||Dengue virus infection in thyroid cancer cells
||Department of Microbiology & Immunology
cancer stem cells
receptors for DENV
Thyroid cancer is the most frequently diagnosed endocrine cancer and causes more deaths than all other endocrine cancers combined. Most thyroid cancers represent good prognosis after surgery, but sometimes the recurrence and metastasis are still occurring. The existence of cancer stem cells (CSCs) has been found to be correlated with the recurrence of thyroid cancers. The properties of CSCs in thyroid cancers have not been thoroughly discussed before. Our recent works on bone marrow and hepatocellular carcinoma (HCC) have shown that the CSCs population can be infected by dengue virus (DENV) efficiently. In the preliminary data, the tissues from tumorous or non-tumorous of thyroid cancer patients were utilized in the current research. Cells obtained in these tissues were subjected to DENV infection as well as FACS analysis was applied to evaluate the levels of cancer stem cell populations in these tissues. The result showed that the cells from thyroid tumors were highly permissive to DENV infection, and the viral titers were significantly higher than those from the non-tumor tissues. Interestingly, we found the cells from thyroid tumor tissues had two different infected performance, permissive and non-permissive. However, the expressions of CSC populations in DENV permissive and non-permissive group were no different. Therefore, we wanted to investigate the characteristics of CSCs in thyroid cancer during DENV infection. Previously, our lab found that CD44 played an important role as receptor for DENV infection in skin stem cells. We then evaluated the expression of CD44 in thyroid CSCs prior to DENV infection. The result suggested that the populations of stem cells in tumor tissues with CD44 were significantly positively correlated with DENV titers. Hence, we used hyaluronic acid (HA), a natural ligand for CD44, to do CD44 blocking assay in thyroid cancer cells. The results showed that blocking of CD44 would not affect the viral titers during DENV infection. Therefore, we speculated that the correlation may only specific in CSCs. However, the primary cells which isolated from thyroid cancer specimen were very rare. In order to testify the CD44 plays a role in thyroid cancer stem cells during DENV infection, we used MDA-T85 cells that is a thyroid cancer cell line for the purpose. MDA-T85 cells expressed high levels of CD44 by FACS analysis, followed by blocking assay with HA. The results suggested that blocking of CD44 in whole cells of MDA-T85 cells would not affect the virus titers during DENV infection, but an opposite outcome of CSCs of MDA-T85 cells was observed. To further confirm the role of CD44 involved in DENV infection, we used siRNA sequences to knockdown CD44 gene in MDA-T85 cells before DENV infection. The results displayed that silencing of CD44 by siRNA could suppress DENV infection in both whole cells and CSCs of MDA-T85 cells. In summary, the primary cells of thyroid cancer specimens are permissive to DENV and CD44 may be a potential receptor for DENV infection in thyroid cancer cell.
Table of contents VII
List of Figures X
Abbreviation Index XI
1. Thyroid cancer 1
2. Treatment resistance and prognosis in thyroid cancer 1
3. Cancer stem cells (CSCs) effect Metastasis and prognosis 2
4. The origin of the CSCs 3
5. The common markers of CSCs 3
6. Dengue virus (DENV) Epidemiology and pathophysiology 4
7. Receptors for DENV 5
8. The structure and functions of CD44 5
9. The role of CD44 in cancer cells 6
10. Hypothesis 7
Materials and Methods 8-25
A. Materials 8
1. Cell lines and virus 8
2. Antibody (thyroid cancer panel) 8
3. Antibody (isotype) 8
4. Magnetic beads 9
5. Medium and Reagent 9
6. Plastic and glass equipments 11
7. Instruments and machines 13
8. Primer 14
9. siRNA 14
10. Ingredients in buffer and medium 15
B. Methods 17
1. Cell line culture (BHK-21, Vero and MDA-T85) 17
2. Thyroid cancer specimens 17
3. Multicolor FACS analysis 18
4. Dengue virus expansion 18
5. Ex vivo and in vitro infection of DENV 19
6. Magnetic Bead isolation 20
7. Ex vivo and in vitro blocking assay of Hyaluronic acid (HA) 20
8. RNA interference assay with siRNA 21
9. qPCR 22
10. Western blot 23
11. Plaque assay 24
12. Statistical analysis 25
1. Tumor cells of thyroid cancer patients were highly permissive to DENV infection. 26
2. Cell populations of cancer stem cells were no difference between DENV permissive and non-permissive groups. 27
3. The percentage of CD44 in stem cells of tumor tissues was significantly positively correlated with DENV titers 27
4. Blocking of CD44 with HA did not inhibit the infection of DENV in thyroid cancer cells. 28
5. MDA-T85, a thyroid cancer cell line containing high percentage of CD44+ cell population, were permissive to DENV infection. 29
6. Blocking CD44 with HA could inhibit the infection of DENV in cancer stem cell of MDA-T85 cells. 29
7. Inhibition of CD44 by siRNA could suppress DENV infection in both whole cells and cancer stem cells of MDA-T85 cells. 30
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