Proteomic analysis of thyrocytes carrying H64 (rs4644) homo-zygotes polymorphism within galectin-3 unveils a carcinogenic profile

Genomic wide association studies and intensive bioinformatic searches of databases have identified several loci associated with genetic predisposition to thyroid cancer [1,2]. Among these, the LGALS3 gene, which encodes galectin-3 protein (gal-3), is a potential candidate to be considered. Within LGALS3 gene, a common single nucleotide polymorphism (SNP) encoding for the variant Proline (P64) to Histidine (H64) at codon 64 (rs4644) has been found to affect function of galectin-3. The aim of this work was to determine proteomes of rs4644 SNP variants in human thyroid cell line (Nthy-Ori). Nthy-Ori cells have a heterozygote genotype C/A allowing the conversion into homozygote genotypes (either A/A and C/C) using the CRISPR/Cas9 gene editing system, as described by Corrado et al. [3]. Gene edited cells were named ORI-CC and ORI-AA, also called gal-3P64 and gal-3H64, respectively. Proteomic analysis of genetic variants of Nthy-Ori cell was performed using two-dimensional gel electrophoresis coupled to mass spectrometry. A total of about 1300 spots were detected on gels from different samples. Then, after com-putational comparison of images, 37 protein spots were found to be differentially expressed in ORI-AA with comparison to ORI-CC cells. In order to identify the proteins potentially correlated with rs4644 mutation, differentially expressed spots were cut from gels, trypsinized and analyzed by nano-LC ESI MS/MS. Ingenuity pathways analysis (IPA) was performed to recognize molecular and cellular functions correlated to differentially expressed proteins. Some of the identified proteins found differentially expressed in gal-3H64 respect to gal3P64 variants, belong to chaperones class as HSPA6, HSPAB1 and to actin-binding protein class, as the calponin 3. IPA analysis suggested the involvement of cancer-related upstream regulators such as TP53, MYCN, and HIF1A1. For the first time, rs4644 SNP variants in thyroid cell were disclosed by a proteomic analysis approach to cause cancer-related molecular changes in non-malignant thyroid cells. [1] Jendrzejewski, J.P.; Sworczak, K.; Comiskey, D.F.; de la Chapelle A. Endokrynol Pol. (2019), 70, 423- 429. [2] Saenko, V.A.; Rogounovitch, T.I. Endocrinol Metab., (2018), 33,164-174. [3] Corrado, A.; Aceto, R.; Silvestri, R.; Dell'Anno, I.; Ricci, B.; Miglietta, S.; Romei, C.; Giovannoni, R.; Poliseno, L.; Evangelista, M.; Vitiello, M.; Cipollini ,M.; Garritano ,S.; Giusti ,L.; Zallocco, L.; Elisei ,R.; Landi, S.; Gemignani, F. Thyroid. , (2021), doi: 10.1089/thy.2020.0366