Dataset Information

The Suppression of miR-199a-3p by Promoter Methylation Contributes to Papillary Thyroid Carcinoma Aggressiveness by Targeting RAP2a and DNMT3a.

ABSTRACT:

Background

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. 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.

Methods

qRT-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.

Results

Our 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.

Conclusion

Our 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.

SUBMITTER: Wu F

PROVIDER: S-EPMC7750465 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The Suppression of miR-199a-3p by Promoter Methylation Contributes to Papillary Thyroid Carcinoma Aggressiveness by Targeting RAP2a and DNMT3a.

Wu Feng F Lin Xiao X Shan Su-Kang SK Li Fuxingzi F Xu Feng F Zhong Jia-Yu JY Guo Bei B Zheng Ming-Hui MH Wang Yi Y Mo Zhao-Hui ZH Yuan Ling-Qing LQ

Frontiers in cell and developmental biology 20201207

<h4>Background</h4>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. 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 <i>in vivo</i> and <i>in vitro</i>.<h4>Methods</h4>qRT-PCR and Western blot assays were used to determine t ...[more]

PMID: 33365310

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Project description:Papillary thyroid carcinoma (PTC) is the fastest-growing disease caused by numerous molecular alterations in addition to previously reported DNA mutations. There is a compelling need to identify novel transcriptomic alterations that are associated with the pathogenesis of PTC with potential diagnostic and prognostic implications. Methods: We gathered and compared 242 expression profiles between paired PTC and adjacent normal tissues and identified and validated the coding and long non-coding RNAs (lncRNAs) associated with the extrathyroidal extension (ETE) of 655 PTC patients in two independent cohorts, followed by predicting their interactions with drugs. Co-expression, RNA interaction, Kaplan-Meier survival and multivariate Cox proportional regression analyses were performed to identify dysregulated lncRNAs and genes that correlated with clinical outcomes of PTC. Alternative splicing (AS), RNA circularization, and editing were also compared between transcriptomes to expand the repertoire of molecular alterations in PTC. Results: Numerous genes related to cellular microenvironment and steroid hormone response were associated with the ETE of PTC. Drug susceptibility predictions of the expression signature revealed two highly ranked compounds, 6-bromoindirubin-3'-oxime and lovastatin. Co-expression and RNA interaction analysis revealed the essential role of lncRNAs in PTC pathogenesis by modulating extracellular matrix and cell adhesion. Eight genes and two novel lncRNAs were identified that correlated with the aggressive nature and disease-free survival of PTC. Furthermore, this study provided the transcriptome-wide landscape of circRNAs in PTC and uncovered dissimilar expression profiles among circRNAs originating from the same host gene, suggesting the functional complexity of circRNAs in PTC carcinogenesis. The newly identified AS events in the SERPINA1 and FN1 genes may improve the sensitivity and specificity of these diagnostic biomarkers. Conclusions: Our study uncovered a comprehensive transcriptomic signature associated with the carcinogenesis and aggressive behavior of PTC, as well as presents a catalog of 10 potential biomarkers, which would facilitate PTC prognosis and development of new therapeutic strategies for this cancer.

Project description:A significant proportion of patients with papillary thyroid cancer (PTC) present with extrathyroidal extension (ETE) and lymph node metastasis (LNM). However, the molecular mechanism of tumor invasiveness in PTC remains to be elucidated. The aim of this study is to understand the role of Hedgehog (Hh) signaling in tumor aggressiveness in patients with PTC. Subjects were patients who underwent thyroidectomy from 2012 to 2013 in a single institution. Frozen or paraffin-embedded tumor tissues with contralateral-matched normal thyroid tissues were collected. Hh signaling activity was analyzed by quantitative RT-PCR (qRT-PCR) and immunohistochemical (IHC) staining. Datasets from Gene Expression Omnibus (GEO) (National Center for Biotechnology Information) were subjected to Gene Set Enrichment Analysis (GSEA). BRAFT1799A and telomerase reverse transcriptase promoter mutation C228T were analyzed by direct sequencing. Among 137 patients with PTC, glioma-associated oncogene homolog 1 (GLI1) group III (patients in whom the ratio of GLI1 messenger ribonucleic acid (mRNA) level in tumor tissue to GLI1 mRNA level in matched normal tissue was in the upper third of the subject population) had elevated risk for ETE (odds ratio [OR] 4.381, 95% confidence interval [CI] 1.414-13.569, P = 0.01) and LNM (OR 5.627, 95% CI 1.674-18.913, P = 0.005). Glioma-associated oncogene homolog 2 (GLI2) group III also had elevated risk for ETE (OR 4.152, 95% CI 1.292-13.342, P = 0.017) and LNM (OR 3.924, 95% CI 1.097-14.042, P = 0.036). GSEA suggested that higher GLI1 expression is associated with expression of the KEGG gene set related to axon guidance (P = 0.031, false discovery rate < 0.05), as verified by qRT-PCR and IHC staining in our subjects.GLI1 and GLI2 expressions were clearly related to aggressive clinicopathological features and aberrant activation of GLI1 involved in the axon guidance pathway. These results may contribute to development of new prognostic markers, as well as novel therapeutic targets.

Dataset Information

The Suppression of miR-199a-3p by Promoter Methylation Contributes to Papillary Thyroid Carcinoma Aggressiveness by Targeting RAP2a and DNMT3a.

Background

Methods

Results

Conclusion

Publications

The Suppression of miR-199a-3p by Promoter Methylation Contributes to Papillary Thyroid Carcinoma Aggressiveness by Targeting RAP2a and DNMT3a.

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