Localizomics study in TPA-induced HL-60 differentiation into macrophages and the effects of microRNAs miR130a and miR27b.
Date Issued
2007
Date
2007
Author(s)
Chang, Yu-Wei
DOI
zh-TW
Abstract
在過去的幾十年問，HL一60這株細胞株是一種非常廣泛應用來研究分化治療應用在M3次分類的的急性原骨髓型白血病以及造血幹細胞分化成巨噬細胞的模式細胞。12一鄰一四葵酸一佛波醇一13一乙酸（TPA）是一種甘油二醋（DAG）的類似物，能f夠活化蛋白質激醃C進而促使HL一60細胞進行分化並失去分裂增生的能力。過去幾年有不少研究透過去氣核醣核普酸微陣列以及蛋白質體學來探討找尋什麼樣的機制牽涉到12一鄰一四葵酸一佛波醇一13一乙酸誘導HL一60細胞株分化的過程。在此，我們利用分離細胞膜與細胞質片段的蛋白質透過二維電泳及質譜儀的技術將之分離來研究蛋白質體學中非常重的次細胞區域學。我們在二維電泳膠上發現在細胞質片段有三個點其表現量是受到12一鄰一四葵酸一佛波醇一13一乙酸抑制，有四個在細胞質片段上以及八個在細胞膜片段上的點表現量受到12一鄰一四葵酸一佛波醇一13一乙酸誘導。利用穿膜區域預測軟體以及次細胞區域預測軟體進行預測，結果顯示有四種蛋白是位在粒線體、有一種蛋白質是位在內質網還有一種穿膜蛋白質則位在溶醃體。我們推測粒線體在HL一60分化的過程中扮演很重要的角色。一款micr0RNA預測軟體Milink也許能夠提供一些線索來探討這些蛋白表現變化量，透過即時定量聚合醃連鎖反應我們偵測到miR27b及miR13oa在HL一60分化過程中受到抑制。
The human myeloid leukemia cell line HL-60 is a useful model to study the molecular mechanisms of differentiation therapy in M3 subtype acute promyelocytic leukemia (APL) and hematopoietic development of macrophage in decades. 12-O-tetradecanoy-phorbol-13-acetate (TPA), acting via its cellular receptor protein kinase C (PKC), induce HL-60 cells to differentiate into macrophages and lose their proliferative activity. Many approaches such as DNA microarray and proteomics study have been used to find out what kinds of mechanisms are involved in the TPA-induced HL-60 differentiation process. Here we show the results of localizomics study, a very important part of proteomics study, by extracting cytoplasimc and membrane proteins from TPA-treated HL-60 cells and then following by 2DGE and mass spectrometry techniques. We found that three down-regulated and four up-regulated proteins localized in cytoplasm and eight up-regulated proteins localized in membrane. Using transmembrane domain prediction programs and subcellular localization prediction program, pWolf, 60% proteins are found in mitochondria as non-transmembrane proteins, and 40% proteins are found in lysosome and ER as non-transmembrane and transmembrane proteins, respectively. As the results showed above, we suggest that proteins in mitochondria may play an important role in the process of differentiation. Milink, a microRNA prediction program, may provide us some hints. We also show that miR27b and miR130a were down-regulated in TPA-induced HL-60 differentiation into macrophage.
Subjects
HL60
TPA
二維電泳
微小核醣核
磷酸化蛋白
HL-60
Two-dimension gel electrophoresis
microRNA
subcellular location
Type
other
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