough functional cellular assays. Surprisingly, heterozygous clones showed a significant reduction in ECT2 expression compared to controls, contradicting the expectation of overexpression. This unexpected result highlights the need for further investigation into ECT2 expression dynamics and its role in cellular behaviour, with the aim of better understanding the role of ECT2 in PTC and contributing to deeper insights into familial thyroid cancer.
2023
Genesis and functional analysis of CRISPR-Cas9 engineered cellular models to study a genomic rearrangement involving the ECT2 gene in Familial Papillary Thyroid Carcinoma predisposition
Papillary Thyroid Carcinoma (PTC) is the most common thyroid malignancy, with well-established genetic factors in sporadic cases, but poorly-understood mechanisms in familial forms. This project of thesis investigates a family affected by PTC, focusing on a 199,034 bp deletion in the ECT2 gene (Epithelial Cell Transforming 2), which is involved in cell transformation and cancer progression. This deletion affects the miR-223 binding site, a microRNA that normally downregulates ECT2 expression. The inability of miR-223 to bind could lead to increased ECT2 levels, promoting tumour progression. To assess the impact of this ECT2 deletion, CRISPR-Cas9 was used to create ECT2-mutated models in a thyroid cell line by transfecting Nthy-ori-3-1 cells with specific sgRNAs. Stable clones were then selected and ECT2 expression was measured at both mRNA and protein levels. Finally, the significantly altered mutated clones are further analysed trough functional cellular assays. Surprisingly, heterozygous clones showed a significant reduction in ECT2 expression compared to controls, contradicting the expectation of overexpression. This unexpected result highlights the need for further investigation into ECT2 expression dynamics and its role in cellular behaviour, with the aim of better understanding the role of ECT2 in PTC and contributing to deeper insights into familial thyroid cancer.
Familial PTC
ECT2
CRISPR-Cas9
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12608/81067