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CD151 as a potential molecular marker of cholangiocarcinoma stem cells

Other Titles
 담도암 줄기 세포의 분자표지자로서 가능성 있는 CD151 
Authors
 전태주 
Issue Date
2017
Description
Dept. of Medicine/박사
Abstract
Cancer stem cells are currently hypothesized to be responsible for tumorigenesis, metastasis, recurrence, and chemoresistance in the various types of cancer.
CD151 is a member of the transmembrane 4 superfamily, which enhances cell motility, invasion, and metastasis of cancer cells. In intrahepatic cholangiocarcinoma, overexpression of CD151 associated with c-Met is implicated in invasion and metastasis, which result in poor prognosis. However, there are few studies that focus on CD151 in cholangiocarcinoma, and the role of CD151 in cancer stem cells, including cholangiocarcinoma stem cells, which have hardly been documented.
The aim of this study is therefore to characterize the role of CD151 in cholangiocarcinoma stem cell.
To evaluate cholangiocarcinoma stem cell, the author prepared cancer stem like cells using sphere cultivation (SNU-245, SNU-1196). A cDNA microarray was used to gain up-regulated genes between tumorsphere and biliary carcinoma. The expressions of stemness genes and the proteins related to c-Met and Epithelial Mesenchymal Transition (EMT) were evaluated through RT-PCR and western blot to find if tumorsphere has characteristics of stem cell. Expression of CD151 was compared between adherent cells of SNU-1196 and tumorsphere. To evaluate the role of CD151 in cholangiocarcinoma stem cells, CD151 knockdown was done by CD151 siRNA in SNU-1196 cells and then sphere formation and cytotoxicity assay were performed. In order to evaluate the mechanism of CD151, c-Met, EMT pathway related molecules, β-catenin, and Oct 4 were evaluated through western blot. Proliferation and metastatic capabilities were also evaluated after transfection of CD151 siRNA in SNU-1196.
As a result, CD151 gene was highly up-regulated in both tumorspheres and biliary carcinoma on cDNA microarray. Cultivated tumorsphere had cancer stem cell-like properties, showing highly expressed Notch, Hedgehog, and Wnt pathway related to stem cells in RT-PCR. Expressions of the proteins that related to c-Met, PI3K, EMT pathway, β-catenin, and Oct 4 were more highly increased and CD151 was more highly expressed in tumorsphere than in adherent cells.
After CD151 knockdown, tumorsphere formation was inhibited and chemoresistance was reduced. The expression of the molecules associated c-Met, PI3K, EMT pathways, β-catenin and Oct4 was reduced. Therefore, the characteristics of proliferation and migration were also reduced after transfection of CD151 siRNA in SNU-1196 cells.
The author could suggest that CD151 was more expressed in cholangiocarcinoma stem-like cells and regulated self-renewal, chemoresistance, proliferation and metastatic capabilities. It might interact with c-Met and activate PI3K, EMT pathway, β-catenin, and Oct4. So, it would be a potential cholangiocarcinoma stem cell related marker and therapeutic target in the future.


최근 암줄기 세포가 다양한 암에서 종양형성, 전이, 재발, 항암제내성을 일으킨다고 가정하고 있다.
CD151은 transmembrane 4 superfamily의 하나로 세포의 이동뿐 아니라 암세포의 침습이나 전이에 관여한다. 간내 담도암에서 CD151과 c-Met의 과발현이 암의 침습과 전이와 연관이 있어 불량 예후를 보인다는 연구와 같이 담도암에서 CD151의 역할에 대한 연구는 있었으나 담도암 줄기세포에서의 역할에 대한 연구는 현재 없다. 이에 본 연구에서는 CD151의 담도암 줄기세포에서 역할을 대해서 알아보고자 하였다.
담도암 줄기세포에 대한 평가를 위해 본 연구에서는 2개의 담도암 세포주(SNU-245, SNU-1196)를 이용하여 tumorsphere를 배양하여 담도암 줄기 양 세포를 얻었다. cDNA microarray를 이용하여 tumorsphere와 담도암에서 과발현된 유전자를 얻었다. Tumorsphere가 줄기세포의 특성을 갖는 지를 보기 위해 RT-PCR과 western blot을 통하여 줄기세포 관련 mRNA와 c-Met과 EMT 관련 단백질을 확인하였다. CD151의 발현을 SNU-1196 세포주의 부착 세포와 tumorsphere에서 비교하였다. CD151의 담도암 줄기 세포에서의 역할을 확인하기 위하여 SNU-1196을 CD151 siRNA로 처리하여 CD151 발현을 억제한 후에 sphere formation assay, cytotoxicity assay를 시행하였다. 또한 c-Met, EMT pathway 관련 인자 및 β-catenin, Oct4의 발현을 western blot을 통하여 확인하였으며 proliferation assay 및 migration assay를 시행하였다.
결과적으로 cDNA microarray에서 CD151이 tumorsphere와 담도암 모두에서 과발현됨을 알 수 있었다. 배양된 tumor sphere는 RT-PCR에서 줄기세포와 관련된 Notch, Hedgehog, Wnt 관련 인자들이 과발현되고 있고, western blot에서 c-Met, PI3K, EMT 관련 인자, β-catenin, Oct4의 발현이 증가되어 있어서 암줄기 세포의 특성을 가지고 있었다. CD151은 부착 세포보다 tumors...
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Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Internal Medicine (내과학교실) > 3. Dissertation
Yonsei Authors
Jeon, Tae Joo(전태주)
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/154926
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