| 摘要: | 內質網是一個重要的胞器，主要功能為蛋白質合成、修飾即透過分泌路徑將蛋白質運送至目的地。而當內質網受到刺激時，使得蛋白質不完全摺疊而累積在內質網中，造成內質網中的壓力失去平衡，當累積到細胞無法承受則會產生所謂的內質網壓力(Endoplasmic reticulum stress)。內質網壓力主要會透過三個主要的訊息傳遞路徑，分別是ATF-6、PERK、Ire1去誘導下游基因的表現幫助蛋白質摺疊。而除了這些，也會有許多轉錄因子會參與內質網壓力反應，如NF-κB、AP-1及CHOP等。過去研究顯示，內質網壓力與某些疾病的發生極為相關，如糖尿病第二型、病毒感染、腫瘤等。因此在本次研究中，我們想要去確認在內質網壓力下NUCB-2在腫瘤細胞中過量表現的角色以及調控機制。Nucleobindin 2(NUCB-2/NEFA;DNA binding protein/EF-hand/acidic protein)，是由24個胺基酸的訊息胜肽和396個結構蛋白所組成的。根據過去文獻顯示，NUCB-2已經被證實與食慾有關，在臨床上也發現乳癌中可觀察到有過量NUCB-2的表現，且在這些高NUCB-2表現的患者在治療效果上並不佳，所以我們在本次研究想要去確認NUCB-2在腫瘤細胞中過量表現所扮演的角色以及機制。初步結果，利用tunicamycin以及brefledin A這兩種內質網壓力誘導劑在Huh-7、HepG2、MCF-7這三株細胞株以及在動物模式下，都發現有明顯NUCB-2 mRNA過量的表現。另一方面，我們進一步分析NUCB-2分佈的情況，發現NUCB-2在內質網壓力下可以入核，可能在其中調控著基因的表現，此外我們也利用shRNA來降低NUCB-2 mRNA表現量來進一步去探討在內質網壓力下誘導細胞凋亡的情況。我們會繼續朝著分析NUCB-2在內質網壓力中所扮演的角色以及確認在內質網壓力下NUCB-2的誘導機制和探討過量的NUCB-2對於化學治療處理的影響。最後期待NUCB-2對於在未來治療癌症上有所幫助。
The endoplasmic reticulum (ER) is a major compartment for proteins destined to the endo/exocytotic pathway. It is responsible for the synthesis, modification and delivery of proteins to their proper target sites within the secretory pathway and the extracellular space.. Accumulation of unfolded protein will activate ATF-6, PERK and Ire1 signaling pathways in response to ER stress. Otherwise, many transcription factors are involved inregulation of ER stress, such as NF-κB, cleaved form ATF-6, AP1, CHOP and ATF4. Previous studies have shown that many physiological conditions or disease were identified in which related to ER stress, e.g. differentiation of B-cells into plasma cells, tumor, viral infection, degenerative neuronal disorders and type II diabetes. Nucleobindin-2(NUCB-2/NEFA; DNA binding/EF-hand/acidic protein) is composed of signal peptide of 24 amino acids and a protein structure containing 396 amino acids, and NUCB-2 is distributed in brain tissue, pancreatic islet and adipose tissue. NUCB-2 has been implicated in the regulation of feeding and metabolism. Recently studies have indicated that overexpression of NUCB-2 is observed in clinical tumor samples such as gastric cancer and breast cancer. However, the effect of NUCB-2 overexpression on tumor cells is still unclear. In this study, we want to identification of the novel role and mechanism of NUCB-2 overexoression during ER stress in hepatoma cells. Our preliminary results indicated that tunicamycin or brefeldin A, two ER stress inducers, increased NUCB-2 expression in cell lines (Huh-7/MCF-7/HepG2) and animal model. NUCB-2 mRNA expression level was induced by ER stress as determined with RT-PCR and real-time PCR, and induction of NUCB-2 protein level was significantly increased by ER stress. Furthermore, nuclear translocation of NUCB-2 was observed during ER stress. The role of NUCB-2 in mediating tunicamycin-induced apoptosis was also investigated, and downregulation of NUCB-2 expression by NUCB-2 shRNA significantly promoted tunicamycin-induced apoptosis in Huh-7 cells. Taken together, we hope to identify relationship between NUCB-2 and tumor development in response to ER stress, and these results could provide an important insight in tumorgenesis and chemotherapy. Consequently, this mechanistic research may provide a molecular basis to develop a potent pharmaceutical agent in clinical use. |
