Anticancer Effect of Attenuated Salmonella enterica serovar Typhimurium Harboring β2-Syntrophin MicroRNA in Melanoma-Bearing Mice
- 주제(키워드) syntrophin , salmonella , melanoma , miRNA
- 발행기관 고려대학교 대학원
- 지도교수 박용근
- 발행년도 2013
- 학위수여년월 2013. 8
- 학위구분 석사
- 학과 일반대학원 생명과학과
- 세부전공 분자의생명과학 전공
- 원문페이지 65 p
- 실제URI http://www.dcollection.net/handler/korea/000000045308
- 본문언어 영어
- 제출원본 000045764566
초록/요약
β2-Syntrophin (SNTB2) is an adapter protein that organizes the specific intracellular localization of variety of membrane proteins. The protein plays a central role in the regulation of secretory granules via its interaction like insulin secretion. But SNTB2 has not been well known about cancer disease. In previous study, it was found that STNB2 protein of human colon cancer tissue was highly expressed compared to human normal tissue. So, I investigated whether SNTB2 is highly expressed in other cancer tissues. I found that SNTB2 was more expressed in mouse melanoma cancer tissue than in mouse normal skin tissue. To down-regulate of SNTB2 expression, I constructed the attenuated Salmonella enterica serovar Typhimurium expressing SNTB2 microRNA (miRNA). The attenuated S. Typhimurium expressing SNTB2 miRNA can invade specific into melanoma cancer cells and SNTB2 expression of infected melanoma cells was effectively down-regulated. I observed the melanoma cancer cell lysis and melanoma growth suppression in infected melanoma cells with the constructed Salmonella. In addition, the attenuated S. Typhimurium harboring SNTB2 miRNA vector induced apoptosis of melanoma cells and leaded to cell death. When the combination of the attenuated S. Typhimurium and radiation were together treated into melanoma cells, cytotoxicity and apoptosis of melanoma cells increased a little. Furthermore, reactive oxygen species (ROS) was significantly increased in infected cells with the constructed Salmonella. In vivo test, the group of mice treated attenuated S. Typhimurium harboring SNTB2 miRNA showed the suppression of tumor volume in melanoma and prolonged the survival period These results suggest that anticancer therapy by S. Typhimurium harboring SNTB2 miRNA vector is a promising strategy for melanoma models.
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CONTENTS
ABSTRACT ………………………………………………… 1
INTRODUCTION ………………………………………… 3
MATERIALS AND METHODS
Bacterial strains, media and growth conditions ……………… 7
Design & Cloning of miRNA expression vector ……………… 7
Construction of Salmonella strains harboring miRNA vector …… 8
Transfection of Salmonella into cells and radiation …………… 8
Salmonella invasion assay …………………………………… 9
LDH assay ………………………………………………… 10
SR-FLICA (Poly caspases) assay ……………………………… 10
Protection extraction & Immunoblot analysis …………………… 11
Reactive oxygen species (ROS) assay ………………………… 12
Melanoma tumor challenge ………………………… 13
Salmonella inoculation in melanoma-bearing mice ……… 13
RESULTS
Construction of β2-Syntrophin (SNTB2) microRNA (miRNA)
expression vector and confirmation of expression of the constructed
Salmonella in prokaryotic and eukaryotic cells. …………………… 15
B16F10 melanoma cells invasion rate of the constructed Salmonella …23
Cytotoxicity of B16F10 melanoma cells by attenuated S. Typhimurium
harboring SNTB2 miRNA vector ……………………………… 25
Attenuated S. Typhimurium harboring SNTB2 miRNA vector
induced apoptosis and ROS in B16F10 melanoma cells ……… 29
The constructed Salmonella inhibited tumor growth and prolong
survival rate in melanoma-bearing mice. ……………………… 35
The combination therapy with attenuated S. Typhimurium harboring
SNTB2 miRNA vector and radiation enhanced anticancer effect in
Melanoma models. ………………………………………… 38
DISCUSSION
REFERENCES
ABSTRACT IN KOREAN
