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Understanding the dosage-dependent role of Dicer1 in thyroid tumorigenesis

ABSTRACT: Multiple studies suggest Dicer1 as a haploinsufficient tumor suppressor gene: while the loss of one allele promotes tumorigenesis, the complete loss of Dicer1 prevents tumor formation. To study the impact of Dicer1 partial or total loss in papillary thyroid carcinoma cells in vitro, we generated stable Dicer1 (+/-) cell lines by CRISPR-Cas9 from TPC1 cell line. No Dicer1 (-/-) cell lines could be generated. Therefore, siRNA against Dicer1 was transfected into Dicer1 (+/-) cell lines to further decrease its expression. RNA sequencing and transcriptomic analysis revealed alterations in proliferation, cell cycle and cell locomotion. BrdU staining revealed a slow-down of the cell cycle, with lower percentages of cells in S-phase and higher percentages of cells in G0-G1-phase. Furthermore, transwell invasion and migration assays showed a decrease of invasive and migrating cells following transfection of Dicer1 siRNA. These results were confirmed in two supplementary thyroid cell lines, BCPAP and H-Tori3. Additionally, to achieve high protein levels, we performed Dicer1 plasmid transfection and we observed reduced proliferation and invasion as well as increased apoptosis. Globally, our results allow us to better understand the function of Dicer1 in thyroid cancer tumorigenesis and suggest Dicer1 as an attractive target for novel therapeutic strategies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE273129 | GEO | 2024/10/09

REPOSITORIES: GEO

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Project description:MicroRNAs (miRNAs) are a class of endogenous, non-coding RNAs that mediate post-transcriptional gene silencing by inhibiting mRNA translation and promoting mRNA decay. DICER1, an RNAse III endonuclease encoded by Dicer1, is required for processing short 21-22 nucleotide miRNAs from longer double-stranded RNA precursors. Here, we investigate the loss of Dicer1 in mouse postnatal male germ cells to determine how disruptions in the miRNA biogenesis pathway may contribute to infertility. Reduced levels of Dicer1 transcripts and DICER1 were confirmed in germ cell knock-out (GCKO) testes by postnatal day 18 (P18). Compared to wild-type (WT) at 8 weeks, GCKO males had no change in body weight, yet showed significant reductions in testis mass and sperm number. Histology and fertility tests confirmed spermatogenic failure in GCKO males. Array analyses at P18 showed 96% of miRNA genes were down-regulated and 37% of protein-coding genes were differentially expressed in GCKO testes. Interestingly, we observed preferential overexpression of genes on the sex chromosomes in GCKO testes, with more than 80% of the genes overlapping those proposed to undergo meiotic sex chromosome inactivation (MSCI) in the germ cells. Compared to WT, GCKO mice showed higher percentages of cells at early meiotic stages (leptotene and zygotene) but lower percentages at later stages (pachytene, diplotene and metaphase I), providing evidence that deletion of Dicer1 leads to disruptions in meiotic progression. Furthermore, we observed fewer elongating spermatids with proper translational activation of transition protein 2 (Tnp2), protamine 1 and 2 (Prm1 and Prm2) in GCKO testes after step 12-14. Therefore, deleting Dicer1 in early postnatal germ cells causes misregulation of transcripts encoded by genes on the sex chromosomes, impairs meiotic progression and post-meiotic translational control and results in spermatogenic failure and infertility. Total RNA, including miRNAs, were purified from a total of six individual mouse samples. The tissue collected was obtained from wild-type (control; n=3) and Dicer1 germ cell knockout (mutant; n=3) testes on P18.

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Understanding the dosage-dependent role of Dicer1 in thyroid tumorigenesis

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