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Regulation of HIF1 and IL-8 using RNAi induces anti-tumor effects in hepatocellular carcinoma

Other Titles
 간암에서의 저산소활성인자 조절을 통한 항종양 효과 연구 
Authors
 최성훈 
Issue Date
2015
Description
Dept. of Medical Science/박사
Abstract
Hypoxia is the condition where tumor cells have been deprived of oxygen and has been shown to have a role of tumor development in the hepatocellular carcinoma (HCC). The hypoxia inducible factors (HIFs) are transcriptional regulators that affect a homeostatic response to oxidative stress and have been identified as a key transcription activator of angiogenesis, survival, and metabolism. IL-8 also controlled endothelia cells survival and angiogenesis. Combination effects of HIF1α and IL-8 or HIF-1β were not fully supported by currently available evidences. In these studies, I evaluated the effects of HIFs and IL-8 knockdown on angiogenesis, apoptosis and tumor growth in HCC.HCC cell lines were infected or transfected with adenoviruses expressing small-hairpin RNA (shRNA)/small interference RNA (siRNA) specific for HIFs or IL-8, cultured under hypoxic conditions (1% O2), and examined for their levels of HIFs, IL-8, angiogenesis factors and apoptotic factors using immunoblot. The effects of adenovirus-mediated shRNA-induced HIF-1α and IL-8 knockdown on tumor growth and angiogenesis were also investigated in a subcutaneous Hep3B-tumor xenograft mouse model. The expression levels of HIFs and apoptotic and growth factors were examined by real-time quantitative PCR and western blot. We also investigated apoptosis by TUNEL assay (FACS and immunofluorescence) and measured concentration of cytochrome C.HIF-1α knockdown directly repressed tumor growth, whereas IL-8 knockdown indirectly repressed tumor growth. Combined knockdown of HIF-1α and IL-8 increased survival rates of mice. Conditioned media of Combined knockdown in HCC cells also decreased microvessel density and tumor volume in vivo. Similarly, HIF-1 α and IL-8 knockdown inhibited the angiogenic effects of HCC cell-conditioned media on tube formation and invasion by endothelial cells in vitro. Inhibition of HIF-1α and IL-8 up-regulated the expression of apoptotic factors while down-regulating anti-apoptotic factors simultaneously. Knockdown of HIF-1α and IL-8 increased concentration of cytosolic cytochrome C and enhanced DNA fragmentation in HCC cell lines and HUVECs. Moreover, culture supernatant collected from the knockdown of HIF-1α and IL-8 in HCC cell lines induced apoptosis in HUVECs under hypoxia. Silencing of HIF-1β expression suppressed tumor cell growth and inhibited the expression of tumor growth-related factors, such as vascular endothelial growth factor, epidermal growth factor, and hepatocyte growth factor. Suppression of tumor cell invasion and migration was also demonstrated in HIF-1β-silenced HCC cell lines.These findings indicate that knockdown of HIFs and IL-8 inhibit angiogenesis, anti-apoptosis and tumor growth in HCC. Further development of HIFs and IL-8 shRNA/siRNA technologies could lead to effective therapies for HCC.
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Appears in Collections:
1. College of Medicine (의과대학) > Others (기타) > 3. Dissertation
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/145723
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