[Frontiers in Bioscience, Landmark, 25, 564-576, Jan 1, 2020]

Silencing NEAT1 suppresses thyroid carcinoma via miR-126/NEAT1/VEGFA axis

Weiwei Zeng1, Yan Lin1, Hai Lin, Xuemei Wu1

1Department of Endocrinology, Rui’an People’s Hospital and The Third Affiliated Hospital of Wenzhou Medical University, rui’an, Zhejiang, 325200, P. R. China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Material and methods
    3.1. Reagents
    3.2. Cell culture
    3.3. Cell transfection
    3.4. qPCR
    3.5. Luciferase reporter assay
    3.6. BrdU labeling and staining
    3.7. Transwell culture
    3.8. Scratch test
    3.9. Flow cytometry
    3.10. Western blotting
    3.11. RNA Binding-Protein Immunoprecipitation (RIP) assay
    3.12. Pull down assay
    3.13. Establishment of xenograft mouse model
    3.14. Immunohistochemistry
    3.15. Statistic analysis
4. Results
    4.1. NEAT1 is highly expressed in thyroid cancer cells and knockdown of NEAT1 inhibits cell growth and promotes cell apoptosis
    4.2. Knockdown of NEAT1 inhibits migration and epithelial-mesenchymal transition (EMT)
    4.3. miR-126 targets and negatively regulates NEAT1
    4.4. miR-126 regulates VEGFA expression, cell growth and motility and apoptosis
    4.5. Silencing NEAT1 down-regulates VEGFA level, increases apoptosis and inhibits tumor cell growth in vivo
5. Discussion
6. Acknowledgments
7. References

1. ABSTRACT

The incidence of papillary thyroid carcinoma (PTC) has steadily increased over the recent years, making this cancer a common malignant tumor world-wide. We tested the hypothesis that Nuclear Enriched Abundant sh-NEAT1 knock-down ranscript 1 (NEAT1) is involved in the pathogenesis of PTC in vitro and in vivo. We show that NEAT1 is highly expressed in Papillary Thyroid Carcinoma cell line (PTC-1) and anaplastic thyroid cancer cell line (SW1736) as compared with the human thyroid follicular epithelial cell line (Nthy-ori 3-1). shRNA knockdown of NEAT1 led to the inhibition of cell growth, invasion, migration and Epithelial to Mesenchymal Transition (EMT) of thyroid cancer cells. This treatment increased the rate of apoptosis in SW1736 cells. Silencing of NEAT1 increased the level of its regulator, miRNA-126 and down-regulated VEGFA that sets the density of tumor vasculature. Administrtation of sh-NEAT1 also inhibited tumor growth in vivo, increased the miRNA-126 level and down-regulated VEGFA. Taken together, these results indicate that silencing NEAT1 suppresses thyroid carcinoma via miR-126/NEAT1/VEGFA axis.

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Abbreviation: TC, thyroid carcinoma; PTC, Papillary thyroid carcinoma; ATC, Anaplastic thyroid cancer; NEAT1, Nuclear Enriched Abundant Transcript 1; lncRNAs; long-chain non-coding RNAs; EMT, epithelial-mesenchymal transition

Key Words: Thyroid carcinoma, Nuclear Enriched Abundant Transcript 1, miR-126, tumorigenesis, VEGFA

Send correspondence to: Xuemei Wu, Department of Endocrinology, Rui’an People’s Hospital and The Third Affiliated Hospital of Wenzhou Medical University, Jiyun mountain road, Anyang street, Ruian, Zhejiang, 325200, P. R. China, Tel: 86- 13906646000, Fax: 0577-65866339, E-mail: z53xb5zj@sina.com