Klhl14-AS is a long noncoding RNA expressed since early specification of thyroid bud and is the most enriched gene in the mouse thyroid primordium at E10.5. Here, we studied its involvement in thyroid carcinogenesis by analyzing its expression in cancer tissues and different models of neoplastic transformation. Compared with normal thyroid tissue and cells, Klhl14-AS was significantly downregulated in human thyroid carcinoma tissue specimens, particularly the anaplastic histotype, thyroid cancer cell lines, and rodent models of thyroid cancer. Downregulating the expression of Klhl14-AS in normal thyroid cells decreased the expression of thyroid differentiation markers and cell death and increased cell viability. These effects were mediated by the binding of Klhl14-AS to two miRNAs, Mir182-5p and Mir20a-5p, which silenced Pax8 and Bcl2, both essential players of thyroid differentiation. MIR182-5p and MIR20a-5p were upregulated in human thyroid cancer and thyroid cancer experimental models and their effects on Pax8 and Bcl2 were rescued by Klhl14-AS overexpression, confirming Klhl14-AS as a ceRNA for both Pax8 and Bcl2. This work connects deregulation of differentiation with increased proliferation and survival in thyroid neoplastic cells and highlights a novel ceRNA circuitry involving key regulators of thyroid physiology.Significance: This study describes a new ceRNA with potential tumor suppression activity and helps us better understand the regulatory mechanisms during thyroid differentiation and carcinogenesis.

<div>Abstract<p><i>Klhl14-AS</i> is a long noncoding RNA expressed since early specification of thyroid bud and is the most enriched gene in the mouse thyroid primordium at E10.5. Here, we studied its involvement in thyroid carcinogenesis by analyzing its expression in cancer tissues and different models of neoplastic transformation. Compared with normal thyroid tissue and cells, <i>Klhl14-AS</i> was significantly downregulated in human thyroid carcinoma tissue specimens, particularly the anaplastic histotype, thyroid cancer cell lines, and rodent models of thyroid cancer. Downregulating the expression of <i>Klhl14-AS</i> in normal thyroid cells decreased the expression of thyroid differentiation markers and cell death and increased cell viability. These effects were mediated by the binding of <i>Klhl14-AS</i> to two miRNAs, <i>Mir182-5p</i> and <i>Mir20a-5p</i>, which silenced <i>Pax8</i> and <i>Bcl2</i>, both essential players of thyroid differentiation. <i>MIR182-5p</i> and <i>MIR20a-5p</i> were upregulated in human thyroid cancer and thyroid cancer experimental models and their effects on <i>Pax8</i> and <i>Bcl2</i> were rescued by <i>Klhl14-AS</i> overexpression, confirming <i>Klhl14-AS</i> as a ceRNA for both <i>Pax8</i> and <i>Bcl2</i>. This work connects deregulation of differentiation with increased proliferation and survival in thyroid neoplastic cells and highlights a novel ceRNA circuitry involving key regulators of thyroid physiology.</p>Significance:<p>This study describes a new ceRNA with potential tumor suppression activity and helps us better understand the regulatory mechanisms during thyroid differentiation and carcinogenesis.</p></div>

<p>Supplementary tables S1-S5. Tab S1: Oligonucleo1des used as primers for qRT-PCR; Tab S2: Oligonucleo1des used for cloning in either reporter or expression vectors. Only gene-specific sequences are reported; Tab S3: Klhl-14-AS specific oligonucleo1des used in RNA pull-down experiments; Tab S4: PITA analysis results of miRNA binding sites. The conserved region of Klhl14-AS was analyzed by using PITA algorithm for the presence of binding sites for miRNAs. Only miRNAs predicted to bind on the conserved sequences in all the species are shown; Tab S5: Rat Pax8 and Bcl-2 MREs. PutaZve MREs on the Bcl2 and Pax8 transcripts were idenZfied by searching for rno-miR-182 and rno-miR-20a-5p canonical (7-8- nt) and marginal (6-nt) seed-matched sites using a custom Perl script.</p>

<p>Supplementary figures S1-S7. Fig S1. Klhl14-AS is down-regulated in thyroid cancer in a MAPK-dependent manner; Fig S2. A. Rat Klhl14-AS alternative transcripts as reported by Ensembl (Rnor_6). B, C Klhl14-AS was knocked-down in FRTL-5 using LNA Gapmers targeting the region indicated in A by the dashed box (Khl14-AS LNA) or control LNA (ctr LNA). Klhl14-AS and Ki67 expression were measured by qRT-pcr. D Human KLHL14-AS transcript as reported by Ensembl (GRCh38.p12). E KLHL14-AS was knocked-down in Nthy-ori 3-1 using LNA Gapmers targeting the region indicated in D by the dashed box (KHL14-AS LNA) or control LNA (ctr LNA). KLHL14-AS expression was measured through qRT-pcr. All data are shown as means {plus minus} SD. *** p < 0.001; Fig S3: miR-182-5p and miR-20a-5p Responsive Elements in the highly conserved region of Klhl14-AS; Fig S4: Klhl14-AS does not bind let7a; Fig S5. Pax8 expression is repressed in thyroid transformaRon models; Fig S6. Pax8 or Bcl2 overexpression have no effects on thyroid differenRaRon gene expression; Fig S7. GEPIA-annotated thyroid cancer data.</p>

<p>Supplementary figures S1-S7. Fig S1. Klhl14-AS is down-regulated in thyroid cancer in a MAPK-dependent manner; Fig S2. A. Rat Klhl14-AS alternative transcripts as reported by Ensembl (Rnor_6). B, C Klhl14-AS was knocked-down in FRTL-5 using LNA Gapmers targeting the region indicated in A by the dashed box (Khl14-AS LNA) or control LNA (ctr LNA). Klhl14-AS and Ki67 expression were measured by qRT-pcr. D Human KLHL14-AS transcript as reported by Ensembl (GRCh38.p12). E KLHL14-AS was knocked-down in Nthy-ori 3-1 using LNA Gapmers targeting the region indicated in D by the dashed box (KHL14-AS LNA) or control LNA (ctr LNA). KLHL14-AS expression was measured through qRT-pcr. All data are shown as means {plus minus} SD. *** p < 0.001; Fig S3: miR-182-5p and miR-20a-5p Responsive Elements in the highly conserved region of Klhl14-AS; Fig S4: Klhl14-AS does not bind let7a; Fig S5. Pax8 expression is repressed in thyroid transformaRon models; Fig S6. Pax8 or Bcl2 overexpression have no effects on thyroid differenRaRon gene expression; Fig S7. GEPIA-annotated thyroid cancer data.</p>