Abstract
BackgroundIt was previously demonstrated that miR-199a-3p plays an important role in tumor progression; especially, its down-regulation in papillary thyroid cancer (PTC) is associated with cancer cell invasion and proliferation. In the present report, we investigated the mechanism involved in the down-regulation of miR-199a-3p in PTC and how miR-199a-3p regulates PTC invasion both in vivo and in vitro.MethodsqRT-PCR and Western blot assays were used to determine the expression of the investigated genes. Bisulfite sequencing PCR was used to investigate miR-199a-3p methylation. The functions of miR-199a-3p were investigated by a series of in vitro and in vivo experiments.ResultsOur results showed hypermethylation of the miR-199a-3p promoter, which resulted in decreased miR-199a-3p expression both in PTC cell lines and PTC tissues. DNA-methyltransferase 3a (DNMT3a), a target gene of miR-199a-3p, was increased both in PTC cell lines and PTC tissues, while 5-aza-2′-deoxycytidine (methyltransferase-specific inhibitor) or knock-down using DNMT3a Small-Interfering RNA could restore the expression of miR-199a-3p, and the over-expression of miR-199a-3p could decrease the expression of DNMT3a; this suggests that miR-199a-3p/DNMT3a constructs a regulatory circuit in regulating miR-199a-3p/DNMT3a expression. Moreover, gain- and loss-of-function studies revealed that miR-199a-3p is involved in cancer cell migration, invasion, and growth. Meanwhile, we found that RAP2a was also a direct target of miR-199a-3p, which might mediate the tumor-growth-inhibiting effect of miR-199a-3p. To further confirm the tumor-suppressive properties of miR-199a-3p, stable overexpression of miR-199a-3p in a PTC cell line (BCPAP cells) was xenografted to athymic BALB/c nude mice, resulting in delayed tumor growth in vivo. In clinical PTC samples, the expression of RAP2a and DNMT3a was increased significantly, and the expression of RAP2a was inversely correlated with that of miR-199a-3p.ConclusionOur studies demonstrate that an epigenetic change in the promoter region of miR-199a contributes to the aggressive behavior of PTC via the miR-199a-3p/DNMT3a regulatory circuit and directly targets RAP2a.
Highlights
DNA methylation, which involves the translocation of a methyl group to the number 5 carbon of the cytosine ring in a CpG dinucleotide, plays a crucial role in regulating gene expression in cancer development (Raynal et al, 2012)
Our data imply that an epigenetic change in the promoter region of miR-199a contributes to the aggressive behavior of papillary thyroid cancer (PTC) via a regulatory circuit involving miR-199a-3p/DNMT3a and targets RAP2a directly
We found an obvious increase in the expression of DNMT3a by IHC in PTC tissues in comparison with normal thyroid tissues (Figures 1C,D)
Summary
DNA methylation, which involves the translocation of a methyl group to the number 5 carbon of the cytosine ring in a CpG dinucleotide, plays a crucial role in regulating gene expression in cancer development (Raynal et al, 2012). Transcriptional modulation of tumor suppressor genes (TSGs) or oncogenes by DNA methylation of promoter-associated CpG islands is a hallmark of carcinogenesis (Fernandez et al, 2012). It has been reported that miR-199a-3p plays tumor suppressor functions in the carcinogenesis of PTC (Liu et al, 2017), and miR-199a-3p was generally hypermethylated in malignant testicular tumors (Cheung et al, 2011; Gu et al, 2013; Chen et al, 2014) and ovarian cancer (Deng et al, 2017), which correlated with its down-regulation. It was previously demonstrated that miR-199a-3p plays an important role in tumor progression; especially, its down-regulation in papillary thyroid cancer (PTC) is associated with cancer cell invasion and proliferation. We investigated the mechanism involved in the down-regulation of miR-199a-3p in PTC and how miR-199a-3p regulates PTC invasion both in vivo and in vitro
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