The use of microRNA inhibitors to restore the expression of the tumor suppressor, the SLC5A8 gene, and the determination of their applicability in the treatment of papillary thyroid cancer.

Aim of the research / research hypothesis: Thyroid neoplasms are the most common group of endocrine neoplasms, and their number is constantly increasing. Papillary thyroid cancer (PTC) is the most common, accounting for 85% of the total. The basis of its treatment is surgical resection of the thyroid gland with regional lymph nodes and complementary therapy consisting of the administration of radioactive iodine 1131. Some patients, however, are resistant to therapy with radioactive iodine. Due to the limited effectiveness of other forms of treatment, e.g. teleradiotherapy, chemotherapy, these patients die because of the aggressive course of the disease. Hence, it is necessary to search for new therapies. The protein product of the SLC5A8 gene was originally identified as an iodide transporter present in the apical membrane of thyrocytes and therefore called AIT (Apical Iodide Transporter). It is also a tumor suppressor and its expression is reduced in many cancers, including PTC. Most studies on its suppressor function concern the large intestine and are based on the ability of AIT to transport short-chain fatty acids. Only a few studies mention the modulation by SLC5A8 of other genes involved in the tumorigenesis process, and these are genes as fundamental as TP53, FASL, FASR, Bcl-2, TRAIL, TRAILR1 and 2, BIRC5 It is therefore surprising that no one has made the changes induced by SLCSA8 overexpression at the level of the whole transcriptome. There are only a few studies on the regulation of SLC5A8 expression, which directed our attention to the role of microRNAs in this process. MicroRNAs (miRNAs) are about 22 nucleotide non-coding RNA molecules that inhibit gene expression by binding to a complementary sequence in their transcripts. In our preliminary research we have shown that miR-181a-5β, -182-5p and -494 regulate SLC5A8 expression, and their increased level in PTC can lead to a decrease in SLCSA8 expression. We also showed the influence of microRNAs on the radioactive iodine transport process. Changes in the expression of microRNAs can be modulated by specific inhibitors, therefore we confirmed that transfection with a synthetic miR-181a-5p inhibitor increases the expression of SLC5A8 mRNA and we hypothesized that by inhibiting the activity of the identified microRNAs we would increase the expression of SLC5A8, which will lead to increased accumulation of radioactive iodine and to a change in the level of genes regulated by SLC5A8, which may increase the effectiveness of anti-cancer therapies.The aim of the project is to use specific microRNA inhibitors to restore the proper level of SLC5A8 in cells derived from thyroid cancer. We will conduct a double analysis of the effect of increased SLC5A8 expression on cell physiology: using next-generation sequencing, we will determine the total transcripts of cell lines in which the action of selected microRNAs will be inhibited (analysis of potential cytotoxicity of inhibitors). Next, we will examine the total transcripts of cell lines in which we overexpress the SLCSA8 gene (analysis of the role of SLC5A8 in the regulation of gene expression). Thanks to this analysis, we will identify the pathways in which SLC5A8 exerts its suppressor role. MiR-181a-5p, -182-5p and -494-3p are also overexpressed in other neoplasms, so we can call them onkomiRami. Therefore, it is even more justified to study the influence of their inhibition on changes taking place at the level of the entire transcriptome. The next step will be to compare gene expression changes caused by SLC5A8 overexpression and microRNA inhibition. Not only will this provide a list of target genes, but it will also bring us closer to the question of whether tissue-specific inhibition of these microRNAs could find therapeutic application in the future. Research method / methodology used: During the project implementation, we will create a plasmid expressing the coding sequence of the SLC5A8 gene and the so-called sponge for miR-181a-5p, -182-5p and -494, expressing a transcript with sequence complementary to 3 microRNAs, acting as a specific inhibitor of these molecules. Optimal transfection conditions will be determined by measuring the expression of SLC5A8 and at least one of its target genes described by using real-time PCR. After determining the optimal protocol, we will isolate RNA from the cells, prepare a cDNA library and subject it to next-generation sequencing (NGS). We will create lists of genes with altered expression due to SLC5A8 overexpression and (separately) microRNA silencing. We will then compare these lists with each other to determine changes caused by gene overexpression, changes caused by silencing of selected microRNAs, and consequently we will determine the potential utility of microRNA inhibitors in restoring SLC5A8 expression in PTC cancer. The impact of the expected results on the development of science, civilization, society: This will be the first project to characterize the influence of SLC5A8 on the expression of other genes at the level of the entire transcriptome. We will also determine the possibility of restoring its expression by microRNA inhibition. Importantly, thanks to the use of the NGS technique, we can identify other target genes useful from a therapeutic point of view for these microRNAs. We will compare the changes caused by overexpression of the SLC5A8 gene with those caused by silencing of selected microRNAs, which also regulate other target genes, including those involved in tumor suppression. Thanks to this, we will determine the potential utility of silencing selected microRNAs in patients suffering from papillary thyroid cancer.