BACKGROUND: Galectin-3 (LGALS3) is an important glycoprotein involved in the malignant transformation of thyrocytes acting in the extracellular matrix, cytoplasm and nucleus where it regulates TTF-1 and TCF4 transcription factors. Within LGALS3 gene, a common genetic polymorphism (c.191C>A, p.Pro64His; rs4644) encoding for the variant Proline to Histidine at codon 64 has been extensively studied. However, data on rs4644 in the context of thyroid cancer are lacking. Thus, the aim of the present work was to evaluate the role of the SNP rs4644 as risk factor for differentiated thyroid cancer (DTC) and to verify the effect on the transcriptome in thyrocytes.METHODS: A case-control association study on 1223 controls and 1142 unrelated consecutive DTC patients was carried out to evaluate the association between rs4644-P64H and the risk of DTC. Moreover, we employed the non-malignant cell line Nthy-Ori (rs4644-C/A) and the CRISPR/Cas9 technique to generate isogenic cells carrying either the rs4644-A/A or rs4644-C/C homozygosis. Then, the transcriptome of the derivative and unmodified parental cells was analyzed by RNA-seq. Genes differentially expressed in a statistically significant way were validated by RT-qPCR and further tested in the parental Nthy-Ori cells after LGALS3 gene silencing, to investigate whether the expression of target genes was dependent also upon the galectin-3 levels.RESULTS: Rs4644 AA genotype was associated with a reduced risk of DTC (compared to CC, ORadj=0.66; 95%CI=0.46-0.93; Pass=0.02). Moreover, we showed that rs4644 affects galectin-3 as transcriptional co-regulator. Among 34 genes affected by rs4644 HES1, HSPA6, SPC24, and NHS were of particular interest since their expression was rs4644-dependent (CC>AA for the first and AA>CC for the others) also in 574 thyroid tissues of GTex biobank (https://gtexportal.org/home/index.html). Moreover, the expression of these genes was sensible to LGALS3-silencing. We hypothesized that rs4644 could alter the transcription of target genes by modulating the interaction with TTF-1. Indeed, the proximity ligation assay in Nthy-Ori cells showed that the interaction was genotype-dependent.CONCLUSIONS: Our data showed that in thyroid rs4644 is a trans-expression quantitative trait locus that can modify the transcriptional expression of downstream genes, through the modulation of TTF-1 and, likely, also of other transcriptional factors such as TCF4.

Pro64His (rs4644) polymorphism within galectin-3 is a risk factor of differentiated thyroid carcinoma and affects the transcriptome of thyrocytes engineered via CRISPR/Cas9 system

Giusti, Laura;
2021-01-01

Abstract

BACKGROUND: Galectin-3 (LGALS3) is an important glycoprotein involved in the malignant transformation of thyrocytes acting in the extracellular matrix, cytoplasm and nucleus where it regulates TTF-1 and TCF4 transcription factors. Within LGALS3 gene, a common genetic polymorphism (c.191C>A, p.Pro64His; rs4644) encoding for the variant Proline to Histidine at codon 64 has been extensively studied. However, data on rs4644 in the context of thyroid cancer are lacking. Thus, the aim of the present work was to evaluate the role of the SNP rs4644 as risk factor for differentiated thyroid cancer (DTC) and to verify the effect on the transcriptome in thyrocytes.METHODS: A case-control association study on 1223 controls and 1142 unrelated consecutive DTC patients was carried out to evaluate the association between rs4644-P64H and the risk of DTC. Moreover, we employed the non-malignant cell line Nthy-Ori (rs4644-C/A) and the CRISPR/Cas9 technique to generate isogenic cells carrying either the rs4644-A/A or rs4644-C/C homozygosis. Then, the transcriptome of the derivative and unmodified parental cells was analyzed by RNA-seq. Genes differentially expressed in a statistically significant way were validated by RT-qPCR and further tested in the parental Nthy-Ori cells after LGALS3 gene silencing, to investigate whether the expression of target genes was dependent also upon the galectin-3 levels.RESULTS: Rs4644 AA genotype was associated with a reduced risk of DTC (compared to CC, ORadj=0.66; 95%CI=0.46-0.93; Pass=0.02). Moreover, we showed that rs4644 affects galectin-3 as transcriptional co-regulator. Among 34 genes affected by rs4644 HES1, HSPA6, SPC24, and NHS were of particular interest since their expression was rs4644-dependent (CC>AA for the first and AA>CC for the others) also in 574 thyroid tissues of GTex biobank (https://gtexportal.org/home/index.html). Moreover, the expression of these genes was sensible to LGALS3-silencing. We hypothesized that rs4644 could alter the transcription of target genes by modulating the interaction with TTF-1. Indeed, the proximity ligation assay in Nthy-Ori cells showed that the interaction was genotype-dependent.CONCLUSIONS: Our data showed that in thyroid rs4644 is a trans-expression quantitative trait locus that can modify the transcriptional expression of downstream genes, through the modulation of TTF-1 and, likely, also of other transcriptional factors such as TCF4.
2021
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11581/449636
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