DLK1-DIO3 region as a source of tumor suppressor miRNAs in papillary thyroid carcinoma

Abstract

Background: In previous studies, we demonstrated the downregulation of several miRNAs from the DLK1-DIO3 genomic region in papillary thyroid carcinoma (PTC). Due to the large number of miRNAs within this region, the individual contribution of these molecules to PTC development and progression remains unclear.

Objective: In this study, we aimed to clarify the contribution of DLK1-DIO3-derived miRNAs to PTC.

Methods: We used different computational approaches and in vitro resources to assess the biological processes and signaling pathways potentially modulated by these miRNAs.

Results: Our analysis suggests that, out of more than 100 mature miRNAs originated from the DLK1-DIO3 region, a set of 12 miRNAs accounts for most of the impact on PTC development and progression, cooperating to modulate distinct cancer-relevant biological processes, such as cell migration, extracellular matrix remodeling, and signal transduction. The restoration of the expression of one of these miRNAs (miR-485-5p) in a BRAFT199A-positive PTC cell line impaired proliferation and migration, suppressing the expression of GAB2 and RAC1, validated miR-485-5p targets.

Conclusions: Overall, our results shed light on the role of the DLK1-DIO3 region, which harbors promising tumor suppressor miRNAs in thyroid cancer, and open prospects for the functional exploration of these miRNAs as therapeutic targets for PTC.

Keywords: DLK1-DIO3 region; MicroRNA; Papillary thyroid cancer; miR-485–5p.

Fig. 1

Abnormal expression of DLK1-DIO3-derived miRNAs in cancer and target prediction in papillary thyroid carcinoma. (a) The miRNAs from the DLK1-DIO3 region are deregulated in different types of cancer. The mean fold change between tumor and non-tumor samples for each miRNA in each cancer type is represented in the heatmap on the left. The statistical significance is represented in the heatmap on the right. The miRNA expression data were downloaded from the ENCORI database, which compiles data from the TCGA project. (b) The DLK1-DIO3-derived miRNAs were ranked according to the total number of predicted targets involved in cancer-related processes. (c) Scheme of the computational strategy adopted for identification of DLK1-DIO3-derived miRNAs with tumor suppressor potential.

Fig. 2

DLK1-DIO3-derived miRNAs cooperate to modulate cancer-related processes in PTC. (a) Gene set enrichment analysis of the list of the predicted targets of all miRNAs in the DLK1-DIO3 region compared to the list of predicted targets of the top 12 miRNAs combined and individually. miRNAs with similar enrichment patterns were marked as group 1 (miR-495–3p, miR-485-p, miR-665) and 2 (miR-539–5p, miR-543, miR-381–3p, miR-300, miR-544a, miR-432–5p). (b) Venn diagrams show a small number of common targets between miRNAs in groups 1 and 2. The mature sequences of the miRNAs in the diagrams are shown at the bottom, with seed sequences in bold. (c) Network of protein-protein interactions between predicted targets of miRNAs from group 1. The color code represents the targeting of each node by one or more miRNAs. The network was built using STRING protein in Cytoscape 3.7.1. Confidence score = 0.8 and no additional interactions were allowed.

Fig. 3

miR-485–5p as a candidate tumor suppressor in PTC. (a) miR-485–5p downregulation in human thyroid cancer samples from miRNA-seq data (TCGA project). Mean expression of miR-485–5p in 437 PTC samples versus 59 normal thyroid tissues. (b) “Waterfall-plot” graph of miR-485–5p expression in 59 PTC samples compared to matched normal thyroid samples from miRNAseq data (TCGA project). Pink bars represent upregulation and blue bars represent downregulation. Hatched bars represent BRAFT1799A-positive samples. (c) Functional enrichment of miR-485–3p predicted targets. GO categories relevant to this study are shown in colors. Dashed line marks P-value = 0.05. (d) miR-485–5p is downregulated in PTC-derived cell lines compared to a normal thyroid follicular immortalized cell line. For each cell line 1 × 10⁵ cells were cultivated for 72 h, total RNA was extracted and cDNA was synthesized. (e) Two PTC cell lines (TPC-1 and BCPAP) were transfected with miR-485–5p-expressing plasmid or transfected with the empty vector. For each cell line, the transfectants with higher miR-485–5p expression were selected for further analysis. (f) TPC-1 and BCPAP cells, transfected with pMSCV-485 plasmid or the empty vector, were collected 24, 48, and 72 h after seeding, fixed, and counted. In (d) and (e)RNU6B gene was used as endogenous control. The y-axis shows values in arbitrary units (a.u.). Data is presented as the average of three independent experiments and bars represent the standard deviation. P ≤ 0,01(**). R.P.M: reads per million.

Fig. 4

miR-485–5p suppresses migration and invasion of PTC cells in vitro. (a) TPC-1 and BCPAP cells, overexpressing miR-485–5p or not, were cultured until near total confluence and a lesion was performed on the monolayer using a pipette tip. Photographs were taken at times 0, 16, and 24 h (N = 3). The BCPAP 24 h time point on the graph has no column due to the complete wound closure. (b) For the migration assay, TPC-1 and BCPAP cells, overexpressing miR-485–5p or not, were seeded in the upper compartment of modified Boyden chambers. For the invasion assay, the chambers were covered with a commercial reconstitutable extracellular matrix. Migrated/invaded cells at the lower chamber compartment were fixed, stained, and photographed after 12 h. Data is presented as one representative experiment (from three independent experiments) and bars represent the standard deviation. P ≤ 0,01(**). 40x magnification.

Fig. 5

GAB2, RAC1, and ICAM1 as targets of miR-485–5p in PTC. (a-c, Top) Mean expression of GAB2, RAC1, or ICAM1 in 437 PTC samples versus 59 normal thyroid samples from RNA-seq (TCGA project) and respective correlation with clinical attributes according to the TCGA project. (N = 388); (R.P.M) reads per million; (d, e) Total RNA was extracted from TPC-1 and BCPAP cells transfected with pMSCV-485 plasmid or the empty vector and used for cDNA synthesis. Expression of GAB2, RAC1, ICAM1, TWIST1, SNAI1, SNAI2, ZEB1 and ZEB2 was quantified by qPCR. RPL19 gene was used as endogenous control. The y-axis shows values in arbitrary units (a.u). Data is presented as the average of three independent experiments and bars represent the standard deviation. P ≤ 0,05 (*), P ≤ 0,001 (***).