Abstract 22P
Background
Differentiated thyroid cancers (DTC) are characterized by poorly characterized hereditary susceptibility. Some germline variants augmenting DTC risk (in particular rs11693806) locate in a long non-coding RNA (lncRNA) gene termed disrupted in renal cancer 3 (DIRC3). To date no studies have evaluated the biological function of DIRC3 lncRNA in DTC.
Methods
DIRC3 expression and rs11693806 genotype was tested in 75 patient-matched DTC/normal thyroid tissue pairs. Public RNA-seq data (TCGA) was used to test correlations between expression of DIRC3 and protein-coding genes in DTC. We evaluated DIRC3 expression, its subcellular localization and genotype in a panel of DTC cell lines. Antisense oligonucleotides were used to silence DIRC3; the effect on expression of putative target gene(s), cell viability, migratory potential and invasiveness was tested. The significance of rs11693806 locus was evaluated with CRISPR.
Results
Study performed in DTC/normal thyroid tissue pairs indicates that DIRC3 is downregulated in cancer (p<0.01). Lower DIRC3 levels were noted in DTC manifesting with capsular invasion or metastasis. TCGA data revealed that DIRC3 is co-expressed with IGFBP5 (insulin-like growth factor binding protein 5), a nearby cancer suppressor. The gene co-expression was confirmed in our material (Spearman 0.73). In cancer cell lines, DIRC3 lncRNA preferentially localizes in nuclear cellular fraction. DIRC3 silencing induced IGFBP5 downregulation, and augmented cancer cell viability, migration and invasiveness. A strong chemotactic response to insulin-like growth factor 1 (IGF-1) was also noted. Using CRISPR we produced isogenic MDA-T32 cell line clones harboring small monoallelic deletions in the rs11693806 locus. We observed ∼2-fold reduction in IGFBP5 mRNA in the edited cells as compared to the parental cell line. Transcriptome changes (RNA-seq) induced by DIRC3 silencing will be tested shortly.
Conclusions
DIRC3 emerges as a putative thyroid cancer suppressor producing a cis-acting lncRNA that governs expression of IGFBP5, a modulator of IGF-1 signaling pathway. We propose an interplay between the germline variants, DIRC3 and IGF-1 signaling as a factor that orchestrates thyroid carcinogenesis; the hypothesis is being verified.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
The authors.
Funding
Narodowe Centrum Nauki (National Science Centre, Poland).
Disclosure
All authors have declared no conflicts of interest.