검색 상세

Transcription Factor HOXA9 is Linked to the Calcification and Invasion of Papillary Thyroid Carcinoma

초록/요약

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer, representing 75 to 85 percent of all thyroid cancer cases. Calcification is one of the important characteristic feature for the diagnosis of papillary thyroid carcinoma. Runt-related transcription factor 2 (RUNX2), a master transcription factor associated with osteogenic differentiation, is reportedly related to PTC calcification and invasiveness. However, its regulatory role in this process is somewhat uncharacterized. Here, I attempted to identify gene that regulates RUNX2 and to clarify its function in PTC calcification and carcinogenesis. The expression of RUNX2-upstream genes was evaluated by semi-quantitative reverse-transcriptase PCR and quantitative real-time PCR in normal thyroid cell line Nthy-Ori 3-1 and the PTC cell lines TPC1 and BHP10-3. After cloning the RUNX2 promoter, luciferase assay and chromatin immunoprecipitation assay were performed with the primary candidate gene - homeobox family A9 (HOXA9). I found that RUNX2 promoter activity and binding ability were enhanced by HOXA9. Over-expression of HOXA9 was found to enhance alkaline phosphatase activity, mineralization, and the ability of in vitro cell migration and invasion, whereas downregulation of HOXA9 showed the opposite effects. To confirm the results from in vitro promoter, mineralization and carcinogenesis assays, I evaluated haematoxylin and eosin staining in human thyroid cancer tissue specimens from thyroid carcinoma patients and identified immunohistochemistry to verify the expression of RUNX2 and HOXA9 in serial calcified malignant thyroid cancer tissues. Haematoxylin and eosin staining and immunohistochemistry results showed that the expression of RUNX2 and HOXA9 were found in calcified cancer tissues simultaneously. Next, I performed RUNX2 down regulation experiment in the control cell lines and HOXA9 overexpressed system with the normal cell line Nthy-Ori 3-1 and PTC cell lines TPC1 and BHP10-3. Further, mineralization assay, wound healing assay, and transwell assay were performed in these cells. Mineralization were decreased in all RUNX2 downregulated cells, and alkaline phosphatase activity in Nthy-Ori 3-1 and BHP10-3 cells was enhanced in the RUNX2 knockdowned and HOXA9 overexpressed cells compared to the RUNX2 knockdowned in control cells. Furthermore, migration ability was all decreased in RUNX2 knockdowned cells compared to the control cells, and invasion ability was enhanced in the RUNX2 knockdown groups with HOXA9 overexpression compared to that in respective RUNX2 knockdown with control groups. These results indicated that HOXA9, as a positive regulator of RUNX2, can enhance calcification, migration, and invasion in PTC. These data can improve the understanding of the molecular mechanisms of calcification in papillary thyroid carcinoma as well as tumorigenesis.

more

목차

ABSTRACT i
TABLE OF CONTENTS iv
LIST OF FIGURES vi
LIST OF TABLES vii
LIST OF APPENDIX viii
I. INTRODUCTION 1
II. MATERIALS AND METHODS 4
1. Cell culture 4
2. Candidate regulators of RUNX2 4
3. Semi-quantitative reverse-transcriptase PCR and quantitative real-time PCR 10
4. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay 16
5. Plasmids, lentivirus packaging, and stable cell lines 17
6. Western blotting 20
7. Alkaline phosphatase (ALP) assay and Alizarin Red S (ARS) staining 20
8. Wound healing assay 21
9. Invasion assay 21
10. H&E staining and Immunohistochemisty (IHC) assay 22
11. Statistical analysis 23
III. RESULTS 24
1. HOXA9 regulates RUNX2 gene expression 24
2. HOXA9 mediates the calcification of thyroid cells 31
3. HOXA9 is associated with thyroid cell migration and invasion 34
4. HOXA9 and RUNX2 can be simultaneously detected in calcified thyroid cancer tissues 38
5. HOXA9 increase PTC calcification and tumor invasion directly or indirectly via RUNX2 42
IV. DISCUSSION 51
V. CONCLUSION 56
REFERENCES 58
국문요약 82

more
반출 Meta View 목록