分类： 生物学 >> 生物物理学 提交时间： 2016-05-11
摘要： Long noncoding RNAs (IncRNAs), which have emerged in recent years as a new and crucial layer of gene regulators, regulate various biological processes such as carcinogenesis and metastasis. HOTAIR (Hox transcript antisense intergenic RNA), a lncRNA overexpressed in most human cancers, has been shown to be an oncogenic lncRNA. Here, we explored the role of HOTAIR in HeLa cells and searched for proteins regulated by HOTAIR. To understand the mechanism of action of HOTAIR from a systems perspective, we employed a quantitative proteomic strategy to systematically identify potential targets of HOTAIR. The expression of 170 proteins was significantly dys-regulated after inhibition of HOTAIR, implying that they could be potential targets of HOTAIR. Analysis of this data at the systems level revealed major changes in proteins involved in diverse cellular components, including the cytoskeleton and the respiratory chain. Further functional studies on vimentin (VIM), a key protein involved in the cytoskeleton, revealed that HOTAIR exerts its effects on migration and invasion of HeLa cells, at least in part, through the regulation of VIM expression. Inhibition of HOTAIR leads to mitochondrial dysfunction and ultrastructural alterations, suggesting a novel role of HOTAIR in maintaining mitochondrial function in cancer cells. Our results provide novel insights into the mechanisms underlying the function of HOTAIR in cancer cells. We expect that the methods used in this study will become an integral part of functional studies of IncRNAs.
分类： 生物学 >> 生物物理学 提交时间： 2016-05-11
Huang, Ye
Tian, Miao
Chen, Zhuo
Ma, Yuan
Chen, Yue
Zhang, Lihe
Yang, Zhenjun
Zhang, Yichao
Zhao, Yi-Lei
Sheng, Gang
Wang, Yanli
Peng, Yihong
Huang, Ye
Huang, Ye
摘要： It has been demonstrated that passenger strand cleavage is important for the activation of RNA-induced silencing complex (RISC), which is a crucial step for siRNA-mediated gene silencing. Herein, we report that isonucleotide (isoNA) modification around the cleavage site of the passenger strand would affect the in vitro potency of modified siRNAs by altering the motion pattern of the Ago2-PAZ domain. According to western blotting, q-PCR and antiviral test results, we proved that D-isonucleotide (isoNA) modification at the position 8 of the passenger strand (siMek1-S08D), which is adjacent to the cleavage site, markedly improved the in vitro potency of the modified siRNA, whereas siRNAs with D-isoNA incorporation at position 9 (siMek1-S09D) or L-isoNA incorporation at positions 8 and 9 (siMek1-S08L, siMek1-S09L) displayed lower activity compared to native siRNA. Kinetics evaluation of passenger strand cleavage induced by T. thermophilus Ago (Tt-Ago) showed that D-isoNA modification at position 8 of the passenger strand had no significant influence on the cleavage rate, but L-isoNA modification at position 8 slowed the cleavage rate markedly. Moreover, the results of molecular dynamics simulations showed that D-isoNA modification at position 8 affected the open-close motion of the PAZ domain in the Ago/siRNA complex, which may promote the loading of RISC and release of a passenger strand cleavage product, and consequently accelerate the activation of RISC and enhance silencing activity. However, D-isoNA modification at position 9 or L-isoNA modification at position 8 or 9 exerted opposite influences on the motion of the Ago-PAZ domain.
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